group 3 late embryogenesis abundant proteins from embryos

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Group 3 late Embryogenesis abundant proteinsfrom embryos of Artemia francisana : molecularcharacteristics, expression and functionLeaf Chandra BoswellLouisiana State University and Agricultural and Mechanical College

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GROUP 3 LATE EMBRYOGENESIS ABUNDANT PROTEINS FROM

EMBRYOS OF ARTEMIA FRANCISCANA: MOLECULAR

CHARACTERISTICS, EXPRESSION AND FUNCTION

A Dissertation

Submitted to the Graduate Faculty of the

Louisiana State University and

Agricultural and Mechanical College

in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy

in

The Department of Biological Sciences

by

Leaf Chandra Boswell

B.S. Biological Sciences, Louisiana State University, 2005

December 2013

ii

For my family, for their continuous love and support

in all aspects of my life.

iii

ACKNOWLEDGMENTS

First I would like to thank my mentor, Dr. Steven Hand for his guidance in

performing research and in the presentation of data. I would also like to thank Dr.

Michael Menze (Eastern Illinoise University) for helping me learn the fundamentals of

lab work and for helpful discussions in many areas of this project. Assistance with DNA

sequencing was provided by Dr. Scott W. Herke, manager of the Genomics Facility in the

Department of Biological Sciences at LSU. The Mass Spectrometry Facility in the

Department of Chemistry at LSU is also acknowledged for sample analysis. I would like

to thank Dr. Ted Gauthier for helpful advice regarding circular dichroism experiments. I

also thank Dr. Simon Chang for graciously providing PFK used in the protein

stabilization studies. Staff of the LSU Socolofsky Microscopy Center, especially Dr.

Holly Hale-Donze, is acknowledged for assisting with the imaging studies. Thanks are

extended to Dr. Hal Holloway and the LSU School of Veterinary Medicine Histology

Laboratory for paraffin embedding, sectioning and related processing. This study was

supported by National Science Foundation grant IOS-0920254.

iv

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ............................................................................................... iii

LIST OF TABLES ...............................................................................................................v

LIST OF FIGURES ........................................................................................................... vi

ABSTRACT ..................................................................................................................... viii

CHAPTER 1 INTRODUCTION .......................................................................................10

1.1 Research Aims of This Dissertation ................................................................17

CHAPTER 2 QUANTIFICATION OF CELLULAR PROTEIN EXPRESSION

AND MOLECULAR FEATURES OF GROUP 3 LEA PROTEINS

FROM EMBRYOS OF ARTEMIA FRANCISCANA .............................................19

2.1 Introduction ......................................................................................................19

2.2 Methods............................................................................................................22

2.3 Results ..............................................................................................................29

2.4 Discussion ........................................................................................................38

CHAPTER 3 GROUP 3 LEA PROTEINS FROM EMBRYOS OF ARTEMIA

FRANCISCANA: STRUCTURAL PROPERTIES AND PROTECTIVE

ABILITIES DURING DESICCATION ................................................................43

3.1 Introduction ......................................................................................................43

3.2 Methods............................................................................................................46

3.3 Results ..............................................................................................................51

3.4 Discussion ........................................................................................................61

CHAPTER 4 INTRACELLULAR LOCALIZATION OF GROUP 3 LEA

PROTEINS IN EMBRYOS OF ARTEMIA FRANCISCANA ................................66

4.1 Introduction ......................................................................................................66

4.2 Methods............................................................................................................69

4.3 Results ..............................................................................................................72

4.4 Discussion ........................................................................................................77

CHAPTER 5 SUMMARY AND FUTURE DIRECTIONS ..............................................81

LITERATURE CITED ......................................................................................................87

VITA ................................................................................................................................101

v

LIST OF TABLES

Table 2.1 Mass spectrometry confirms that the two molecular mass forms of

AfrLEA2 share sequence similarity with the bona fide purified protein ...............31

vi

LIST OF FIGURES

Figure 1.1 mRNA expression profile for the gene Afrlea3m from A. franciscana

embryos ..................................................................................................................14

Figure 2.1 Western blot analyses of purified recombinant proteins and various

extracts from Artemia franciscana .........................................................................30

Figure 2.2 PCR products amplified with cDNA prepared from diapause embryos

of A. franciscana ....................................................................................................32

Figure 2.3 Sequence comparisons for the four cDNA sequences amplified with

primers designed against Afrlea3m ........................................................................33

Figure 2.4 Quantification of AfrLEA2 protein in extracts of A. franciscana by

Western blot analysis .............................................................................................34

Figure 2.5 AfrLEA2 concentrations from diapause through 8 h of pre-emergence

development ...........................................................................................................35

Figure 2.6 Quantification of mitochondrial LEA proteins by Western blot analysis

in heat-treated extracts of A. franciscana ..............................................................36

Figure 2.7 Protein concentrations of AfrLEA3m_43, AfrLEA3m, and

AfrLEA3m_29 for diapause, 0-8 h of pre-emergence development, and

24 h nauplius larvae ...............................................................................................37

Figure 3.1 CD spectra of recombinant AfrLEA2 in the hydrated state, in the

presence of SDS and TFE, and after desiccation ...................................................52

Figure 3.2 Structural composition of recombinant AfrLEA2 as calculated from the

respective CD data with algorithms described in the Methods section .................52

Figure 3.3 CD spectra of recombinant AfrLEA3m in the hydrated state, in the

presence of SDS and TFE, and after desiccation ...................................................53

Figure 3.4 Structural composition of recombinant AfrLEA3m as calculated from

the respective CD data with algorithms described in the Methods section ...........53

Figure 3.5 CD analysis of BSA in the hydrated state, in the presence of SDS and

TFE, and after desiccation .....................................................................................55

Figure 3.6 Structural composition of BSA as calculated from the respective CD

data with algorithms described in the Methods section .........................................55

vii

Figure 3.7 Residual LDH activity after desiccation for one week without additives

(control) or in the presence of different protectants ...............................................57



Figure 3.9 Residual PFK activity after desiccation for 24 h without additives


Figure 3.10 Residual PFK activity after desiccation for 24 h without additives


Figure 3.11 Residual CS activity after two 24 h bouts of drying without additives




Figure 4.1 Embryos of A. franciscana visualized at 0 h of pre-emergence

development by differential interference contrast (DIC) microscopy ...................73

Figure 4.2 Subcellular localization of AfrLEA2 in embryos of A. franciscana

visualized at 0 h of pre-emergence development ...................................................74

Figure 4.3 Subcellular localization of AfrLEA2 in embryos of A. franciscana

visualized after 6 h of pre-emergence development ..............................................75

Figure 4.4 Subcellular localization of AfrLEA3m (and its closely related variants

recognized by AfrLEA3m antibody) in embryos of A. franciscana

visualized at 0 h of pre-emergence development ...................................................76

viii

ABSTRACT

Late Embryogenesis Abundant (LEA) proteins are highly hydrophilic,

intrinsically disordered proteins whose expression has been correlated with desiccation

tolerance in anhydrobiotic organisms. Embryos of the brine shrimp, A. franciscana,

contain high titers of group 3 LEA proteins during desiccation-tolerant stages such as

diapause and pre-emergence development. Here I report the sequencing of three novel

variants of AfrLEA3m mRNA (Afrlea3m_47, Afrlea3m_43 and Afrlea3m_29), whose

deduced protein sequences are predicted to localize to the mitochondrion. These mRNAs

are very similar to Afrlea3m, but each has a stretch of sequence that is absent in at least

one of the others. In addition Afrlea3m_43 has five single nucleotide changes scattered

across its sequence, and Afrlea3m_47 and Afrlea3m_43 have three single nucleotide

differences in the section of sequence shared only by these two variants.

Protein expression for AfrLEA2, AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29

is highest in diapause embryos and decreases throughout development to their lowest

levels in desiccation-sensitive nauplius larvae. This pattern of protein expression is in

agreement with previously reported mRNA expression for AfrLEA2 and AfrLEA3m and

supports a role for LEA proteins in desiccation tolerance of embryos. When adjustment

is made for mitochondria matrix volume, the effective concentrations of cytoplasmic

versus mitochondrial group 3 LEA proteins are similar in vivo, and the values provide

guidance for the design of in vitro functional studies with these proteins. Investigations

of protein secondary structure show AfrLEA2 and AfrLEA3m to be intrinsically

disordered in solution and that they gain structure during desiccation and in the presence

ix

of the solvents TFE and SDS. I also show that during drying recombinant AfrLEA2 and

AfrLEA3m confer protection to three desiccation-sensitive enzymes (lactate

dehydrogenase, phosphofructokinase and citrate synthase). The degree of protective

ability was found to depend on the target enzyme chosen. The strongest degree of

stabilization was observed when a given LEA protein was used in the presence of the

stabilizing sugar trehalose, which is naturally accumulated by A. franciscana embryos.

Finally, AfrLEA2 is shown by immunohistochemistry to reside in the cytoplasm and

nucleus of embryonic cells of A. franciscana, and the AfrLEA3m proteins are localized

to the mitochondrion. The presence of LEA proteins in multiple subcellular

compartments suggests a requirement to protect biological structures in many areas of a

cell in order for an organism to survive desiccation stress.

10

CHAPTER 1

INTRODUCTION

Late Embryogenesis Abundant (LEA) proteins are historically predicted to

function in desiccation tolerance, mainly based on the observation that their expression

correlates to desiccation tolerant stages (Tunnacliffe and Wise 2007; Hand et al. 2011).

This proposed role in desiccation tolerance is directly supported by studies such as those

by Gal et al. (2004), and Battista et al. (2001), which show reduced LEA protein

expression, in a nematode and a bacterium respectively, decreases tolerance of the

organism to water stress. Specific roles suggested for LEA proteins include stabilization

of sugar glasses (vitrified, noncrystalline structure in cells promoted by sugars like

trehalose) (Wolkers et al. 2001; Hoekstra 2005; Shimizu et al. 2010), protein stabilization

via protein-protein interaction or ‘molecular shield’ activity (Tompa and Kovacs 2010;

Chakrabortee et al. 2012), membrane stabilization (Tunnacliffe and Wise 2007; Tolleter

et al. 2010), and formation of structural networks (Wise and Tunnacliffe 2004). Of these

predicted functions, the ability of LEA proteins to protect proteins from aggregation and

preserve their activity, stabilize membranes, and strengthen sugar glasses during water

stress are best defined (see current reviews Tunnacliffe and Wise 2007; Tunnacliffe et al.

2010; Hand et al. 2011; Hincha and Thalhammer 2012). Other functions such as ion

sequestration and hydration buffers have been suggested (for review see Tunnacliffe and

Wise 2007), but the biological significance of such functions have been challenged (Hand

et al. 2011). The primary objective of research presented in this dissertation is to better

11

the current understanding for the role of LEA proteins in desiccation tolerance, and to

provide further classification of five Group 3 LEA proteins from embryos of A.

franciscana.

LEA proteins, as named by Galau et al. (1986), were originally identified in

embryos of wheat and cotton over 30 years ago (Cuming and Lane 1979; Dure et al.

1981; Galau and Dure 1981). Since their initial discovery LEA proteins have been

documented not only in plants (Cuming 1999; Hoekstra et al. 2001; Tunnacliffe and Wise

2007; Shih et al. 2008), but also bacteria (Stacy and Aalen 1998; Dure 2001; Battista et

al. 2001), cyanobacteria (Close and Lammers 1993), a slime mold (Eichinger et al. 2005),

fungi (Mtwisha et al. 1998; Sales et al. 2000; Katinka et al. 2001; Abba et al. 2006), and

more recently animals, such as nematodes (Solomon et al. 2000; Browne et al. 2002; Gal

et al. 2003, 2004; Browne et al. 2004; Tyson et al. 2007; Haegeman et al. 2009), rotifers

(Tunnacliffe et al. 2005; Denekamp et al. 2009; Denekamp et al. 2010), embryos of brine

shrimp (Hand et al. 2007; Sharon et al. 2009; Menze et al. 2009; Chen et al. 2009;

Warner et al. 2010; Warner et al. 2012; Marunde et al. 2013), springtails (Clark et al.

2007; Bahrndorff et al. 2009), and a chironomid insect larvae (Kikawada et al. 2006).

The name, Late Embryogenesis Abundant, stems from the observation that LEA proteins

accumulate late in the maturation process of plant seeds (Galau et al. 1986). In addition

many plant LEA proteins accumulate in response to abscisic acid (ABA) and water stress

(Cuming 1999; Bartels 2005). The expression of non-plant LEA proteins typically

correlate to desiccation tolerant stages (for reviews, see Tunnacliffe and Wise 2007;

Hand et al. 2011).

12

Original classification of LEA proteins by Dure et al. (1989) defined three groups

(group 1, 2, and 3) based on common amino acid domains. Dure’s original classification

has been followed by various proposed naming schemes, many of which are

contradictory, including an alternative naming scheme proposed by Dure himself, which

labels each group based on a cottonseed prototype (Dure 1993). Most LEA proteins fall

within group 1 (D-19, PFAM LEA_5), group 2 (D-11, PFAM Dehydrin) and group 3 (D-

7, PFAM LEA_4), but other minor groups have been proposed including group 4 (D-113,

PFAM LEA_1), group 5 (D-29, PFAM LEA_4), group 6 (D-34 PFAM SMP), Lea5 (D-

73, PFAM LEA_3), and Lea14 (D-95, PFAM LEA_2) (Bray 1993; Galau et al. 1993;

Bray 1994; Tunnacliffe et al. 2010). However, currently there is only consensus on

groups 1, 2, and 3. Wise (2003) re-examined LEA protein classification with newly-

developed bioinformatics tools, which led to redistribution of group 4 and 5 members to

groups 2 and 3, thus eliminating groups 4 and 5, and there is argument that group 6,

Lea5, and Lea14 should not be considered part of the LEA protein family (Tunnacliffe

and Wise 2007).

The majority of LEA proteins classified to the three major groups have a biased

amino acid composition resulting in high hydrophilicity and a consequential lack of

secondary structure in solution (Tunnacliffe and Wise 2007). The high hydrophilicity of

LEA proteins also contributes to solubility at elevated temperatures; a characteristic

widely used during purification. LEA protein similarities, beyond hydrophilicity, are

often disparate between major groups. For example LEA proteins often vary in size and

net charge even within a defined group (Tunnacliffe and Wise 2007). According to Dure

et al. (1989) group 1 LEA proteins are characterized by the presence of a hydrophilic 20-

13

amino acid motif, and group 3 proteins by the presence of multiple repeats of an 11-

amino acid motif, although often with low homology (e.g. Grelet et al. 2005; Hand et al.

2007). Group 2 proteins, often referred to as dehydrins, are characterized by the presence

of at least two out of three sequence motifs named Y, S and K by Close (1997).

