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9-1-2013

Functional Analyses of Tiger Salamander (Ambystoma tigrinum) Functional Analyses of Tiger Salamander (Ambystoma tigrinum)

Hemoglobin Hemoglobin

Jorge Polanco Winona State University

Erica Eischens Winona State University

Cody Benedict Winona State University

Gabriel Velez Winona State University

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Recommended Citation Recommended Citation Polanco, Jorge; Eischens, Erica; Benedict, Cody; and Velez, Gabriel, "Functional Analyses of Tiger Salamander (Ambystoma tigrinum) Hemoglobin" (2013). Student Research and Creative Projects 2013-2014. 30. https://openriver.winona.edu/studentgrants2014/30

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Functional analyses of tiger salamander (Ambystoma tigrinum) hemoglobin

Jorge Polanco, Erica Eischens, Cody Benedict, Gabriel Velez, Noah Anderson, and Amy RunckDepartment of Biology, College of Science and Engineering, Winona State University, Winona, MN

Introduction ResultsThe tiger salamander, Ambystoma tigrinum, has two adult forms,terrestrial and neotenic. Like the terrestrial adult, the neotene isreproductively mature. However, the neotene retains a paddle-like tailand gills, and is thus more morphologically similar to the larval lifestage. Usually this species metamorphose into breeding terrestrial adults,but in the population of interest, many neotenes are found.

Like humans, amphibians will produce different hemoglobin proteinsduring different developmental stages. Erythrocytes of the closelyrelated axolotl (Ambystoma mexicanum) contain multiple hemoglobinisoforms, made from two copies of adult α-globin genes (αMajor, andαminor), and one copy of β-globin (1). Previous primary structuralanalyses and phylogenetic comparisons have been reported for thesehemoglobins, but there is a lack of understanding of their structural andfunctional capabilities. Here, we attempt to characterize the hemoglobincomposition of both adult forms of the tiger salamander. We examinedhemoglobin from neotenic and terrestrial salamanders to determinehemoglobin O2-affinity, primary structure, and 3D structure. Wecharacterized the number of isoforms and analyzed 3D structure in orderto see how differences could affect the molecular interactions. Theseresults will provide further insight into the expression of these genes aswell as further characterization of this salamander’s neoteny.

Summary

Future Directions

ACKNOWLEDGMENTS: We would like to Mike Mossman (WI DNR) and Dr. Ragsdale and Erika Vail (WSU) for assistance with lab work and data analyses. All work was conducted under WSU IACUC approval. We thank the WSU Foundation and the College of Science and Engineering for financial support.

Methods

Figure 4. Structural bioinformatics analysis reveals potentialdifferences in intermolecular interactions between the major andminor hemoglobin tetramers (A) Full terrestrial hemoglobin models(major and minor) were analyzed for the presence of stabilizing saltbridge interactions using ESBRI (5) with a maximum distance of 4.0Å. Interacting residues and average distances in Å were reported forresidues listed in Figure 3. These results suggest a difference in thenumber of intermolecular interactions between the major and minortetramers that could potentially explain their differences in oxygenbinding abilities. (B) Visualization of the Glu134 – Arg141 interactionin the terrestrial major tetramer. (C) Lys33 – Asp21 interaction in theterrestrial minor tetramer.

Figure 3. Tiger salamander α- and β-globin amino acid sequences aligned to human sequences using MUSCLE (6). Using RT-PCR, two protein alleles were recovered in αMajor, onewhich was unique to the neotenic individual. Two protein alleles were recovered in αminor, one which was unique to the terrestrial individual. Two β-globin protein alleles were recovered,one which was unique to the neotenic individual. The symbol indicates residues involved in contact of subunits α1 and β1, and the symbol indicates residues involved in α1 β2 contact. Thesymbol ♯ indicates amino acids which participate in forming salt bridges that stabilize the deoxy (T) quaternary structure (Bohr Effect).

Figure 1. Oxygen dissociation curves for blood from neotenic and terrestrial Ambystomatigrinum individuals at native conditions. Measures of P50 indicate the pO2 tension atwhich hemoglobin in 50% saturated at 12.8 ºC (P50 in humans in around 26 mm Hg at 37ºC).

•The P50 of an individual neotene (P50=13.18 mmHg) was lower than an individualmetamorphosed adult salamander (P50=23.9 mmHg) indicating that the neotene’shemoglobin has a higher O2 affinity.•The Hb transition from neotenic to terrestrial adult form may be physiologicallysignificant for inducing a change in the required oxygen affinity.

Hemoglobin oxygen-affinity results

Further work is examining seasonal differences of hemoglobin form andfunction in the neotenes. The seasons are hypothesized to be a major factor,since the neotenic and larval tiger salamanders must remain in the frozen overlakes in the winter, which limits the oxygen availability. To overcome thisobstacle, we are hypothesizing changes in hemoglobin.