Anhydrobiotic embryos of A. franciscana, a model species for studying

desiccation tolerance, possess a multitude of group 1 and group 3 LEA proteins (Hand et

al. 2007; Menze et al. 2009; Sharon et al. 2009; Chen et al. 2009; Warner et al. 2010; Wu

et al. 2011; Warner et al. 2012; Marunde et al. 2013). LEA proteins expressed by A.

franciscana have been localized to both the cytoplasm (Hand et al. 2007) and, for the first

time in animals, the mitochondrion (Menze et al. 2009). Previously the only known

mitochondrial LEA protein had been documented from seeds of the pea plant, Pisum

sativum (Grelet et al. 2005). In Chapter 2 of this dissertation, I characterize three novel

LEA proteins from A. franciscana that are located in the mitochondrion. Chapter 2 also

investigates the protein expression levels for a cytosolic AfrLEA protein (AfrLEA2), and

three mitochondrial AfrLEA proteins (AfrLEA3m, AfrLEA3m_29, and AfrLEA3m_47).

mRNA expression data, previously published for AfrLEA2 (Hand et al. 2007) and

AfrLEA3m (Menze et al. 2009), indicate that the mRNA encoding each of these two

proteins are upregulated in desiccation-tolerant developmental stages when compared to

desiccation-intolerant nauplius larvae (for example see Fig 1.1). Partially discordant

mRNA/protein expression has been reported for LEA proteins expressed by a nematode

(Goyal et al. 2005a), emphasizing the importance of characterizing expression patterns

for both mRNA and protein. In addition to general protein expression levels of four

14

Figure 1.1 mRNA expression profile for the gene Afrlea3m from A. franciscana embryos.

LEA mRNA is maintained 9-11 fold higher in the two desiccation-tolerant embryonic

stages when compared to the desiccation-intolerant nauplius larva. Double asterisks

indicate that the paired means are statistically different (t-test, p < 0.05) (redrawn from

Menze et al. 2009).

group 3 LEA proteins across several developmental time points, Chapter 2 provides

physiological concentrations for one cytosolic and three mitochondrial LEA proteins.

Cellular titers, such as the ones provided, are currently lacking for LEA proteins.

Many LEA proteins are predicted to adopt a primarily α-helical structure (Dure et

al. 1989). However, in solution a number of LEA proteins with predicted α-helical

structure are found to be predominantly unstructured (Tunnacliffe and Wise 2007;

Tunnacliffe et al. 2010; Hand et al. 2011; Hincha and Thalhammer 2012). Attempts to

15

crystallize LEA proteins have been unsuccessful (e.g. McCubbin et al. 1985), which has

been attributed to their intrinsic disorder and high degree of hydration (Tunnacliffe and

Wise 2007). Structural information for a LEA protein in the hydrated state was first

presented by McCubbin et al. (1985) indicating that Em, a group 1 LEA protein isolated

from wheat embryos, is largely unstructured in solution. Ultimately, a small group 3

LEA protein expressed in pollen of Typha latifolia was found to gain structure upon

desiccation (Wolkers et al. 2001). Investigation into the structure of LEA proteins is

important as it may help shed light on LEA protein function. Chapter 3 of this

dissertation uses circular dichroism (CD) spectroscopy in order to deduce the secondary

structure of two recombinant LEA proteins in solution, in the presence of two solvents

[sodium dodecyl sulfate (SDS) and trifluoroethanol (TFE)] and in the dry state.

The gain of structure upon desiccation has bolstered the prediction that LEA

proteins may in fact function in the dry state. Accordingly, numerous LEA proteins have

been shown to protect proteins and/or membranes upon desiccation (for recent reviews

see Tunnacliffe and Wise 2007; Tunnacliffe et al. 2010; Hand et al. 2011; Hincha and

Thalhammer 2012). The ability of a LEA protein to provide protection during water

stress through an anti-aggregation effect was first shown by Goyal et al. (2005b), for two

LEA proteins from Aphelenchus avenae. These proteins were able to prevent

aggregation and protect the activity of both CS and LDH during desiccation and

subsequent rehydration. However, unlike classical molecular chaperones, the same two

proteins afforded no protection to these two enzymes during heat stress. Therefore,

Goyal et al. (2005b) propose that LEA proteins are acting as a novel form of molecular

chaperone, for which they coin the term “molecular shield”. In this context LEA proteins

16

would prevent the aggregation of desiccation sensitive molecules by serving as a physical

barrier among sensitive molecules. The ability of two recombinant group 3 LEA proteins

from A. franciscana embryos to protect multiple enzymes of both cytosolic and

mitochondrial origin is investigated in Chapter 3. The use of LEA proteins and enzymes

from different cellular compartments allows for a novel mix-and-match approach to

distinguish whether a mitochondrial LEA protein preferentially protects mitochondrial

enzymes, while a cytosolic LEA protein preferentially protects cytosolic enzymes, or if

the protective effect of LEA proteins is universal.

Protection of subcellular components, such as the mitochondrion, is undoubtedly

necessary if a cell is to survive desiccation (Liu et al. 2005; Hand and Hagedorn 2008;

Menze and Hand 2009). This need for protection of membrane bound organelles is

supported by subcellular targeting of LEA proteins, which has been documented in many

species, both plant and animal alike (Tunnacliffe and Wise 2007; Hand et al. 2011).

Known subcellular locations for plant LEA proteins include the cytoplasm, nucleus,

chloroplast, endoplasmic reticulum, vacuoles, peroxisomes, and the plasma membrane

(Tunnacliffe and Wise 2007). Mitochondrial localization was first reported in plants for a

LEA protein (PsLEAm) from seeds of Pisum sativum (Grelet et al. 2005), and soon

thereafter in the brine shrimp A. franciscana (Menze et al. 2009). In addition to the

mitochondrion, animal LEA proteins have also been localized to the endoplasmic

reticulum, the Golgi, and secreted into the extracellular space (Tripathi et al. 2012).

Chapter 4 uses immunohistochemistry to confirm the predicted subcellular localization of

AfrLEA2 and AfrLEA3m in embryos of the brine shrimp A. franciscana.

17

1.1 Research Aims of This Dissertation

The overall objective of this dissertation is to improve our current understanding

for the role of LEA proteins in desiccation tolerance through molecular characterization,

expression data, and functional studies of group 3 LEA proteins from embryos of A.

franciscana. In Chapter 2, I sequence and characterize three novel group 3 LEA proteins

from embryos of A. franciscana, which we name AfrLEA3m_29, AfrLEA3m_43, and

AfrLEA3m_47. Through sequencing, I illustrate these three LEA proteins to share

sequence similarities with AfrLEA3m, the original mitochondrial LEA protein reported

from A. franciscana. I performed Western blot analysis on protein extracts from several

developmental time points in order to determine if protein expression corresponded to

mRNA expression. Lastly, using Western blot analysis, I created standard curves with

known amounts of recombinant AfrLEA2 and AfrLEA3m in order to calculate

endogenous protein levels for AfrLEA2, AfrLEA3m, AfrLEA3m_29 and AfrLEA3m_43.

I also provide evidence that endogenous AfrLEA2 exist primarily as a dimer in vivo.

The focus of Chapter 3 is the secondary structure of recombinant AfrLEA2 and

AfrLEA3m, as well as the ability of these two LEA proteins to protect the activity of

desiccation sensitive proteins during drying and subsequent rehydration. I use circular

dichroism spectroscopy to resolve the secondary structure of AfrLEA2 and AfrLEA3m

both in solution and after desiccation, as well as to test the effects of SDS, TFE on LEA

protein structure. I then evaluate the protective ability of AfrLEA2 and AfrLEA3m by

using a novel mix-and-match technique. Through use of this mix-and-match technique I

investigate whether LEA proteins provide universal protection to multiple enzymes or

18

perhaps a cytosolic LEA protein preferentially protects cytosolic enzymes, while a

mitochondrial LEA protein preferentially protects mitochondrial enzymes.

Chapter 4 focuses on the intracellular localization of AfrLEA2, and the four

mitochondrial LEA proteins, AfrLEA3m, AfrLEA3m_29, AfrLEAm_43 and

AfrLEA3m_47. All four of the aforementioned mitochondrial LEA proteins are

recognized by the antibody raised against AfrLEA3m. Therefore, the localization study

investigates the four mitochondrial LEA proteins as a group. Bioinformatics software

predict AfrLEA2 to reside in the cytosol, and AfrLEA3m to translocate to the

mitochondrion. Previous studies have confirmed that AfrLEA3m translocates to the

mitochondrion in mammalian cells (Menze et al. 2009; Li et al. 2012). However, the

intracellular locations predicted for AfrLEA2 and AfrLEA3m have not been

experimentally confirmed in embryos of A. franciscana. I use immunohistochemistry to

confirm the predicted intracellular location for AfrLEA2 and the mitochondrial LEA

proteins recognized by antibody produced against AfrLEA3m.

19

CHAPTER 2

QUANTIFICATION OF CELLULAR PROTEIN EXPRESSION AND

MOLECULAR FEATURES OF GROUP 3 LEA PROTEINS FROM

EMBRYOS OF ARTEMIA FRANCISCANA 2.1 Introduction

When considering the ability to survive water stress, the most extreme examples

are anhydrobiotic organisms, which can survive extended periods of almost complete

desiccation (Keilin 1959; Crowe and Clegg 1973; Crowe and Madin 1974; Crowe and

Clegg 1978; Clegg 2005; Watanabe 2006; Cornette and Kikawada 2011; Welnicz et al.

2011). In nature, anhydrobiotic organisms such as nematodes and tardigrades routinely

experience dehydration down to 2% tissue water (Crowe and Madin 1974; Alpert 2006),

and the brine shrimp embryo can survive even lower residual water content under

aggressive experimental drying in the laboratory (Clegg et al. 1978; Hengherr et al.

2011b, a). As research on this topic progresses, it is becoming clear that desiccation

tolerance relies on a number of different mechanisms and requires the stabilization of

individual organelles in addition to cytosolic components (Pouchkina-Stantcheva et al.

2007; Tunnacliffe and Wise 2007; Hand and Hagedorn 2008; Atkin and Macherel 2009;

Hand et al. 2011; Tripathi et al. 2012). The accumulation of low molecular weight

organic solutes, such as trehalose, is often seen in desiccation tolerant organisms. These

organic solutes aid in macromolecular protection at low water contents (Yancey et al.

1982; Yancey 2005). In addition to organic solutes, several types of protective

macromolecules are correlated with desiccation tolerance, including Late Embryogenesis

Abundant (LEA) proteins and small stress proteins like Artemia P26, Hsp 21 and Hsp 22

20

(Clegg et al. 1994; Liang et al. 1997a; Liang et al. 1997b; Willsie and Clegg 2001; Clegg

2005; Qiu and Macrae 2008a; Qiu and MacRae 2008b). Desiccation-tolerant embryos of

A. franciscana possess a multitude of LEA proteins (Hand et al. 2007; Menze et al. 2009;

Sharon et al. 2009; Chen et al. 2009; Warner et al. 2010; Wu et al. 2011; Warner et al.

2012). In the present study I sequence the mRNA of three novel AfrLEA3m variants,

and quantify protein expression for AfrLEA2, AfrLEA3m, AfrLEA3m_43, and

AfrLEA3m_29 during diapause and development in A. franciscana. I also report

evidence that cytoplasmic-targeted AfrLEA2 exists primarily as a homodimer in vivo.

Western blot analysis of mitochondria isolated from 0 h post-diapause embryos reveals

four distinct bands when probed with antiserum against AfrLEA3m that have been shown

by mass spectrometry to correspond to the three novel mRNA sequenced in this study

and AfrLEA3m (Boswell et al., 2013).

To date all of the animal LEA proteins described have been assigned to group 3

(for classification scheme see Wise 2003), with the exception of group 1 LEA proteins

discovered in A. franciscana (Sharon et al. 2009; Warner et al. 2010; Wu et al. 2011;

Marunde et al. 2013). Group 3 LEA proteins are predicted to have high alpha-helix

content, but have been found experimentally to be unfolded when fully hydrated in

aqueous solution (Goyal et al. 2003). Interestingly, Goyal et al. (2003) found that a

group 3 LEA protein from an anhydrobiotic nematode adopted a α-helical structure upon

desiccation, with a possible coiled-coil formation. Group 3 LEA proteins are

characterized as being highly hydrophilic, intrinsically unstructured proteins with an

overrepresentation of charged and acidic amino acid residues (Tunnacliffe and Wise

2007; Battaglia et al. 2008).

21

Various functions have been proposed for LEA proteins based on their natively

unfolded structure and the correlation of gene expression to desiccation tolerance.

Predicted physiological roles for LEA proteins include stabilization of sugar glasses

(vitrified, noncrystalline structure in cells promoted by sugars like trehalose) (Wolkers et

al. 2001; Hoekstra 2005; Shimizu et al. 2010), protein stabilization via protein-protein

interaction or ‘molecular shield’ activity (Tompa and Kovacs 2010; Chakrabortee et al.

2012), membrane stabilization (Tunnacliffe and Wise 2007; Tolleter et al. 2010), ion

sequestration (Grelet et al. 2005), and formation of structural networks (Wise and

Tunnacliffe 2004). Such networks of LEA proteins have been hypothesized to increase

cellular resistance to physical stresses imposed by desiccation (Goyal et al. 2003).

Experimentally, LEA proteins prevent protein aggregation, protect enzyme function, and

maintain membrane integrity during water stress (for reviews see Tunnacliffe and Wise

2007; Hand et al. 2011; Hincha and Thalhammer 2012). However, the exact mechanisms

for these protective abilities continue to be explored. Few studies attempt to rigorously

estimate the effective cellular concentrations of LEA proteins (e.g., see excellent results

for cotton seeds Roberts et al. 1993). As a consequence, some functional roles projected

from in vitro experiments may not be applicable in vivo because the concentrations used

for in vitro characterization of LEA proteins are often arbitrary and may be unrealistic.

In the present study, the titer of cytoplasmic-localized LEA protein (AfrLEA2) was 0.79

± 0.21 to 1.85 ± 0.15 mg/g cellular water across development, and the combined

mitochondrial-targeted LEA proteins (AfrLEA3m, AfrLEA3m_29, AfrLEA3m_43) was

roughly 1.2-2.2 mg/ml matrix volume for post-diapause embryos. Such estimates suggest

that the effective concentrations of cytoplasmic versus mitochondrial group 3 LEA

22

proteins are similar in vivo and provide guidance for the design of in vitro functional

studies with these proteins.

2.2 Methods

Cloning, expression and antibody production for recombinant AfrLEA2 and AfrLEA3m

The original nucleic acid sequences for Afrlea2 (GenBank accession no.

EU477187) and Afrlea3m (GenBank accession no. FJ592175) cloned from A. franciscana

embryos (Hand et al. 2007; Menze et al. 2009) were amplified from our existing A.

franciscana cDNA library. Each gene was ligated into pET-30a (an expression vector

with a T7 lac promoter; Novagen, Rockland, MA) and then Rosetta™ 2(DE3) Singles™

Competent Cells (Novagen) were transformed with the genes according to the

manufacturer’s instructions. AfrLEA2 was expressed with an N-terminal 6X-His tag,

and AfrLEA3m was expressed with a C-terminal 6X-His tag so as not to interfere with

the mitochondrial localization sequence found at the N-terminus. Expression of

recombinant AfrLEA protein was induced by the addition of 1 mM IPTG for 2-3 h, and

confirmed by SDS PAGE and protein staining with Coomassie Blue. Bacterial cells were

pelleted by centrifugation (5,000 x g, 15 min) at 4ºC and chemically lysed using

Bugbuster® Protein Extraction Reagent (Novagen) in the presence of a protease inhibitor

cocktail, P8849 (Sigma-Aldrich, St Louis, MO). After removal of cellular debris by

centrifugation, the cell lysate was subjected to affinity chromatography on a HisTrap™

FF crude column (GE Healthcare, Waukesha, WI; 1 ml or 5 ml size, depending on

experimental requirements). Affinity purification binding buffer contained 20 mM

sodium phosphate, 0.5 M NaCl, and 20 mM imidazol, pH 7.5. A step elution was

performed using an elution buffer containing 20 mM sodium phosphate, 0.5 M NaCl, and

23

0.5 M imidazol, pH 7.5. Flow rate was set at the maximum rate recommended by the

manufacturer (1 ml/min for 1 ml column, or 5 ml/min for 5 ml column). Fractions

containing recombinant protein were heat treated at 95ºC for 20 min followed by

centrifugation (20,000 x g, 30 min) to separate the soluble fraction. The soluble fraction

was dialyzed overnight against the starting buffer for anion exchange (20 mM

triethanolamine, 10 mM NaCl, pH 7.0). The sample was then applied to an anion

exchange column (HiTrap™ Q FF; GE Healthcare). The elution buffer contained 20 mM

triethanolamine, and 1 M NaCl, pH 7.0. The fractions containing pure recombinant

protein, as assessed by SDS-PAGE and protein staining, were exchanged into LEA

storage buffer (20 mM HEPES, 50 mM NaCl, pH 7.5) and concentrated using Amicon®

Ultra Centrifugal filters (Ultracel®-10K; Millipore, Billencia, MA). Antibodies were

raised in chickens against recombinant AfrLEA2 and AfrLEA3m by Aves Labs, Inc.

(Tigard, OR).

Preparation of cDNA and sequencing of additional Afrlea3m-related genes

In extracts of mitochondria isolated from A. franciscana, four protein bands were

identified with the AfrLEA3m antibody produced above (see Results). Consequently, we

suspected that multiple mRNA species might be detected with cDNA prepared from A.

franciscana embryos. Total RNA was isolated from diapause embryos using an RNeasy

Midi kit (Qiagen, Valencia, CA), and then a DyNAmo cDNA synthesis kit (New England

Biolabs, Ipswich, MA) was used for reverse transcription according to manufacturer’s

instructions. Primers for Afrlea3m amplified four products, which were cloned with a

pENTR™/D-TOPO® Cloning Kit (Invitrogen, Carlsbad, CA) as described in the

manufacturer instructions. One Shot® TOP10 Chemically Competent E. coli

24

(Invitrogen) were transformed with these genes. Direct colony PCR was performed to

screen for transformed colonies. Colonies were identified that contained each of the four

inserts, and a QIAprep 96 Turbo Miniprep Kit (Qiagen) was used to purify plasmid DNA

from each. Sequencing was conducted with BigDye terminator chemistry and an ABI

PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Molecular mass determination by SDS-PAGE

The molecular mass of recombinant and endogenous LEA proteins were

determined by SDS-PAGE as described by Hames (1998). Briefly, the log of molecular

mass for biotinylated protein standards (Cell Signaling Technology, Danvers, MA) was

plotted against relative migration distance (Rf) for the proteins after separation by SDS-

PAGE. Rf was calculated as the migration distance of a protein divided by the migration

distance of the dye front. The Rf values for LEA proteins were used to interpolate their

molecular masses from the standard curves. The reported masses are the result of six

separate measurements on three independent gels. AfrLEA2 samples were analyzed on

7% gels for optimal determination of size.

Mass Spectrometry

In-gel trypsin digestion of proteins separated by SDS-PAGE was performed as

described by Shevchenko et al. (2006). Briefly, bands of interest were excised from a

Coomassie-stained SDS gel and destained in 100 µl of 100 mM ammonium

bicarbonate/acetonitrile (1:1, vol/vol) for about 30 min. After destaining, 500 µl of 100%

acetonitrile was added to the destain mixture to dehydrate the gel pieces. The gel pieces

were then transferred to 50 µl of a trypsin cocktail (13 ng protease/µl of a solution

25

containing 10 mM ammonium bicarbonate and 10% (vol/vol) acetonitrile) and incubated

for 90 min to saturate the gel pieces with trypsin. Sequencing grade trypsin (cat. #

V5111) was obtained from Promega (Fitchburg, WI). Next 10-20 µl of 100 mM

ammonium bicarbonate was added to the trypsin cocktail, and gel pieces were incubated

overnight at 37ºC for complete protein digestion. Peptide products were extracted by

adding 100 µl of extraction solution (5% formic acid/acetonitrile (1:2, vol/vol)) to the

trypsin cocktail and incubated on a shaker for 15 min at 37ºC. The liquid fraction

containing the peptide digestion products was collected and dried in a vacuum centrifuge.

Samples were submitted to the Mass Spectrometry Facility in the Department of

Chemistry (Louisiana State University) and analyzed by LC-MS-MS on a QSTAR XL

quadrupole time-of-flight mass spectrometer (Applied Biosystems). The MS/MS data for

each protein digest was submitted for a database search using Mascot from Matrix

Sciences (Boston, MA).

Preparation of protein extracts from diapause and post-diapause embryos

Diapause embryos were collected from the surface of the Great Salt Lake (Ogden,

UT) in fall 2011. Diapause embryos were maintained at ambient temperature in 1.25 M

NaCl containing 200 units/ml nystatin, 50 mg/ml kanamycin, and 50 mg/ml penicillin-

streptomycin; were protected from light. Prior to use diapause embryos were rinsed and

incubated in 35 ppt artificial seawater (Instant Ocean; Aquarium Systems, Mentor, OH)

at room temperature with shaking (110 rpm) for 4 days (Reynolds and Hand 2004) to

allow hatching of any embryos that had broken diapause. Hatched nauplius larvae were

removed and intact diapause embryos were used for experiments. Post-diapause embryos

of A. franciscana were obtained in the dry state from Great Salt Lake Artemia (Ogden,

26

UT; grade: laboratory reference standard) and stored at -20ºC. Prior to use these

dehydrated embryos were hydrated overnight in ice-cold 0.25 M NaCl. Embryos for the

0 h time point were processed immediately after hydration at 0ºC. Other embryos were

transferred to fresh 0.25 M NaCl at 23ºC and incubated with shaking (110 rpm) to

promote pre-emergence development, and embryos were sampled at or 2, 4, 6, and 8 h.

Prior to homogenization embryos were filtered and then blotted between two sheets of

Whatman no. 41 filter paper to remove interstitial water. Blotting was performed

according to Clegg (1974). To obtain nauplius larvae, hydrated embryos were incubated

in 35 ppt artificial seawater for 24 h at 23ºC with shaking (110 rpm). Nauplius larvae

were separated from unhatched embryos and shells, and then filtered and blotted.

For quantification of AfrLEA2 by Western blot (see below), 100 mg of embryos

or 24 h nauplii were transferred directly into 1.9 ml of Laemmli sample buffer [62.5 mM

Tris-HCl (pH 6.8), 2 % SDS, 10 % glycerol, 5 % 2-mercaptoethanol (Laemmli 1970)]

and homogenized in a ground glass homogenizer for 5-7 min. The homogenate was then

heated at 95ºC for 5 min and centrifuged (10,000 x g, 10 min) to remove the insoluble

debris like shell and chitin fragments. In order to quantify the mitochondrial AfrLEA

proteins, it was necessary to first enrich these protein in extracts, because due to their

mitochondrial location these proteins comprise a smaller percentage of total cellular

protein compared to the cytosolic AfrLEA2. Accordingly, 200 mg of embryos (or 24 h

nauplii) were instead homogenized into the non-denaturing LEA storage buffer described

above. The homogenate was heated at 95ºC for 20 min and centrifuged (20,000 x g, 30

min, 4ºC) to sediment the heat-insoluble fraction. The soluble supernatant, which

contains only heat-stable macromolecules like LEA proteins, was retained and combined

27

3:1 with 4X Laemmli sample buffer. Protein concentration was obtained for all samples

using a Lowry assay as described by Peterson (1977).

Western Blot analysis

For A. franciscana samples 10 µg of total protein was loaded per lane on SDS

acrylamide gels (4 % stacking gel, 11 % resolving gel) and electrophoresed for 80 min at

125 V in a Bio-Rad mini-Protean 3 cell. Proteins were then transferred to a nitrocellulose

membrane (Bio-Rad, Hercules, CA) in a transfer buffer containing 192 mM glycine, 20%

methanol, 0.025% SDS, and 25 mM Tris; acceptable transfer was confirmed by staining

the membranes with Ponceau S. Membranes were then blocked in a 5 % fat free dry milk

solution for 1 h. Incubation with primary antibody raised against recombinant AfrLEA

protein (Aves Labs Inc) was performed overnight at 4ºC, and then blots were washed for

a total of 1 h with four changes of TBS-T (0.1% Tween 20, 20 mM Tris-HCl, 500 mM

NaCl, pH 7.6). Next the membranes were incubated for 1 h at room temperature with the

HRP-linked secondary antibody (goat anti-chicken; Aves Labs Inc) and washed as above.

Protein bands were visualized with LumiGLO chemiluminescent substrate (Cell

Signaling Technology).

Quantity One Basic 4.6.9 software (Bio-Rad Laboratories) was used to quantify

band intensities. Global background subtraction was applied to each image analyzed.

These intensities were converted to ng AfrLEA protein per band by comparing intensity

values to a standard curve generated with known amounts of pure recombinant AfrLEA

protein. Experimental samples for AfrLEA2 were normalized to -tubulin as a loading

control before any further calculations were performed. Then the values for ng

protein/band were converted to mg protein/ml embryo water based on water content data

28

previously published (Glasheen and Hand 1989). Mitochondrial AfrLEA proteins were

quantified similarly except they were not normalized to a housekeeper protein due to the

heat treatment step required prior to Western blotting. Values for mitochondrial AfrLEA

proteins were reported as µg/g wet tissue. Finally, to facilitate comparison to the

effective in vivo concentration of AfrLEA2, mitochondrial AfrLEA proteins were also

expressed as mg protein/ml matrix volume. This calculation is based upon an estimated

mitochondrial volume of 5% of the total cell volume for post-diapause embryos of A.

franciscana (Rees et al. 1989), and an estimate that matrix volume is about 50% of total

mitochondrial volume in the semi-condensed/condensed states (Hackenbrock 1968;

Scalettar et al. 1991).

Detection of glycoproteins in polyacrylamide gels

In-gel detection of glycoproteins was performed using the Periodic Acid Schiff

(PAS) method. Schiff’s fuchsin-sulfite reagent was obtained from Sigma-Aldrich and

detection was performed as described by the supplier. Briefly, gels were incubated in

fixative solution (40% ethanol and 7% acetic acid) for 1.5 h with four changes of solution

and then left in the fixative overnight. Afterwards, the fixative solution was again

refreshed twice, each followed by a 30 min incubation. Oxidation of glycoprotein bands

was accomplished by immersing gels in a solution containing 1% periodic acid and 3%

acetic acid for 60 min. Gels were then washed ten times (10 min each) to remove traces

of periodic acid before incubation in Schiff’s Reagent for 60 min in the dark. Stained

gels were washed in a solution with 0.58% potassium disulfite and 3% acetic acid to

remove any background.

29

2.3 Results

Properties of AfrLEA2 in embryos of A. franciscana

As previously reported, AfrLEA2 is a group 3 LEA protein (38.9 kDa) that is

predicted to reside in the cytoplasmic compartment (Hand et al. 2007). This predicted

location was confirmed in HepG2 cells transfected with the GFP-tagged protein (Li et al,

2012). Recombinant AfrLEA2 has a total molecular mass of 43.1 kDa (38.9 kDa plus 4.2

kDa for a 6X-His tag and associated sequence) but migrates on SDS gels at a calculated

mass of 49.3 ± 0.9 (mean ± SD; n = 3) (Fig. 2.1). This higher apparent molecular mass

with SDS-PAGE may be explained by the observation that some intrinsically disordered

proteins have a reduced binding for SDS and therefore frequently exhibit decreased

migration on SDS gels (Tompa 2002). Based on the migration of recombinant AfrLEA2,

we expect endogenous AfrLEA2 to migrate at about 44 kDa. Surprisingly, when A.

franciscana extracts are electrophoresed on an 11% polyacrylamide gel and probed with

anti-AfrLEA2 polyclonal antibody, a protein band is detected around 75 kDa (data not

shown). Upon closer examination with a 7% polyacrylamide gel to provide better

resolution of larger proteins, three bands are discernible, with calculated apparent

molecular masses of 82.0 ± 0.4, 75.3 ± 1.0, and 72.7 ± 0.5 kDa (means ± SD; n = 3) (Fig

2.1). For most developmental stages of A. franciscana where AfrLEA2 is expressed, the

82 kDa form is predominant. Based on the molecular mass and further evidence below,

we suggest that the 82 kDa protein is a dimer of the 38.9 kDa AfrLEA2, and the smaller

30

Figure 2.1 Western blot analyses of purified recombinant proteins and various extracts

from Artemia franciscana. Lane one was loaded with molecular mass standards, lane

two with 1 µg of recombinant AfrLEA2 protein, and lane three with 10 µg protein of an

extract prepared from diapause embryos. With extracts prepared from diapause embryos,

anti-AfrLEA2 antibody reveals three bands on a 7% polyacrylamide gel with apparent

molecular masses of 82.0 ± 0.4, 75.3 ± 1.0, and 72.7 ± 0.5 kDa. Arrows indicate dimers

and trimers formed by recombinant AfrLEA2.

bands may be degradation products or even processed AfrLEA2 (cf. Goyal et al. 2005a;

Kikawada et al. 2006).

Oligomers of LEA proteins resistant to SDS dissociation have been previously

documented by PAGE (Goyal et al. 2003), and we detected apparent dimers and trimers

of purified recombinant AfrLEA2 by SDS-PAGE with anti-AfrLEA2 antibodies (Fig 2.1;

97.6 kDa and 137.5 kDa), which supports the ability of the protein to form oligomers.

31

Table 2.1 Mass spectrometry confirms that the two molecular mass forms of AfrLEA2

share sequence similarity with the bona fide purified protein. Mascot ions scores are -

10*Log(P), where P is the probability that the observed match is a random event.

Individual ions scores, or combined scores where multiple peptides are identified from a

single protein, greater than 60 indicate identity or extensive homology (p<0.05).

Apparent multimers of recombinant AfrLEA2 are also visualized using an anti-6X-His

antibody (data not shown), which suggests the multimers are not non-specific products

that cross-react with AfrLEA2 antibody. While only small numbers of cytoplasmic

proteins are typically glycosylated, in-gel staining with PAS reagent did not indicate

AfrLEA2 to be glycosylated, based on the absence of PAS-positive bands of appropriate

size in embryo extracts enriched for LEA proteins by heat purification. In-gel trypsin

digests were performed on gel slices from the regions where the dimer and monomer

migrate, and the peptides were analyzed by mass spectrometry (LC-MS-MS). One or

more peptide fragments from both areas were found to share sequence identity to bona

fide AfrLEA2 based on robust scores (Table 2.1). Finally, with Afrlea2-specific

primers, PCR amplification was performed on cDNA prepared from diapause embryos.

Only a single 1.1 kb product was generated, which is the expected size of mRNA

encoding for AfrLEA2 (364 amino acids) (Fig. 2.2). The combined evidence indicates

that the predominant 82 kDa protein visualized on Western blots with AfrLEA2 antibody

is a dimer of the 38.9 kDa AfrLEA2.