Neotene P50: 13.18 mmHg Terrestrial P50: 23.9 mmHg

Isoelectric focusing (IEF) results

• The IEF analysis revealed the presence of at least three Hb isoforms in the neotenic andterrestrial individuals, and these isoforms were recovered at varying amounts.

• Different tetramers can lead to different oxygen binding affinities• Isoelectric point (pI) of the salamander hemoglobins were around ranged from 6.8 to

7.1

Figure 2. Hemoglobin IEF gels (A) Unstained gel from five neotenic individuals (B)Stained gel showing six individuals.

A B1 2 3 4 5 6 7 8

Legend1: Ladder 2: Neotenic 22 clot 3: Neotenic 22 supernatant 4: Terrestrial clot

5:Terrestrial supernatant6: Terrestrial 7 7: Larval8: Ladder

• Successful cloning and sequencing of the globin genes revealthe presence aMajor, aminor, and β-globin protein sequencessimilar to that of Ambystoma mexicanum. From the nucleotidesequences, primary structural data were obtained to showdifferences between these protein alleles.

• Salt bridge prediction data indicate differences in theintermolecular interactions making up the major and minortetramers. These theoretical data could explain functionaldifferences between these forms of hemoglobin.

• Results of the oxygen disassociation curves constructed withthe Hemox Analyzer support the hypothesis that hemoglobinfrom neotenic tiger salamanders will have a higher oxygenaffinity due to less dissolved O2 available in water ascompared to the partial pressure of O2 in air.

• Three tetramers of varying amounts were uncovered throughIEF. These different isoforms may contribute to the differencesin O2-binding abilities between the neotenic and terrestrialsalamanders, which will be more closely investigated usingmolecular dynamics simulations with explicit solvent modelsto mimic physiological conditions.

References: (1) Shishikura F, Takeuchi, H, Nagai T. Axolotl hemoglobin: cDNA-derived aminoacid sequences of two α globins and a β globin from an adult Ambystoma mexicanum. ComparativeBiochemistry and Physiology, Part B 142 (2005) 258–268. (2) N. Eswar, M. A. et al. ComparativeProtein Structure Modeling With MODELLER. Current Protocols in Bioinformatics, 15, 5.6.1-5.6.30,2006. (3) Pettersen EF, et al. UCSF Chimera--a visualization system for exploratory research andanalysis. J Comput Chem. 2004 Oct;25(13):1605-12. (4) Brooks BR et al. (2009). CHARMM: Thebiomolecular simulation program. J Comput Chem, 30(10):1545-614. (5) Costantini et al. ESBRI: Aweb server for evaluating salt bridges in proteins. Bioinformation. 2008; 3(3): 137–138. (6) Edgar, R.C.(2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic AcidsRes. 32(5):1792-1797.

• Hemoglobin O2 affinity– oxygen dissociation curves were generatedfor a neotenic and terrestrial individual at 12.8 ºC using a HemoxAnalyzer (TCS Scientific).

• Identification of hemoglobin isoforms-the number of hemoglobinisoforms were determined using a Pharmacia FBE 3000 isoelectricfocusing (IEF) system with precast gels (pH 3-10).

• Molecular modeling – models of the individual Ambystoma globinchains were generated using MODELLER (2) Full tetramer modelswere generated by aligning individual chains to a model of humanhemoglobin (PDB: 1HBB) using UCSF Chimera (3). Models wereminimized using CHARMM (4) and intermolecular interactions werecalculated using ESBRI (5).

Molecular modeling results

EVIDENCE FOR THE EXISTENCE OF FUNCTIONAL ADAPTATIONS IN THE HEMOGLOBIN OF THE TIGER SALAMANDER, Ambystoma tigrinum

Gabriel Velez, Jorge Polanco, Erica Eischens, and Amy M. Runck (Advisor) Department of Biology, Winona State University, Winona, MN

Introduction

Adaptations to extreme conditions• Fine tuning of proteins throughout evolution

Introduction – Amphibian life cycle

Normal Life Cycle:

Terrestrial salamander lays eggs in pond

Eggs hatch after 2 weeks

Larva grows in pond over summer

Larva goes through metamorphosis andbecomes terrestrial adult

Alternative Life Cycle:

Larva becomes gilled breeding adult(neotenic)

Remains aquatic

The tiger salamander – Ambystoma tigrinum

Most populations – terrestrial breeding form.

The population of interest develops into the neotenic form.

Introduction – Structure and function of hemoglobin

Globular protein consisting of 4subunits stabilized through salt bridgeinteractions. Two α subunits

Two β subunits

Found in red blood cells Involved in oxygen transport

Cooperative binding of hemoglobin

The individual hemoglobin subunits cooperatively bind and interact in order to facilitate transport of oxygen to the peripheral tissues.

Like humans, salamanders produce different forms of the hemoglobin protein during development.

The Globin FamilyDifferent proteins in the alpha and beta globin families are expressed differentially during early to late development (Hoffmann FG. et al, 2010).