AfrLEA2 monomer AfrLEA2 dimer Score Peptide Score Peptide

47 K.SISDAAYFTGK.G 83 K.GIGETVKADADVVEGMASTGYEK.L

56 K.INAIQTPEEMDHER.L

101 K.GIGETVKADADVVEGMASTGYEK.L

32

Figure 2.2 PCR products amplified with cDNA prepared from diapause embryos of A.

franciscana. Lane one was loaded with a DNA ladder (kb) and lanes two and three with

products from PCR reactions as indicated. Primers designed against Afrlea2 yielded a

single product of approximately 1.1 kb (Afrlea2 lane). Primers designed against Afrea3m

amplified four products that migrate at 1.2, 1.0, 0.9 and 0.7 kb (Afrlea3m lane)

Multiple independently-encoded variants of AfrLEA3m

With Afrlea3m primers and cDNA template prepared from diapause embryos,

PCR amplification generated four products (Fig. 2.2). In order to gain more insight into

33

Figure 2.3 Sequence comparisons for the four cDNA sequences amplified with primers

designed against Afrlea3m. The green boxed regions (nucleotide 1 – 84) indicate the

mitochondrial leader sequence that is shared by all four cDNAs. Each of the other four

boxed regions (yellow, red, light blue, dark blue) indicate stretches of virtually-identical

sequence that are present in some of the cDNAs but absent in at least one.

AfrLEA3m_47: yellow box = nucleotide 466 – 801, red box = nt 989 – 1036, dark blue

box = nt 1037 – 1084 and light blue box = nt 1085 – 1183. AfrLEA3m_43: yellow box =

nt 466 – 801 and light blue box = nt 989 – 1087. AfrLEA3m: red box = nt 653 – 700,

dark blue box = nt 701 – 748 and light blue box = nt 749 – 847. AfrLEA3m_29: red box

= nt 653 – 700. None of the mRNA variants contain sequence that was not shared by at

least one other isoform. Solid black lines indicate virtually-identical sequence shared by

all four cDNAs

the nature of these mRNAs, each of the four bands amplified from cDNA was cloned and

sequenced. The protein encoded by the 924 bp cDNA is identical to AfrLEA3m that was

previously reported (Menze et al. 2009). Like AfrLEA3m, all three deduced proteins

(AfrLEA3m_47, AfrLEA3m_43 and AfrLEA3m_29; suffixes indicate masses deduced

from cDNA sequence) possess mitochondrial targeting sequences and thus are predicted

to localize to the mitochondrion with high probability (Target P, MitoProt II, and

Predator). The deduced protein sequences for the three new isoforms are highly

hydrophilic as determined by Kite and Doolittle hydropathy plots (data not shown). The

sequences for all four cDNAs are very similar, but each has a stretch of sequence that is

absent in at least one of the others (Fig. 2.3). In addition Afrlea3m_43 has five single

nucleotide changes scattered across its sequence that do not match the other three

cDNAs(data not shown), and Afrlea3m_47 and Afrlea3m_43 have three single nucleotide

differences in the section of sequence shared by these two variants (Fig. 2.3, yellow bar).

34

Thus we conclude that these mRNA species are independently encoded, but closely

related variants, of Afrlea3m.

Protein expression of AfrLEA2 during development

By comparing band intensities to a standard curve created using recombinant

AfrLEA2 (Fig. 2.4), we were able to estimate the quantity of AfrLEA2 (subforms

combined) present in each sample. These values were then converted to mg LEA protein

per ml embryo water. Because AfrLEA2 is a cytoplasmic-localized protein, this

concentration unit provides a meaningful estimate of its effective titer in vivo. AfrLEA2

is most abundant in diapause and decreases throughout development to undetectable

levels in 24 h nauplius larvae (Fig. 2.4A and Fig 2.5). This pattern is in agreement with

the mRNA expression profile reported for Afrlea2 (Hand et al. 2007) and further supports

Figure 2.4 Quantification of AfrLEA2 protein in extracts of A. franciscana by Western

blot analysis. A) Expression levels for AfrLEA2 are shown at various stages of the life

cycle [diapause; pre-emergence development (hours 0, 2, 4, 6, 8,); and nauplius larvae

(24 h)]. AfrLEA2 is most abundant in diapause and decreases throughout development to

undetectable levels in nauplius larvae. α-tubulin is included as a loading control for each

time point. B) Concentration dependency of recombinant AfrLEA2 as measured with

anti-AfrLEA2 antibody. C) Standard curve for recombinant AfrLEA2 (R2 = .986).

35

Figure 2.5 AfrLEA2 concentrations from diapause through 8 h of pre-emergence

development. All values were normalized to α-tubulin. Asterisks (*) indicate that the

means are statistically different from diapause values (1-way-ANOVA, Tukey, P < 0.05).

Conversion of AfrLEA2 concentrations to ‘per ml embryo water’ is based on water

content data previously published (Glasheen and Hand 1989)

The role for LEA proteins in desiccation tolerance in A. franciscana, a physiological

feature that disappears beginning at the larval stage. Based on these results there are 1.85

± 0.15 mg (mean ± SD; n = 3) of AfrLEA2 per ml embryo water in diapause embryos

(Fig. 2.5), or 5.05 µg of AfrLEA2 per mg total embryo protein.

Quantification of protein expression for AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29

during development

As described above, a standard curve (Fig. 2.6) was used to determine the

concentrations of AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29 present in each

sample. The low amount of expressed AfrLEA3m_47 made it problematic to quantify.

36

Figure 2.6 Quantification of mitochondrial LEA proteins by Western blot analysis in

heat-treated extracts of A. franciscana. A) Expression levels for mitochondrial LEA

proteins are shown at various stages of the life cycle [diapause, pre-emergence

development (hours 0, 2, 4, 6, 8); and nauplius larvae (24 h)]. AfrLEA3m isoforms are

most abundant in diapause and decrease throughout development to the lowest levels

observed, which are found in nauplius larvae. Equal amounts of total protein in extracts

were loaded for each time point. B) Concentration dependency of recombinant

AfrLEA3m as measured with anti-AfrLEA3m antibody. C) Standard curve for

recombinant AfrLEA3m. (R2 = .99)

Because these three proteins are localized to the mitochondrial compartment, the

concentrations of each mitochondrial AfrLEA were initially expressed as µg protein per g

wet tissue (Fig. 2.7). There were significant decreases in content for each of the three

proteins from diapause through pre-emergence development (0 – 8 h), and then even

further decreases occurred in 24 h nauplius larvae (Fig. 2.7). These trends in protein

expression mirror the mRNA expression data reported for Afrlea3m (Menze et al. 2009).

Finally, to estimate an effective in vivo concentration, the amount of combined

mitochondrial-targeted LEA proteins (AfrLEA3m, AfrLEA3m_29, and, AfrLEA3m_43)

was also expressed as mg protein/ml mitochondrial matrix volume. For post-diapause

37

Figure 2.7 Protein concentrations of AfrLEA3m_43, AfrLEA3m, and AfrLEA3m_29 for

diapause, 0-8 h of pre-emergence development, and 24 h nauplius larvae. For each LEA

protein, the asterisks (*) indicate that the means for pre-emergence and larval stages are

statistically different from their respective diapause value (1-way-ANOVA, Tukey, P <

0.05). AfrLEA3m_47 was too faint to reliably quantify across development.

embryos, this value is approximately 1.2 mg protein/ml matrix volume. Considering that

the mitochondrial density in cells is comparable between diapause and post-diapause

embryos (Reynolds and Hand, 2004), the value would be approximately 2.2 mg/ml

during diapause. Interestingly, such estimates suggest that the effective concentrations

of cytoplasmic versus mitochondrial group 3 LEA proteins are comparable in vivo and

provide guidance for the design of in vitro functional studies with these proteins.

38

2.4 Discussion

In the present study we have characterized the protein expression levels for four

group 3 LEA proteins throughout A. franciscana development. The four mitochondrial

LEA mRNA studied here share similar sequence identity, but contain multiple single

base pair differences, so we predict that these mRNA are encoded by separated genes.

We have previously reported mRNA expression for AfrLEA2 (Hand et al. 2007) and

AfrLEA3m (Menze et al. 2009) to be highest in desiccation tolerant stages (diapause and

post-diapause embryos) when compared to desiccation-sensitive nauplius larvae. The

protein expression patterns reported in this study are in agreement with the mRNA

expression and provide further evidence that LEA proteins play a role in desiccation

tolerance.

Finally, we have also experimentally measured physiologically-relevant quantities

of a cytoplasmic (AfrLEA2) and three mitochondrial (AfrLEA3m_43, AfrLEA3m, and

AfrLEA3m_29) LEA proteins across development in A. franciscana. Values of this type

are useful to more accurately evaluate whether concentration-dependent properties

identified for LEA proteins in vitro are relevant for in vivo settings.

Our results provide evidence that endogenous AfrLEA2 exists primarily as a

dimer in A. franciscana embryos. The presence of SDS-resistant oligomers have been

previously reported for LEA proteins and other hydrophilic proteins (Goyal et al. 2003;

Maskin et al. 2007; Bahrndorff et al. 2009), but this is the first report to our knowledge of

a LEA protein existing primarily as a dimer in vivo. In addition to molecular mass, key

evidence for the 82 kDa dimer includes the amplification with Afrlea2-specific primers of

only a single PCR product, and that this product is of the correct size for a mRNA

39

encoding the 38.9 kDa monomer. Further, a few bases upstream of the Afrlea2 gene is

sequence for a translational stop codon. Mass spectrometry of the dimer supports

sequence similarity to the monomer. At high concentrations, our purified recombinant

AfrLEA2 will form dimers and trimers in vitro that are resistant to SDS dissociation. The

smaller bands (75.3 and 72.7 kDa) recognized by the anti-AfrLEA2 antibody could be

processed forms of the AfrLEA2 dimer, as reported by Goyal et al. (2005a) for a Group 3

LEA protein (AavLEA1) from the nematode Aphelenchus avenae. These authors provide

evidence for non-random cleavage that could increase the specific activity of the LEA

protein, whereby two shorter proteins are more effective molecular shields than one

larger one. Alternatively, intrinsically disordered proteins, such as LEA proteins, are

susceptible to random degradation due to their unstructured nature (Receveur-Brechot et

al. 2006; Uversky and Dunker 2010).

Unlike the results for Afrlea2, four distinct bands were amplified from cDNA of

diapause embryos with primers designed for Afrlea3m. While general architectural

features of the coding sequences display similarities that include sequence identity at

their N-termini (Fig. 3), the multiple single-nucleotide differences distributed across the

sequences preclude splice variants as an explanation and suggest these four mRNAs are

products of separate, independent genes. A similar case is seen in rotifers, where two

LEA mRNAs are very similar but arise from two individual genes on different

chromosomes (Pouchkina-Stantcheva et al. 2007). Also, very similar variants of Group 1

LEA proteins have been documented in A. franciscana and attributed to independent

genes (Sharon et al. 2009; Warner et al. 2010; Warner et al. 2012; Marunde et al. 2013).

40

As research into the function of LEA proteins continues it is important to consider

their endogenous cellular titer, and how LEA protein concentration relates to proposed in

vivo functions. Considering that an individual LEA protein family can represent up to

3.86% of total cytosolic protein in plant seeds (Roberts et al. 1993), and that most

organisms express a multitude of LEAs, it is becoming apparent that LEA proteins can

embody a large proportion of total cellular protein. We have shown that AfrLEA2, one

of the multiple cytosolic LEA proteins expressed in A. franciscana, has a cellular

concentration of 1.85 mg protein/ml cell water, and three of the known mitochondrial

LEA proteins from A. franciscana have a combined concentration of 2.2 mg protein/ml

mitochondrial matrix volume. These values can be used to re-evaluate previous

predictions for LEA protein function, as well as to guide the design of future

experiments. For example, Tolleter et al. (2007) predict that LEAM, a mitochondrial

protein expressed in pea seeds, provides protection to the inner mitochondrial membrane

during desiccation. According to their calculations, LEAM would need to represent

about 0.6% of total matrix protein in order to provide protection to about one-third of the

inner membrane surface (an estimate of the protein-free area). With total matrix protein

in the range of 400 mg/ml, this predicted amount of 2.4 mg/ml LEAM is not

unreasonable based on our estimation that three mitochondrial LEA proteins from A.

franciscana embryos are present at a combined concentration of 2.2 mg/ml.

Another important consideration is the design and interpretation of in vitro studies

used to attribute functional characteristics to various LEA proteins. Knowledge of the

endogenous titers of LEA proteins is fundamental because it can be used as a starting

point for estimating mass ratios of LEA protein to target molecule in vivo. The ability of

41

LEA proteins to stabilize sugar glasses (Wolkers et al. 2001), model membranes (Tolleter

et al. 2010) and proteins (Goyal et al. 2005b) have been investigated in vitro at mass

ratios of LEA protein to target as high as 2:1, 1:3 and 40:1, respectively. Although there

may be situations where the use of endogenous levels of either LEA proteins or target

molecules are not practical (due to cost or availability), it is important to take the cellular

titers of LEA proteins into consideration when interpreting results, especially when

translating functional characteristics of LEA proteins from in vitro to in vivo conditions.

Furthermore, an open question exists as to why the presence of LEA proteins without

protective sugars is sufficient for desiccation tolerance in some anhydrobiotic species,

while in other tolerant animals, high concentrations of glass-forming sugars (e.g.,

trehalose) are preferentially accumulated during drying together with LEA proteins (cf.

Hand et al. 2011). Perhaps the absolute cellular titer of LEA proteins expressible in a

given cell type/organism governs the apparent need for trehalose.

Several reports now indicate that a multitude of LEA proteins can be expressed in

a given anhydrobiotic organism, which brings into question why it is necessary for one

organism to express so many different LEA variants. Differential subcellular targeting

may be one reason for the presence of so many LEA proteins in a single organism. To

date, plant LEA proteins have been found in the cytoplasm, mitochondrion, nucleus,

chloroplast, endoplasmic reticulum, vacuole, peroxisome, and plasma membrane

(Tunnacliffe and Wise 2007). Animal LEA proteins have also been found commonly in

the cytoplasm (for review see Hand et al. 2011) as well as multiple subcellular locations

including the mitochondrion (Grelet et al. 2005; Menze et al. 2009; Warner et al. 2010;

Warner et al. 2012), nucleus (Warner et al. 2012), endoplasmic reticulum, golgi, and

42

extracellular space (Tripathi et al. 2012). In addition to differential subcellular targeting,

LEA proteins have been shown to stabilize different classes of macromolecules during

water stress. For example, some LEA proteins provide protection to target proteins and

do not protect lipid membranes, while others stabilize lipid membranes and afford no

protection to proteins (Pouchkina-Stantcheva et al. 2007). Considered together it is

possible that differential subcellular targeting and the ability of individual LEA proteins

to stabilize different types of macromolecules may explain the necessity for multiple

LEAs within a single organism.

In conclusion, our results for differential ontogenetic expression of LEA proteins

support their involvement in desiccation tolerance in A. franciscana, and we provide

physiological protein concentrations for LEA proteins in two different cellular

compartments, the cytoplasm and the mitochondrion. Appreciating the cellular titers of

LEA proteins in different organisms is important to further our understanding of how

these proteins function, and can be used as a guide to design future in vitro experiments.

This data contributes to our growing understanding of the multitude of LEA proteins

expressed in A. franciscana (Hand et al. 2007; Menze et al. 2009; Sharon et al. 2009;

Chen et al. 2009; Warner et al. 2010; Wu et al. 2011; Warner et al. 2012), which arguably

should be considered an animal extremophile (Clegg 2011).

43

CHAPTER 3

GROUP 3 LEA PROTEINS FROM EMBRYOS OF ARTEMIA

FRANCISCANA: STRUCTURAL PROPERTIES AND PROTECTIVE

ABILITIES DURING DESICCATION

3.1 Introduction

Group 3 Late Embryogenesis Abundant (LEA) proteins are a family of proteins

accumulated by organisms, both plant and animal alike, in relation to water stress

(Tunnacliffe and Wise 2007; Tunnacliffe et al. 2010; Hand et al. 2011). Major features

of LEA proteins include high hydrophilicity and low sequence complexity (Cuming

1999; Tunnacliffe and Wise 2007; Hand et al. 2011). One well characterized function

attributed to LEA proteins is their ability to protect the activity of desiccation-sensitive

enzymes against multiple types of water stress (for recent reviews see Tunnacliffe and

Wise 2007; Tunnacliffe et al. 2010; Hand et al. 2011; Hincha and Thalhammer 2012).

Most LEA proteins are predicted to adopt a predominantly α-helical structure in the dried

state. However, investigations into LEA secondary structure reveal that the majority of

LEA proteins are predominantly disordered in solution (Wolkers et al. 2001; Goyal et al.

2003; Shih et al. 2004; Pouchkina-Stantcheva et al. 2007; Tolleter et al. 2007;

Thalhammer et al. 2010; Popova et al. 2011; Hundertmark et al. 2012; Shih et al. 2012).

Gain of structure by LEA proteins during dehydration has led to the hypothesis that LEA

proteins may function specifically in the dry state (e.g. Li and He 2009). Alternatively,

there is also evidence that LEA proteins might function as unstructured proteins in the

hydrated state. Several studies have shown that LEA proteins are able to reduce the

aggregation of polyglutamine (polyQ) or amyloid β-peptide when co-expressed in

44

mammalian cells (Chakrabortee et al. 2007; Liu et al. 2011; Chakrabortee et al. 2012a).

Marunde et al. (2013) showed that a group 1 LEA protein can improve cell viability and

mitochondrial function at very modest levels of water stress, which are unlikely to

promote substantial coiling of LEA proteins. Regardless of whether LEA proteins

function in both hydrated and dry states, structural characterization is an important step in

a comprehensive assessment of individual LEA proteins. In this chapter I investigate the

secondary structure of two group 3 LEA proteins from embryos of A. franciscana

(AfrLEA2 and AfrLEA3m) dried and in solution, as well as their capacity to adopt

secondary structure after the addition of sodium dodecyl sulfate (SDS), and

trifluoroethanol (TFE). In addition to structural studies, I tested the ability of

recombinant AfrLEA2 and AfrLEA3m, both alone and in concert with trehalose, to

afford protection to three different target enzymes during desiccation and subsequent

rehydration.

It is well documented that group 2 LEA proteins have the capability to protect

proteins against freezing (for review see Tunnacliffe and Wise 2007), and protection

during freezing has also been reported for group 3 LEA proteins, although not as

extensively as for group 2 (Honjoh et al. 2000; Goyal et al. 2005). In addition to

protection against water stress imposed by freezing, LEA proteins from groups 1, 2, and

3 can afford protection to enzymes during desiccation (Sanchez-Ballesta et al. 2004;

Grelet et al. 2005; Reyes et al. 2005; Goyal et al. 2005). The ability of LEA proteins to

protect the activity of desiccation-sensitive enzymes from the deleterious effects of

dehydration can, at least partially, be attributed to an ability to prevent enzyme

aggregation. AavLEA1, from the nematode A. avenae, prevents the desiccation induced

45

aggregation of both CS and LDH, thereby protecting the activity of these two enzymes

(Goyal et al. 2005). Goyal et al. (2005) propose that the unordered flexible structure of

LEA proteins allows them to function as a physical barrier between aggregation prone

molecules, a function which they term “molecular shield” activity. Although protection

against protein aggregation is imperative if an organism is to survive desiccation,

prevention of protein denaturation must also be considered (Tompa and Kovacs 2010).

Compared to classic chaperones, direct interactions between LEA proteins and target

molecules are not as well characterized, but evidence for loose interaction has been

reported. Cor15am from Arabidopsis thaliana is capable of direct association with LDH

in vitro as shown by crosslinking experiments (Nakayama et al. 2007). In vivo

experiments were also performed and although no stable interactions were detected,

Cor15am was found to consistently co-purify with the large and small subunits of

Rubisco after crosslinking. Chakrabortee et al. (2012b) provide further evidence for

loose interaction between AavLEA1 tagged with mCherry and a polyQ protein using

quantitative Förster resonance energy transfer.

In addition to protective macromolecules such as LEA proteins, anhydrobiotic

organisms typically accumulate organic solutes such as trehalose, which aid in

macromolecular protection during water stress (Yancey et al. 1982; Yancey 2005).

Trehalose constitutes as much as 20% dry weight of A. franciscana embryos (Crowe et

al. 1987). LEA proteins and trehalose in combination are capable of providing a

synergistic protection to target molecules (Goyal et al. 2005). It is pertinent to note here

that trehalose is not an absolute requirement for desiccation tolerance because it is not

accumulated by bdelloid rotifers (Lapinski and Tunnacliffe 2003; Caprioli et al. 2004) or

46

various tardigrades (Hengherr et al. 2008); however, trehalose undoubtedly plays a role in

the organisms in which it is accumulated. The importance of trehalose is exemplified in

one organism which accumulates the sugar by the observation that A. avenae is not able

to survive desiccation unless ample time is provided for the conversion of glycogen to

trehalose, as occurs during slow drying (Madin and Crowe 1975; Crowe et al. 1977).

3.2 Methods

Recombinant LEA Proteins from A. franciscana

Preparation and purification of recombinant AfrLEA2 and AfrLEA3m were

accomplished according to the procedures described in Boswell et al. (2013). Briefly, the

original nucleic acid sequences were amplified from our existing cDNA library from A.

franciscana, ligated into expression vectors, and then competent bacterial cells were

transformed with the genes. AfrLEA2 was expressed with an N-terminal 6X-His tag, and

AfrLEA3m was expressed with a C-terminal 6X-His tag so as not to interfere with the

mitochondrial localization sequence found at the N-terminus. Bacterial cells were lysed

in the presence of protease inhibitors, cellular debris were removed by centrifugation, and

the resulting supernatant subjected to affinity chromatography on a HisTrap™ FF crude

column (GE Healthcare, Waukesha, WI). Fractions containing recombinant protein were

heat treated and centrifuged to separate the soluble fraction. The protein samples were

then applied to an anion exchange column (HiTrap™ Q FF; GE Healthcare). After

elution, the fractions containing pure recombinant protein were exchanged into LEA

storage buffer and concentrated using Amicon® Ultra Centrifugal filters (Ultracel®-10K;

Millipore, Billencia, MA).

47

Recombinant AfrLEA2 has a total molecular mass of 43.1 kDa (38.9 kDa plus 4.2

kDa for a 6X-His tag and associated vector sequence). AfrLEA3m is a mitochondrial

LEA protein with a deduced molecular mass of 34.1 kDa, which includes the 3.2 kDa

mitochondrial targeting sequence.

Far-UV Circular Dichroism Spectroscopy

CD spectra were recorded on a Jasco J-815 spectropolarimeter (Jasco Analytical

Instruments, Easton, MD). The pathlength was 0.1 cm and measurements were taken for

wavelengths from 190 – 250 nm. Spectra were measured at a protein concentration of

0.14 mg/ml for recombinant AfrLEA2, 0.164 mg/ml for recombinant AfrLEA3m and

0.164 for bovine serum albumin (BSA). The buffer (blank) spectrum was subtracted

from each sample spectrum. After blank subtraction each spectrum was converted to

mean residue ellipticity and smoothed using the Savitzky-Golay method (Savitzky and

Golay 1964) with a convolution width of nine. Buffer subtraction, conversion to mean

residue ellipticity, and smoothing was performed using the Spectra Manager Software

(Jasco Analytical Instruments). For measurements of dried proteins, 50 µl of protein

solution (at the respective concentrations above) were dried on one side of a demountable

cuvette overnight in a drybox containing the desiccant Drierite (W.A. Hammond Drierite

Co. Ltd., Xenia, OH). A pathlength of 0.01 cm was used for conversion of dry data to

mean residue ellipticity.

Secondary structure analyses were performed with the DICHROWEB Web server

(Whitmore and Wallace 2004, 2008) using the following algorithms: CONTINLL

(Provencher and Glockner 1981; van Stokkum et al. 1990), and SELCON3 (Sreerama et

al. 1999; Sreerama and Woody 2000). Reference dataset 7 was used for all analyses

48

because this dataset is optimized for 190 – 240 nm wavelengths and contains denatured

proteins (Sreerama and Woody 2000). Protein disorder was predicted using the

PONDR® VL-XT predictor (Molecular Kinetics Inc., Indianapolis, IN) (Romero et al.

1997; Li et al. 1999; Romero et al. 2001).

Desiccation and Activity Assay of Lactate Dehydrogenase (LDH)

LDH from rabbit muscle was obtained from Sigma Aldrich (St Louis, MO;

product code L2500). Before use, LDH was exchanged into LEA storage buffer (20 mM

Hepes, 50 mM NaCl, pH 7.5) using Amicon® Ultra Centrifugal filters (Ultracel®-10K;

Millipore, Billencia, MA). Then 10 µl droplets of 50 µg/ml LDH, with or without

protectants, were dried in 1.5 ml microcentrifuge tubes at room temperature in a drybox

containing Drierite for one week. Samples were re-hydrated with 20 µl of LEA storage

buffer (diluted two-fold) for 1 h on ice. Control assays of LDH activity were performed

prior to desiccation by adding 5 µl of LDH sample (50 µg/ml) to a final reaction volume

of 1.0 ml, which contained 0.2 M Tris-HCl buffer (pH 7.3), 660 µM NADH and 3 mM

sodium pyruvate. LDH activity after desiccation was measured as described for controls,

except that 10 µl of LDH sample were added to account for the two-fold dilution of the

enzyme during rehydration. Change in A340 was recorded for 1.5 min, and LDH activity

was reported as a percentage of the rate measured for non-dried controls. Each sample

was compared to control values that contained the same mixture of protectants in order to

account for an observed increase in LDH activity in the presence of higher concentrations

of protectant protein. It is appropriate to note that the use of LDH in droplets at ≤ 20

µg/ml yields artifactual results because of non-specific binding of LDH protein to vial

surfaces and time-dependent inactivation that can readily be detected with control

49

samples. Reported values are the average of three separate drying trials each with three

nested replicates (n = 9).

Desiccation and Activity Assay Phosphofructokinase (PFK)

The PFK used in this study was a purified recombinant form of the rabbit muscle

enzyme and was a generous gift from Dr. Simon Chang. Prior to use, PFK was

exchanged into a 100 mM sodium phosphate buffer (pH 8.0) containing 1 mM EDTA

and 5 mM dithiothreitol (DTT) using Amicon® Ultra Centrifugal filters. Then 10 µl

droplets of 150 µg/ml PFK, with or without protectants, were dried in 1.5 ml

microcentrifuge tubes at room temperature in a drybox containing Drierite for 24 h.

Samples were then rehydrated with 20 µl of 50 mM sodium phosphate buffer (pH 8.0)

containing 0.5 mM EDTA and 2.5 mM DTT for 1 h on ice.

Activity was assayed essentially as described by Bock and Frieden (1974).

Briefly, reactions (1 ml assay volume) were performed at 25ºC in a 42 mM Tris-acetate

buffer (pH 8.0) containing 51 mM KCl, 5.1 mM NH4Cl with final concentrations of 0.16

mM NADH, 2 mM ATP, 2 mM fructose-6-phosphate, 2 mM magnesium acetate, 2.5

units glycerol-3-phosphate dehydrogenase, 25 units triosephosphate isomerase, and 2

units aldolase. Prior to use, all accessory enzymes (Sigma Aldrich) were exchanged into

a 100 mM Tris-acetate buffer (pH 8.0, at 6ºC) with 0.1 mM EDTA. PFK activity was

measured for control samples prior to desiccation by adding 5 µl of PFK sample (150

µg/ml) to a total reaction volume of 1 ml reaction mixture described above; for dried

samples 10 µl of PFK was added to account for the two-fold dilution of the enzyme

during rehydration. Change in A340 was recorded for 2 min, and PFK activity was

reported as a percentage of the rate measured for non-dried controls. Each sample was

50

compared to control values that contained the same mixture of protectants in order to

account for an observed increase in PFK activity in the presence of higher concentrations

of protectant protein. Reported values are the average of three separate drying trials each

with three nested replicates (n = 9).

Desiccation and Activity Assay of Citrate Synthase (CS)

CS from porcine heart was obtained from Sigma Aldrich (product code: C3260).

Before use, CS was exchanged into 1 M Tris-HCl (pH 8.1) using Amicon® Ultra

Centrifugal filters. Then 10 µl droplets of 50 µg/ml CS, with or without protectants, were

dried at room temperature in a drybox containing Drierite for 24 h. After one round of

drying, each sample was resuspended with 10 µl H2O and dried a second time for 24 h.

Double dried samples were re-hydrated with 20 µl of 0.5 M Tris-HCl (pH 8.1) for 1 h on

ice. Control CS activity assays were performed prior to desiccation by adding 5 µl of CS

sample (50 µg/ml) to a final reaction volume of 1 ml, which contained 0.1 M Tris-HCl

buffer (pH 8.1), 0.1 mM 5,5’-dithiobis-(2-nitrobenzoic acid) and 0.2 mM acetyl-CoA.

The reaction was initiated by the addition of 0.5 mM oxaloacetate. CS activity after

desiccation was measured as described for controls, except that 10 µl of CS sample were

added to a final reaction volume of 1 ml in order to account for the two-fold dilution of

the enzyme during rehydration. Change in A340 was recorded for 2.0 min, and CS

activity was reported as a percentage of the rate measured for non-dried controls. Each

sample was compared to control values that contained the same mixture of protectants in

order to account for an observed increase in CS activity in the presence of higher

concentrations of protectant protein. Reported values are the average of three separate

drying trials each with three nested replicates (n = 9).

51

3.3 Results

Secondary structure of LEA proteins

Intrinsic disorder predicted by PONDR was 39.84% for AfrLEA2 and 37.13% for

AfrLEA3m. These predictions of disorder as well as secondary structure predictions for

AfrLEA2 and AfrLEA3m reported previously (Hand et al. 2007; Menze et al. 2009) were

tested using CD spectroscopy. In solution the CD spectrum of AfrLEA2 exhibits features

typical of a predominantly disordered, random coiled protein with a minimal ellipticity at

around 200 nm (Fig. 3.1). The presence of either 70% TFE or 2% SDS promoted

AfrLEA2 to adopt substantial α-helical structure as indicated by spectra containing a

double minimum near 208 and 222 nm and a strong positive band at 191 nm (Fig 3.1).

Secondary structure estimates confirm the apparent gain of α-helix content by AfrLEA2

in the presence of 2% SDS and 70 % TFE, with an increase from 4% α-helix content in

solution, to 24% and 41% respectively (Fig. 3.2). Dry AfrLEA2 gains structure and

exhibits a spectrum similar to that induced by the presence of TFE, indicating that

AfrLEA2 adopts a predominantly α-helical conformation upon desiccation (Fig. 3.1). In

fact desiccation of AfrLEA2 causes an increase in α-helix content from 4% in solution to

46% dry as determined from the CD spectra (Fig. 3.2).

Similar to what was observed for AfrLEA2, AfrLEA3m was found to be

predominantly disordered in solution with a propensity to adopt a more α-helical

structure in 2% SDS or 70% TFE solutions (Fig. 3.3); α-helix content increases from 2%

52

Figure 3.1 CD spectra of recombinant AfrLEA2 in the hydrated state, in the presence of

SDS and TFE, and after desiccation.

Figure 3.2 Structural composition of recombinant AfrLEA2 as calculated from the

respective CD data with algorithms described in the Methods section.

53

Figure 3.3 CD spectra of recombinant AfrLEA3m in the hydrated state, in the presence of

SDS and TFE, and after desiccation.

Figure 3.4 Structural composition of recombinant AfrLEA3m as calculated from the

respective CD data with algorithms described in the Methods section.

54

for the hydrated protein to 41% and 36 % respectively (Fig. 3.4). Drying of AfrLEA3m

does not seem to induce as large of a structural shift in the CD spectra as that seen for

AfrLEA2 (Fig. 3.3), but the secondary structure estimation indicates that α-helix content

does increase notably from 2% in solution to 18% dry (Fig. 3.4). AfrLEA3m possesses a

greater percentage of β-sheet in the dry state compared to AfrLEA2, which could explain

the lower α-helix of AfrLEA3m. Finally, it is appropriate to note that both recombinant

proteins contain approximately 10% sequence that is atypical of mature LEA protein.

AfrLEA2 contains a 4.2 kDa segment that represents the 6X-His tag and associated

vector sequence, while the AfrLEA3m protein includes a hydrophobic targeting sequence

of 3.2 kDa.

CD spectroscopy measurements were also gathered for the globular protein BSA.

BSA spectra served two purposes: first as a control protein to check the accuracy of both

CD measurements and deconvolution software, and second as a control protein to which

we could compare the structural changes of AfrLEA2 and AfrLEA3m. The spectrum of

BSA in solution was that of a predominantly α-helical protein (Fig. 3.5). Takeda et al.

(1987) estimated the secondary structure of BSA to contain 66% α-helix, 3% β-sheet and

31% random coil. Secondary structure estimates from my CD data are similar -- 56% α-

helix, 6% β-sheet, and 26% random coil. The presence of either 2% SDS or 70% TFE

did not cause a substantial shift in the BSA spectrum (Fig. 3.5). However, drying did

shift the CD spectra of BSA (Fig. 3.5), but towards a pattern that indicates a greater

percentage of random coil (26% in solution to 52% when dry; Fig. 3.6). This spectra

change is consistent with denaturation of the globular protein in the dried state.

55

Figure 3.5 CD analysis of BSA in the hydrated state, in the presence of SDS and TFE,

and after desiccation.

Figure 3.6 Structural composition of BSA as calculated from the respective CD data with

algorithms described in the Methods section.

56

Protection of enzyme activity by LEA proteins during desiccation

Drying studies with target enzyme-LEA protein combinations were performed to

test the protective abilities of the two recombinant AfrLEA proteins against dehydration-

induced damage. To evaluate whether a cytoplasmic LEA protein preferentially protects

cytoplasmic enzymes, while a mitochondrial LEA protein preferentially protects

mitochondria-localized enzymes, we used a mix-and-match design by choosing target

enzymes that reside in each of these two cellular compartments. Cytoplasmic enzymes

chosen were LDH and PFK, and the mitochondrial enzyme was CS.

After desiccation and storage in the dry state for one week, LDH when rehydrated

exhibited a residual activity of 31 ± 8% (mean ± SD) compared to non-dried control

activity (Fig. 3.7 and 3.8). The residual activity of LDH increased to 66 ± 6% (mean ±

SD) when dried in the presence of 100 mM trehalose. The protection afforded LDH by

the two LEA proteins was similar, but not statistically different from the protection seen

with BSA (1-way-ANOVA, Tukey, P > 0.05), both in the presence and absence of 100

mM trehalose (Fig. 3.7 and 3.8).

The second cytoplasmic enzyme tested was PFK. This enzyme is considered one

of the most dehydration-sensitive enzymes known (Crowe et al. 1987). It is completely

and irreversibly inactivated during freeze drying (Carpenter et al. 1987; Carpenter and

Crowe 1989) and during air drying to less ≤ 3% initial sample water (superfused with

CaSO4-dried nitrogen at 33-35oC; Carpenter and Crowe 1988), perhaps due to the

formation of inactive dimers (Crowe et al. 1992). After drying at room temperature for

24 h, PFK displayed a residual activity of 18 ± 3% (mean ± SD), which was only

57


(control) or in the presence of different protectants. The protective ability of AfrLEA2

both with and without 100 mM trehalose is compared to equivalent BSA solutions and to

100 mM trehalose alone. Values are reported as percent of the initial LDH activity

measured prior to desiccation (mean ± SD, n = 9).


(control) or in the presence of different protectants. The protective ability of AfrLEA3m


100 mM trehalose alone. Values are reported as percent of the initial LDH activity


58

Figure 3.9 Residual PFK activity after desiccation for 24 h without additives (control) or

in the presence of different protectants. The protective ability of AfrLEA2 both with and

without 100 mM trehalose is compared to equivalent BSA solutions and to 100 mM

trehalose alone. Values are reported as percent of the initial PFK activity measured prior

to desiccation (mean ± SD, n = 9).

Figure 3.10 Residual PFK activity after desiccation for 24 h without additives (control) or

in the presence of different protectants. The protective ability of AfrLEA3m both with

and without 100 mM trehalose is compared to equivalent BSA solutions and to 100 mM

trehalose alone. Values are reported as percent of the initial PFK activity measured prior

to desiccation (mean ± SD, n = 9).

59

increased to 24 ± 5% in the presence of 100 mM trehalose (Fig. 3.9 and 3.10). AfrLEA2

and AfrLEA3m again performed similarly, but both LEA proteins protected PFK far

better than BSA with or without trehalose. In fact, BSA alone did not afford any

protection to PFK, while a remarkable 98 ± 4% of control (non-dried) activity was

preserved when the enzyme was dried in the presence of 400 µg AfrLEA2 plus 100 mM

trehalose (Fig. 3.9), and 103 ± 8% of control activity was preserved when PFK was dried

in the presence of 400 µg AfrLEA3m plus 100 mM trehalose (Fig. 3.10). To our

knowledge this is the first time the protective ability of LEA proteins has been tested with

a target protein possessing such high sensitivity to desiccation.

Lastly I tested the mitochondrial enzyme CS, which was double dried prior to

each assay for residual activity. CS retained 20 ± 3% of control activity after the final

rehydration (Fig. 3.11 and 3.12). Trehalose provided a high level of protection to CS, as

evidenced by a residual activity of 76 ± 5%. As seen with the target enzyme LDH, the

protective abilities of the two LEA proteins plus 100 mM trehalose were similar to one

another, but not statistically different from the protection observed with BSA plus

trehalose (1-way-ANOVA, Tukey, P > 0.05). However, the presence of 400 µg

AfrLEA3m alone protected 69 ± 4 % of the control CS activity (Fig. 3.12), which is

significantly more protection than was provided by the same concentration of either

AfrLEA2 or BSA (1-way-ANOVA, Tukey, P < 0.05; Fig. 3.11).

60


(control) or in the presence of different protectants. The protective ability of AfrLEA2


100 mM trehalose alone. Values are reported as percent of the initial CS activity



(control) or in the presence of different protectants. The protective ability of AfrLEA3m


100 mM trehalose alone. Values are reported as percent of the initial CS activity


61

3.4 Discussion

Despite the high content of α-helix predicted by bioinformatics software (Hand et

al. 2007; Menze et al. 2009), both recombinant AfrLEA2 and AfrLEA3m are

predominantly disordered in solution. This intrinsic disorder in the hydrated state is a

common theme for LEA proteins and is attributed to their highly hydrophilic nature

(Tunnacliffe and Wise 2007). Both AfrLEA2 and AfrLEA3m adopt a higher percentage

of α-helical structure in the presence of the solvents SDS and TFE, and also are found to

gain structure in the dried state. The ability of LEA proteins to gain structure after drying

was first documented by Wolkers et al. (2001). We also show that both AfrLEA2 and

AfrLEA3m can protect sensitive enzymes from the damages imposed by desiccation,

thereby preserving enzyme activity that is typically lost after drying.

The ability of AfrLEA2 and AfrLEA3m to gain structure during drying is in

agreement with many recent studies on LEA proteins (for review see Tunnacliffe et al.

2010; Hand et al. 2011; Hincha and Thalhammer 2012; Tunnacliffe and Wise 2007). A

predominant lack of secondary structure in solution places LEA proteins within a large

class of proteins most commonly called intrinsically disordered proteins or IDPs (for

review see Uversky and Dunker 2010). Buffer solutions containing 70% TFE were used

in order to probe the conformational propensities of recombinant AfrLEA2 and

AfrLEA3m. Both recombinant LEA proteins were found to gain α-helical structure in

the presence of 70% TFE, while the secondary structure of the control protein BSA

remained effectively unchanged. Although the exact mechanism through which this

desolvating agent promotes secondary structure is debated, α-helix induction is thought to

be due largely to desolvation of the polypeptide backbone (Kentsis and Sosnick 1998).

62

Therefore, it can be argued that the promotion of α-helical structure in AfrLEA2 and

AfrLEA3m by TFE is pertinent to secondary structure gained during desiccation. SDS

was also found to also induce α-helical structure in both AfrLEA2 and AfrLEA3m,

although not as effectively as TFE.

The spectra for AfrLEA2 and AfrLEA3m in the dried state indicate that both

proteins gain structure during desiccation. AfrLEA2 adopts a substantial α-helical

structure while AfrLEA3m gains both α-helix and turns. In contrast, BSA becomes more

disordered during drying. This observed loss of structure suggests denaturation, which

would not be unusual for a globular protein like BSA. Of the three conditions evaluated

by CD in the present study (TFE, SDS, dried), the α-helix content of 59% predicted for

AfrLEA2 by bioinformatics software best matches the content observed for the dried

state (45.6%). In addition, intrinsic disorder predicted by PONDR for AfrLEA2

(39.84%) also best matches secondary structure calculated for the dry state (35.7%

random coil). This outcome is consistent with the general case that predictive algorithms,

when applied to LEA protein, often predict the structure of the dried molecule (Tolleter et

al. 2007; Thalhammer et al. 2010; Popova et al. 2011). Similar to what was seen for

AfrLEA2, intrinsic disorder predicted for AfrLEA3m (37.13%) best matches secondary

structure percent calculated for the dry state (39.05%). However, in contrast to

AfrLEA2, AfrLEA3m is predicted to contain 73% α-helix, but based on CD spectroscopy

only adopts 18% α-helix when desiccated. Secondary structure of a group 3 LEA protein

from pollen was shown to be dependent on both the speed of drying and the presence of

sucrose (Wolkers et al. 2001). Rapid drying, as opposed to the slow drying performed for

AfrLEA2 and AfrLEA3m, promoted a higher proportion of α-helical structure; the

63

presence of sucrose caused the LEA protein to adopt an almost entirely α-helical structure

(Wolkers et al. 2001). How such factors might alter the final dried structures of

AfrLEA2 and AfrLEA3m should be tested.

As discussed earlier, gain of structure upon desiccation has led to the prediction

that LEA proteins may function preferentially in the dry state. However, the ability of

LEA proteins to prevent protein aggregation has been shown in solution, also indicating

functionality in the hydrated state (e.g. Chakrabortee et al. 2007). It is likely that both

scenarios are possible. An individual LEA protein could function as a molecular shield

in solution, and the same LEA protein could gain structure as water is removed to further

protect the cell in the dry state by interacting with membranes, stabilizing sugar glasses,

and forming filamentous networks (for extended reviews, see Tunnacliffe and Wise 2007;

Hand et al. 2011).

Experiments evaluating the capacity of AfrLEA2 and AfrLEA3m to protect

desiccation-sensitive, target enzymes from damage during drying showed that this ability

depends on the target protein chosen. For LDH, neither AfrLEA2 nor AfrLEA3m were

able to afford better protection than that provided by BSA, which is in apparent contrast

with reports for other LEA proteins in the literature. However, it should be noted that

AfrLEA2 and AfrLEA3m did afford a high degree of protection to LDH similar to that

seen with other LEA proteins (e.g. Goyal et al. 2005); the difference is that BSA

stabilized LDH in my study far more than previously reported. Small differences in the

final water content of dried samples could explain this inconsistency. Reyes et al. (2005)

reported that in the presence of BSA, LDH exhibited 75% residual activity after being

dried to 2% water content, but activity dropped below 40% at a water content < 2%.

64

Another aspect that has differed substantially among studies is the concentration of LDH

in the test mixture. In the present study LDH was dried at an initial concentration of 50

µg/ml because preliminary observations showed that at lower concentrations the enzyme

lost activity in a time-dependent fashion simply stored on ice for 1 h during rehydration

(data not shown). In comparison, multiple groups have dried or frozen LDH at

concentrations lower than 10 µg/ml (Miller et al. 1998; Honjoh et al. 2000; Sanchez-

Ballesta et al. 2004; Reyes et al. 2005; Goyal et al. 2005; Nakayama et al. 2007). The use

of such low concentrations of LDH could result in artifactual results due to non-specific

adsorption of LDH to vial surfaces.

In the present study AfrLEA2 and AfrLEA3m, when combined with trehalose, are

able to protect nearly 100% of control PFK activity when rehydrated 24 h after

desiccation, and this protection is far greater than that seen with BSA plus trehalose.

Furthermore, the combined effect of LEA protein plus trehalose seems to be synergistic

compared to either of the agents alone. The stabilization of PFK with trehalose alone is

virtually identical to that reported by Carpenter et al. (1987) under similar slow drying

conditions as used herein. Results for the two cytosolic enzymes tested (LDH, PFK) do

not indicate that a cytosolic LEA protein is able to provide better protection to cytosolic

enzymes than is a mitochondrial LEA protein.

Compared to PFK, CS shows significant resistant to desiccation damage and

therefore was subjected to two 24 h bouts of drying in order to cause more damage to the

enzyme. This double drying technique has been utilized previously with CS (for e.g. see

Pouchkina-Stantcheva et al. 2007). The protective abilities of the two LEA proteins

when combined with trehalose are similar to each other and also to BSA plus trehalose.

65

However, AfrLEA3m alone, at the highest concentration tested, provided significantly

more protection to CS than did AfrLEA2 or BSA. A somewhat similar situation of

preferential protection has been reported for LEA protein-lipid membrane interaction,

where a mitochondrial-targeted LEA protein preferentially protected liposomes with a

lipid composition that mimicked the endogenous composition of the inner mitochondrial

membrane, as compared to liposomes with a more generic composition (Tolleter et al.

2010). Previously, a group 3 LEA protein in the absence trehalose was reported to

protect almost 100% of CS activity after two rounds of desiccation (Goyal et al. 2005).

The apparent discrepancy between this previous study and the values presented here

could be due to differences in drying protocols between experiments. Although Goyal et

al. (2005) perform two rounds of desiccation, each round consisted of vacuum drying for

1 h as opposed to our method of air drying for 24 h.

In conclusion AfrLEA2 and AfrLEA3m are intrinsically disordered proteins that

gain a considerable amount of structure upon desiccation. In addition both proteins are

able to protect desiccation-sensitive enzymes from the deleterious effects of desiccation

and subsequent rehydration. These findings serve to not only further define the

molecular characteristics and possible functions of AfrLEA2 and AfrLEA3m, but also

add to the pool of evidence that supports a role for LEA proteins in desiccation tolerance.

66

CHAPTER 4

INTRACELLULAR LOCALIZATION OF GROUP 3 LEA PROTEINS

IN EMBRYOS OF ARTEMIA FRANCISCANA

4.1 Introduction

Anhydrobiotic organisms, such as embryos of A. franciscana, are capable of

surviving almost complete loss of cellular water (Crowe and Clegg 1973; Crowe and

Madin 1974; Crowe and Clegg 1978; Crowe et al. 1992; Clegg 2005; Watanabe 2006;

Welnicz et al. 2011; Cornette and Kikawada 2011). Desiccation tolerance is

accompanied by the accumulation of one or more types of protective molecules that aid

in the survival of water stress. Included here are low molecular weight solutes such as

trehalose (Yancey et al. 1982; Yancey 2005), Late Embryogenesis Abundant (LEA)

proteins (Cuming 1999; Tunnacliffe and Wise 2007; Shih et al. 2008; Tunnacliffe et al.

2010; Hand et al. 2011), and other small stress proteins such as p26 and Artemin (Clegg

et al. 1994; Liang et al. 1997a; Liang et al. 1997b; Willsie and Clegg 2001; Clegg 2005;

Qiu and Macrae 2008a; Qiu and MacRae 2008b). Intracellular localization of protective

molecules to subcellular compartments is important during desiccation, or other forms of

stress, in order to provide complete functional protection to a cell (Hand and Hagedorn

2008; Menze and Hand 2009). In accordance with the need to protect subcellular

structures during stresses such as desiccation, LEA proteins have been identified that

localize to membrane bound organelles such as the mitochondrion in both plants (Grelet

et al. 2005) and animals (Menze et al. 2009; Warner et al. 2010). The protective sugar

67

trehalose has also been documented to reside in mitochondria of A. fransciscana embryos

(J. Reynolds, M. Menze, and S. Hand, unpublished data). In the current study I will

investigate the subcellular localization of multiple group 3 LEA proteins from embryos of

A. franciscana. The proteins to be evaluated are AfrLEA2, which is predicted to be

cytoplasmic (Hand et al. 2007), and AfrLEA3m (Menze et al. 2009), along with closely-

related isoforms AfrLEA3m_47, AfrLEA3m_43 and AfrLEA3m_29 (Boswell et al.

2013) predicted to localize to the mitochondrion. The AfrLEA3m isoforms are studied as

a group because they all are recognized by the primary antibody raised against

AfrLEA3m. Although localization studies for AfrLEA2 and AfrLEA3m have been

performed previously in mammalian cells transfected with these proteins or specific

constructs (Menze et al. 2009; Li et al. 2012), the subcellular distribution of these two

LEA proteins has never been confirmed in A. franciscana.

The intracellular localization of AfrLEA3m was first investigated experimentally

by Menze et al. (2009) who created a nucleotide construct consisting of the first 70 N-

terminal amino acids of AfrLEA3m ligated to GFP. The chimeric protein was transfected

into HepG2/C3A cells and found to co-localize with Mitotracker red, thus verifying a

mitochondrial targeting capability within the first 70 amino acids of AfrLEA3m.

Another interesting conclusion from this study is that mitochondrial import machinery, as

well as the N-terminal mitochondrial targeting sequence of AfrLEA3m, must be highly

conserved between invertebrates and mammals. Li et al. (2012) provided further

confirmation for mitochondrial localization of AfrLEA3m. In this study, full length

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AfrLEA3m expressed in HepG2 Tet-On cells was localized to mitochondria when

viewed using immunocytochemistry.

AfrLEA2 is a group 3 LEA protein that exists primarily as a homodimer in

embryos of A. franciscana (Boswell et al. 2013). The predicted cytoplasmic location of

AfrLEA2 (Hand et al. 2007) was investigated by transient transfection of a plasmid

encoding for a chimeric protein of full length AfrLEA2 plus GFP (Li et al. 2012).

Chimeric AfrLEA2-GFP was found to reside in the cytoplasm when expressed in HepG2

Tet-On cells. The chimeric AfrLEA2-GFP was also visualized in the nucleus 74 h after

transfection. Although nuclear localization is not predicted for AfrLEA2, small GFP-

tagged proteins have been documented to enter the nucleus without the presence of a

nuclear localization signal (Seibel et al. 2007). Nuclear translocation has also been

documented for p26, a small non-LEA stress protein accumulated by A. franciscana

(Clegg 2011).

In addition to the evidence for subcellular localization discussed above, two LEA

proteins from the bdelloid rotifer, Adineta ricciae, have been documented in the

endoplasmic reticulum, golgi and extracellular space (Tripathi et al. 2012). These two

proteins, ArLEA1A and ArLEA1B, are predicted by bioinformatics to contain an N-

terminal ER localization signal and a C-terminal retention signal ATEL. The presence of

the two proteins in multiple intracellular compartments and their excretion into the

extracellular space could enable widespread protection for this desiccation tolerant

organism through the expression of a limited number of protective proteins (Tripathi et

al. 2012).

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4.2 Methods

Cloning, expression and antibody production for recombinant AfrLEA2 and AfrLEA3m

Preparation and purification of recombinant AfrLEA2 and AfrLEA3m was

accomplished according to the procedures described in Boswell et al. (2013). Briefly, the

original nucleic acid sequences amplified from our existing A. franciscana cDNA library,

ligated into expression vectors, and then competent bacterial cells were transformed with

the plasmids. AfrLEA2 was expressed with an N-terminal 6X-His tag, and AfrLEA3m

was expressed with a C-terminal 6X-His tag so as not to interfere with the mitochondrial

localization sequence found at the N-terminus. Expression of recombinant LEA protein

was induced by the addition of IPTG, and confirmed by SDS PAGE. Bacterial cells were

pelleted and chemically lysed in the presence of a protease inhibitor cocktail. After

removal of cellular debris by centrifugation, the cell lysate was subjected to affinity

chromatography on a HisTrap™ FF crude column (GE Healthcare, Waukesha, WI).

Fractions containing recombinant protein were heat treated to separate the soluble

fraction, which was then dialyzed overnight against the starting buffer for anion

exchange. The sample was then applied to an anion exchange column (HiTrap™ Q FF;

GE Healthcare). The fractions containing pure recombinant protein, as assessed by SDS-

PAGE, were exchanged into LEA storage buffer (20 mM HEPES, 50 mM NaCl, pH 7.5)

and concentrated using Amicon® Ultra Centrifugal filters (Ultracel®-10K; Millipore,

Billencia, MA). Antibodies were raised in chickens against recombinant AfrLEA2 and

AfrLEA3m by Aves Labs, Inc. (Tigard, OR).

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Preparation of A. franciscana embryos for sectioning

Post-diapause embryos of A. franciscana were obtained in the dry state from

Great Salt Lake Artemia (Ogden, UT; grade: laboratory reference standard) and stored at

-20ºC. Prior to use dehydrated embryos were hydrated overnight in ice-cold 0.25 M

NaCl. Embryos were either processed immediately following rehydration, or transferred

to fresh 0.25 M NaCl at 23ºC and incubated with shaking (110 rpm) to promote pre-

emergence development. In preparation for sectioning, embryos were dechorionated as

previously described (Kwast and Hand 1993; Reynolds and Hand 2004) by treatment

with antiformin solution (1% hypochlorate, 0.4 M sodium hydroxide, and 60 mM sodium

carbonate) for 15-20 min at room temperature, followed by three rinses with ice-cold

0.25 M NaCl. In order to deactivate the hypochlorite, embryos were incubated in ice-

cold 1% (w/v) sodium thiosulfate for 5 min and rinsed two times with ice-cold 0.25 M

NaCl followed by an incubation for 3-5 min in ice-cold 40 mM hydrochloric acid

prepared in 0.25 M NaCl, and a final three rinses with 0.25 M NaCl. Prior to further

processing it was necessary to nick the embryo wall because it is only permeable to water

and low molecular weight gasses (Clegg and Conte 1980). Briefly, embryos were

incubated in 2 M sucrose in order to reduce the internal turgor pressure of the embryo,

which prevents extrusion and damage of tissue that would otherwise occur when the cyst

wall is punctured to allow entry of fixative (Hofmann and Hand 1990; Reynolds and

Hand 2004); then embryos were nicked with a needle while being viewed under a

dissecting microscope Next embryos were transferred into 0.2 M potassium phosphate,

pH 7.8, with 4 % paraformaldehyde and 1000 mOsm sucrose for fixation. Fixed samples

were then embedded in paraffin, and 2 µm sections were prepared.

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Immunofluorescent staining

Embryo sections mounted on slides were deparaffinized by two 10 min

incubations in xylene and then gradually rehydrated with the following incubations: 10

min in 100% ethanol, 10 min in 100% ethanol repeated, 5 min in 95% ethanol, 5 min in

70% ethanol, 5 min in 50% ethanol. Finally, sections were rinsed and incubated for 5

min in phosphate buffered saline (PBS) to achieve complete rehydration. Hydrated

sections of embryos were blocked with 5% (vol/vol) normal rabbit serum (Jackson

ImmunoResearch, West Grove, PA) in PBS for 1 h. Primary antibody incubation was

performed overnight at 4ºC. Chicken anti-AfrLEA2 and chicken anti-AfrLEA3m IgY

(Aves Labs, Inc., Tigard, OR) were used at a dilution of 1:400 in 5% normal rabbit

serum, and goat anti-VDAC IgG (Santa Cruz Biotechnology, Dallas, TX) was used at a

dilution of 1:100 in normal rabbit serum. Slides were then washed six times (10 min

each) with PBS followed by incubation with the appropriate secondary antibody for 1 h.

Secondary antibodies were Alexa Fluor® 488-conjugated AffiniPure Rabbit Anti-

Chicken IgY (IgG) (H+L) (excitation, 493; emission, 519; Jackson ImmunoResearch),

and Rhodamine (TRITC)-conjugated AffiniPure Rabbit Anti-Goat IgG (H+L) (excitation,

550 nm; emission, 570 nm; Jackson ImmunoResearch) used at a dilution of 1:500 in 5%

(v/v) normal rabbit serum. After secondary antibody incubation, slides were washed six

times (10 min each) with PBS. To visualize nuclei, sections were treated with ProLong

Gold antifade reagent with DAPI (Life Technologies, Carlsbad, CA) in the case of

deconvolution microcopy. When confocal microscopy was to be used, nuclei were

stained with DRAQ5, which was added to the secondary antibody solution at a final

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concentration of 5 µM. After incubation with secondary antibody containing DRAQ5,

cells were washed as above and treated with ProLong Gold antifade reagent.

Microscopic imaging

For deconvolution microscopy, embryo sections were viewed using a Leica DM

RXA2 upright microscope (Leica Microsystems Inc., Buffalo Grove, IL) and images

were captured with a SensiCam QE 12-bit, cooled CCD camera (PCO-TECH Inc.,

Romulus, MI). Image analysis was performed using SlideBook™ 4.1.0 software (3I

Intelligent Imaging Innovation Inc., Denver, CO). Deconvolution of images was

performed with SlideBook™ 4.1.0 software and used the no neighbors algorithm.

For confocal imaging, embryos were viewed using a Leica TCS SP2 spectral

confocal microscope with Leica LCS Software (Leica Microsystems Inc.). Three lasers,

488 nm at 50% power, 543 nm at 70% power, and 633 nm at 45% power, were used to

sequentially excite Alexa Fluor 488, TRITC, and DRAQ5. Emission passes of 500-535,

558-613, and 655-746 were used to detect the signals.

4.3 Results

Embryos of A. franciscana (Fig. 4.1) are a partial syncytium of about 4,000

nuclei. These encysted gastrula are surrounded by a complex shell that is composed of

cross-linked protein and subtended by an impermeable cuticular membrane of chitin

(Clegg and Conte 1980). Yolk platelets (lipoprotein storage organelles) are the most

abundant structure in the crowded and volume-restricted cytoplasm (Warner et al. 1972).

Mitochondria at this stage are in low abundance and only occupy ~5% of total cellular

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Figure 4.1 Embryos of A. franciscana visualized at 0 h of pre-emergence development by

differential interference contrast (DIC) microscopy. Nuclei are stained with DAPI.

volume (Rees et al. 1989). Trehalose comprises 15% of embryo dry weight (Clegg

2005). The multilayer shell, numerous yolk platelets, and nuclei are clearly visualized

with differential interference contrast (DIC) microscopy (Fig. 4.1).

Endogenous AfrLEA2 is documented with immunohistochemistry to reside in

both the cytoplasm and nucleus of embryonic cells of A. franciscana and to be absent

from mitochondria. The nuclei, visualized with either DAPI or DRAQ5, appear to

contain AfrLEA2 when visualized at 0 h (Fig 4.2) and 6 h (Fig 4.3) of pre-emergence

development. However, the nuclear-localized AfrLEA2 did appear to increase in

embryos viewed after 6 h pre-emergence development compared to 0 h. Merged images

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Figure 4.2 Subcellular localization of AfrLEA2 in embryos of A. franciscana visualized

at 0 h of pre-emergence development. AfrLEA2 (A, D and E, green) is present in the

cytoplasm as well as the nucleus (C and E, pseudocolor blue) and does not appear to co-

localize with VDAC (B and D, red).

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Figure 4.3 Subcellular localization of AfrLEA2 in embryos of A. franciscana visualized

after 6 h of pre-emergence development. AfrLEA2 (A and C, green) is present in the

cytoplasm as well as the nucleus (B and C, blue).

show a co-localization of AfrLEA2 and nuclear staining (Fig. 4.2, E and Fig. 4.3, C) and

a lack of co-localization for AfrLEA2 and VDAC (Fig. 4.2, D). A cytoplasmic location

for AfrLEA2 is in agreement with previously reported subcellular predictions (Hand et al.

2007), and translocation to the nucleus is in agreement with the detection of a chimeric

AfrLEA2-GFP protein in the nucleus of HepG2 Tet-On cells 3 d after transfection (Li et

al. 2012).

The predicted mitochondrial location of AfrLEA3m proteins is confirmed in

embryos of A. franciscana (Fig. 4.4). The co-localization of AfrLEA3m with VDAC,

indicated by the yellow color in merged images, supports a mitochondrial location (Fig.

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4.4, D). I note that co-localization of AfrLEA3m proteins and VDAC was not always

uniform, which could perhaps be a consequence of fluorescence from below the plane of

focus or heterogeneous amounts of AfrLEA3m across the mitochondrial population. It is

also possible that the VDAC antibody has easier access to antigen due to the proteins

location in the outer mitochondrial membrane.

Figure 4.4 Subcellular localization of AfrLEA3m (and its closely related variants

recognized by AfrLEA3m antibody) in embryos of A. franciscana visualized at 0 h of

pre-emergence development. The co-localization of AfrLEA3m proteins (A and D,

green) with VDAC (B and D, red) indicates mitochondrial targeting; areas of co-

localization appear yellow (D). Nuclei (C, pseudocolor blue) are stained with DRAQ5.

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4.4 Discussion

This study provides evidence that supports the cellular locations for AfrLEA2 and

AfrLEA3m proteins in A. franciscana embryos as predicted by bioinformatic analyses.

Although the cellular location of recombinant AfrLEA2 and AfrLEA3m have been

investigated previously when transfected into mammalian cells (Menze et al. 2009; Li et

al. 2012), the cellular localization of these two proteins, as well as of the newly identified

AfrLEA3m_47, AfrLEA3m_43, and AfrLEA3m_29 had yet to be confirmed in embryos

of A. franciscana. Multiple subcellular localizations of LEA proteins, in combination

with the capacities for individual LEA proteins to stabilize different classes of

macromolecules, may explain why a multitude of LEA proteins are expressed in a single

organism. For example, 51 different LEA proteins have been identified in the plant

Arabidopsis thaliana (Hundertmark and Hincha 2008; Bies-Etheve et al. 2008).

Predicted subcellular localization for LEA proteins from A. thaliana includes localization

to the cytosol, chloroplast, mitochondria, and entry into the secretory pathway

(Hundertmark and Hincha 2008).

Immunohistochemistry performed with AfrLEA2 antibody provides evidence for

nuclear localization of this antigen in A. franciscana embryos. This observation is in

agreement with the previously reported nuclear translocation for a chimeric AfrLEA2-

GFP protein expressed in HepG2 Tet-On cells (Li et al. 2012). AfrLEA2-GFP was found

to be cytoplasmic when visualized 24 h after transfection, but after 3 d was also detected

in the nucleus. GFP itself is known to enter the nucleus over time so care must be taken

when concluding a nuclear location based on a GFP-fusion protein (Seibel et al. 2007).

However, the visualization of AfrLEA2 in the nuclei of A. franciscana embryos supports

78

the nuclear localization observed by Li et al. (2012). Warner et al. (2012) identified five

putative LEA proteins in the nuclei of A. franciscana embryos via western blotting with

antibodies raised against group 3 LEA proteins from A. ricciae and A. avenae. However,

to our knowledge AfrLEA2 is the first rigorously-identified LEA protein to be localized

in the nucleus of animals. The accumulation of AfrLEA2 in both the cytoplasm and the

nucleus could allow a single LEA protein to protect a broader range of cellular

components. A similar example exists for a bdelloid rotifer (Tripathi et al. 2012), where

two LEA proteins were documented in the endoplasmic reticulum, golgi, and

extracellular space.

As previously stated, AfrLEA3m is predicted to localize to the mitochondrion

(Menze et al. 2009), and the newly-identified AfrLEA3m_47, AfrLEA3m_43 and

AfrLEA3m_29 are also predicted to be targeted there with the same probabilities based

on bioinformatics. AfrLEA3m_43, which has one amino acid substitution within the

predicted leader sequence, has a probability that is even higher. Immunohistochemistry

generally supports a mitochondrial localization for the proteins, although some limited

discrepancies between the AfrLEA3m proteins and VDAC were observed. As

emphasized earlier, the small cell volume (5%) occupied by mitochondria in embryos of

A. franciscana is several-fold lower than for many other cell types; for example, rat

hepatocytes are estimated to have a value of 23% (Beauvoit et al. 1994). 1994). This

feature, coupled with the limited amount of free cytoplasmic space due to numerous yolk

platelets (cf. Fig 4.1), makes resolution of the mitochondrial distribution challenging at

the level of fluorescence microscopy. Mitotracker red is the most conventional

fluorescent probe for mitochondrial imaging, but its use was unsuccessful here. Although

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Mitotracker is retained in the mitochondrion after fixation, mitochondrial accumulation is

dependent upon an electrical potential across the inner mitochondrial membrane. Post-

diapause embryos used in this study are a partial syncytium of about 4,000 nuclei

surrounded by a complex shell impermeable to all molecules but water and low

molecular weight gasses (Clegg and Conte 1980). The shell must be nicked in order for

substances such as Mitotracker and fixative to enter A. franciscana embryos (see

Methods). Lack of Mitotracker accumulation by embryos for which the permeability

barrier has been ruptured may be due to the loss of oxidative substrates that support

electron transport and the associated membrane potential. Therefore, the addition of

substrates such as succinate to the medium during incubation with Mitotracker in the

future may improve the results with this probe.

It is probable that embryos of A. franciscana contain additional, and as yet

unidentified, LEA proteins that localize to additional subcellular compartments. For

instance, the ER and golgi of A. ricciae contain LEA proteins (Tripathi et al. 2012), and

plant LEA proteins have been found in the cytoplasm, mitochondrion, nucleus,

chloroplast, endoplasmic reticulum, vacuole, peroxisome, and plasma membrane

(Tunnacliffe and Wise 2007). Antibodies raised against two group 3 LEA proteins from

A. ricciae and Aphelenchus avenae each recognize a multitude of putative LEA proteins

in heat-soluble protein fractions prepared from isolated organelles of A. franciscana

(Warner et al. 2012). While interesting, further work is needed to verify that the proteins

recognized are in fact LEA proteins. Identification and subsequent characterization of

the full complement of LEA proteins expressed by embryos of A. franciscana awaits

release of the Artemia genome scheduled very soon.

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In conclusion, I provide evidence supporting the subcellular localization of

AfrLEA2 to both the cytoplasm and nucleus and of the AfrLEA3m proteins to the

mitochondrion. The presence of LEA proteins in multiple subcellular compartments

underscores an apparent need for protective molecules in all areas of a cell, and perhaps

even in the extracellular space, in order for an organism to survive the stresses imposed

by desiccation.

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CHAPTER 5

SUMMARY AND FUTURE DIRECTIONS

The results presented in this dissertation add to the pool of evidence that supports

a role for LEA proteins in desiccation tolerance by providing molecular characteristics,

expression data, and functional studies for group 3 LEA proteins from embryos of A.

franciscana. In chapter 2 three new Afrlea mRNA (Afrlea3m_47, Afrlea3m_43, and

Afrlea3m_29) are sequenced and characterized (Boswell et al. 2013). Deduced protein

sequences from these mRNA are very similar to the previously identified AfrLEA3m

(Menze et al. 2009) and are predicted to reside in the mitochondrion. Due to multiple

base pair differences these mRNA are predicted to arise from different genes. Protein

expression levels were investigated for four group 3 LEA proteins (AfrLEA2,

AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29) and found to be highest in diapause

embryos (a desiccation-tolerant stage) and decrease throughout pre-emergence

development to lowest levels in desiccation-intolerant nauplius larvae. Protein

expression levels for AfrLEA2, which exists primarily as an dimer in A. franciscana,

agree with mRNA expression levels reported by Hand et al. (2007), and AfrLEA2 is

present in diapause embryos at a concentration of 1.85 mg/ml embryo water. Protein

expression levels for AfrLEA3m also agree with previous mRNA reports by Menze et al.

(2009) and mitochondrial AfrLEA proteins have a combined concentration of 2.2 mg/ml

matrix volume.

Structural investigations determine AfrLEA2 and AfrLEA3m to be intrinsically

disordered proteins (Chapter 3) that gain structure after desiccation, and in the presence

of TFE or SDS. Recombinant AfrLEA2 and AfrLEA3m are also able to provide

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protection to sensitive enzymes during desiccation. The ability of LEA proteins such as

AfrLEA2 and AfrLEA3m to gain structure in the dry state leads to a predicted dry

function (e.g. Li and He 2009), but evidence also exists that LEA proteins function in

solution (Chakrabortee et al. 2007; Liu et al. 2011; Chakrabortee et al. 2012). As

mentioned in chapter 3, it is possible that a single LEA protein could function both in the

hydrated and dry state, thereby providing maximum protection to cellular components

throughout the entire drying process.

This hypothesis that LEA proteins may show dual functionality, both in the

hydrated and dry state, can be supported by combining various pieces of literature in

attempt to present a more complete picture of LEA protein protection as a cell is

subjected to the desiccation process. LEA protein functionality in the hydrated state can

be at least partially attributed to molecular shield activity as defined by Goyal et al.

(2005). In this context LEA proteins prevent aggregation of sensitive enzymes by acting

as a physical barrier between molecules during the initial stages of dehydration.

Subsequently, as the desiccation process progresses past a water content of approximately

20% LEA proteins should begin to adopt secondary structure (Li and He 2009). At this

point LEA proteins could begin to perform functions suggested in the “dry” state. For

example, membrane interaction would require that a LEA protein first form amphipathic

α-helices, which could then insert into membranes parallel to their plane (Tolleter et al.

2007). The potential of various LEA proteins to form coiled coils during drying (Goyal

et al. 2003) provides support for the hypothesis that some LEA proteins may form

intracellular filaments during desiccation that would serve to increase the mechanical

strength of desiccated cells (Wise and Tunnacliffe 2004). This increase in mechanical

83

strength would provide protection against the physical stresses experienced by a cell

during desiccation (Wolfe et al. 1986). These hypothetical LEA protein filaments could

also work in concert with sugar glasses in a manner that has been compared to “steel-

reinforced concrete” (Goyal et al. 2003). Structure gained by LEA proteins during drying

is fully reversible (Wolkers et al. 2001), allowing return to the intrinsically disordered

state during rehydration, and prevention of protein aggregation during sub-optimal water

contents experienced until full hydration is achieved.

The possibility of dual functionality, or “moonlighting” of LEA proteins at

differing water contents is exemplified by LEA proteins such as LEAM (also called

PsLEAm) found in pea seed mitochondria (Grelet et al. 2005). LEAM is a group 3 LEA

protein that has been shown to protect both proteins and membranes from desiccation

stress (Grelet et al. 2005; Tolleter et al. 2007). There will of course be exceptions to the

dual functionality hypothesis, as is exemplified by ArLEA1B from a bdelloid rotifer

(Pouchkina-Stantcheva et al. 2007). ArLEA1B is an atypical group 3 LEA protein that is

predominantly α-helical in solution. This particular LEA protein is not able to act as a

molecular shield, as shown by an inability to protect CS from the deleterious effects of

desiccation, but does interact with membranes significantly decreasing the gel-to-liquid

crystalline phase-transition temperature (Tm) of dry liposomes.

Another structural element adopted by some LEA proteins is a solvent-exposed,

left-handed extended helix also called poly (ʟ-proline)-type (PII) conformation (Soulages

et al. 2002; Soulages et al. 2003; Mouillon et al. 2006). These groups show LEA proteins

from groups 1 and 2 to adopt increasing PII helices as a function of decreasing

temperature, but the possible implications of this observation could be more widespread.

84

Mouillon et al. (2006) suggest that the PII helix could act as an intermediate structure for

example: in between random coil and the adoption of α-helix or β-sheet during

desiccation. This group speculates that the intermediate PII helix could hold more tightly

to bound water during desiccation increasing the capability of LEA proteins to act as a

hydration buffer during desiccation. The physiological relevance of LEA proteins as a

hydration buffer has since been challenged (Hand et al. 2011); however, formation of PII

helices could still hold relevance, possibly by prolonging the molecular shield activity of

some LEA proteins to lower water contents before adoption of structure and related

function as discussed above. It is pertinent to note here that Shih et al. (2008) suggest

temperature scans performed by Goyal et al. (2003) on AavLEA1 indicate the formation

of PII helices, although this was not a conclusion of the original study. Thus, there is

evidence that LEA proteins from all three major groups are capable of forming PII

helices.

Finally, the predicted cellular location of AfrLEA2 (cytoplasm) and the four

AfrLEA proteins recognized by anti-AfrLEA3m antiserum (mitochondrial) have been

confirmed in embryos of A. franciscana (Chapter 4). In addition to the predicted

cytoplasmic location, AfrLEA2 was also located in embryo nuclei. Localization of

protective molecules such as LEA proteins to membrane bound organelles is fundamental

if an organism is to survive desiccation stress (Hand and Hagedorn 2008; Menze and

Hand 2009). In order to achieve a complete understanding of LEA protein function, the

entire complement of LEA proteins expressed in an individual organism first needs to be

documented. This possibility is on the horizon regarding A. franciscana, for which the

genome will be published shortly [Artemia Genome Day, September 2, 2013, Laboratory

85

of Aquaculture & Artemia Reference Center, Ghent University, Belgium]. As mentioned

earlier, differential cellular localization is one reason for an organism to express multiple

LEA proteins. In this case sequencing of the A. franciscana genome could allow

characterization of all AfrLEA proteins that are located to different subcellular locations.

However, differential subcellular localization alone does not fully explain the expression

of multiple LEA proteins because more than one LEA protein can be found in a single

subcellular location. Take the mitochondria from embryos of A. franciscana for

example. Currently, multiple group 1 (Warner et al. 2010; Marunde et al. 2013) and

group 3 (Menze et al. 2009; Boswell et al. 2013) LEA proteins have been identified that

localize to the mitochondrion of these desiccation-tolerant embryos. It is possible that the

presence of multiple LEA proteins in a single subcellular compartment is necessary for

the simultaneous protection of different classes of macromolecules. As the complete

array of LEA proteins in each subcellular location is discovered, we can begin to

investigate differential protection. Some LEA proteins like LEAM (Grelet et al. 2005)

may moonlight, while others like ArLEA1B (Pouchkina-Stantcheva et al. 2007) may

perform specific roles.

The mechanisms behind desiccation tolerance are not only a fundamental mystery

that we strive to comprehend for the most basic reason of increasing our ever growing

understanding of biology, but also are mechanisms that we want to be able to confer to

desiccation sensitive molecules and even entire tissue systems. In order to reach long

term goals, like stabilization of mammalian cells for example (cf., Crowe et al. 2005;

Huang and Tunnacliffe 2007; Hand and Hagedorn 2008; Li et al. 2012), we need to be

able to apply the mechanisms of desiccation tolerance learned from anhydrobiotic

86

organisms to desiccation-sensitive systems. Basic characterization of protectant

molecules, as was performed in this dissertation, is necessary in reaching goals such as

the stabilization of dry cells.

Another possible application for LEA and other intrinsically disordered proteins

lies within the medical field. Diseases like Alzheimer’s are linked to conformational

changes in certain proteins leading to their aggregation. Multiple groups have shown that

LEA proteins are capable of limiting the aggregation tendencies of proteins in vivo

(Chakrabortee et al. 2007; Liu et al. 2011; Chakrabortee et al. 2012). Therefore, it is

possible that LEA proteins could be used to prevent, or at least delay disease causing

protein aggregation.

87

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101

VITA

Leaf Chandra Boswell was born and raised in Covington Louisiana. As a child,

Leaf’s enthusiasm for science was enhanced by a lack of cable television and love of the

outdoors. Leaf’s father taught high school biology, chemistry, and environmental

science, which afforded her many opportunities such as attending plant identification

field trips with her father’s classes and raising a menagerie of orphaned wild animals.

After completing high school at Saint Scholastica Academy, Leaf attended Louisiana

State University and was awarded a bachelor’s degree in Biological Sciences.

group 3 late embryogenesis abundant proteins from embryos

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