3’5’

ρ globin

βH globin

βA globin

ε globin

Chicken β-globin gene cluster

3’5’ αE globin

αD globin

αA globin

Chicken α-globin gene cluster

3’5’

Salamander α-globin gene cluster

αM αm

Previous research

The axolotl, Ambystoma mexicanum Adult (Neotenic):

αM β – Major Hb αm β – Minor Hb

Juvenile:αD β – Expressed after 1 month

Major Hb Minor Hb

Presenter

Presentation Notes

Will we see the same pattern of gene expression pattern in the tiger salamander given that it can metamorphose into the two adult forms and what are the functional consequences of these two distinct adult forms of hemoglobin.

Research Approach

Questions to be addressed:Which hemoglobin isoforms are present in these salamanders? And at

what levels? – RT-PCR

Is there a difference in the functional performance of these hemoglobins between the two adult forms that is related to their environmental conditions? – Oxygen-binding studies

If so, then are there residues/structural differences that could potentially explain this phenomenon? – Sequencing, molecular modeling

Sampling

Badger Army Ammunition PlantWork has always been done under standardized lab conditionsSept + Feb sampling and at least 2 other samplingsWe have been tracking temperature of the water and level of

dissolved O2

Protein Alignment

• Individual globin chains were amplified using RT-PCR.• Proteins were sequenced and aligned to visualize residue changes (above).

Protein Alignment

Three amino acid residues, α40(Lys), β94(Asp), β146(His), form two salt bridges which stabilize the T quaternary structure.

Terrestrial αminor allele a

Terrestrial αmajor

Human

Neotene αmajor allele bNeotene αmajor allele a

Neotene αminor allele

Terrestrial αminor allele b

α minor 40(Lys Gln)

α

Comparative Molecular Modeling and Structural Bioinformatics

Homology models generated

Homology modeling reveals little change in the tertiary structure of the individual α subunits.

0

1

2

3

4

5

6

7

8

0 20 40 60 80 100 120 140

RMSF

Residue

Alpha Maj vs Minor Alpha Minor a vs b

Alpha minor Minor Hb

Human Hemoglobin Model

Intermolecular InteractionsTerrestrial Major Globin

Salt Bridge Residue Interacting Residues Average Distance (Å)*

αArg31 αGlu36; αAsp99 2.13; 1.64

αGlu36 αArg100 3.34

αLys40 αAsp21; αAsp34; αGlu50 2.78; 2.80; 2.75

αHis112 αAsp94 3.20

αHis122 αAsp126 3.12

αArg141 αAsp6; αGlu134 3.16; 3.08

βArg30 αAsp94; αAsp126 3.12; 3.67

βHis125 βAsp21; αAsp34; αGlu50 2.09; 2.24; 3.99

βHis146 βGlu6; βGlu7; βAsp10; βGlu121 3.29; 2.72; 1.96; 3.79

Terrestrial Minor Globin

αLys33 βAsp21 2.60

αHis103 αAsp26; αAsp35; αAsp93; βAsp99 2.44; 3.63; 2.80; 2.77

βArg30 αAsp26; αAsp35; αAsp93; βAsp99 3.02; 1.98; 3.76; 2.14

βHis125 βAsp21 1.68

βHis146 βGlu6; βGlu7; βAsp10; βGlu121 3.29; 2.72; 1.96; 3.79

* - calculated from multiple interactions on same residue pair.

Intermolecular Interactions

Alpha Major – Residue Contact Alpha Minor – No Contact

25

50

75

100

10080604020

PO2 (mm Hg)

% O

xyhe

mog

lobi

n

Neotene P50: 13.18 mmHg

Terrestrial P50: 23.9 mmHg

O2 binding in neotenes and terrestrials

High-affinity Hbs bind more O2 at a given PO2

Human P50: 27 mmHg

Summary

Key structural differences in alpha major and minor Potential differences in stabilizing interactions Could explain destabilization of T state in minor Hb

Future Studies

More samplingGene expression studies – qRT-PCRContinued hemoglobin oxygen affinity studies

BioinformaticsMolecular dynamics simulationsStructural conservation studies

Acknowledgements

Financial support provided by WSU Foundation and the College of Science and Engineering

Noah Anderson – UW Baraboo Sauk County

Francis Ragsdale – Winona State University

Mike Mossman – Wisconsin DNR

Erika Vail – Winona State University

Erin O’ Leary-Jepsen – Idaho State University

Current and past lab members:

Tara Juresh

Cody Benedict

Katelyn Madigan

Erin Gilliland

Elijah Velasquez

Zachary Coffey

Isoelectric Focusing

Ladder

terrestrial

neotene

neotene

neotene

terrestrial

qRT-PCR

Provides insight into relative expression levels of globin genes at different environmental conditions.

References

1. Shishikura F, Takeuchi, H, Nagai T. Axolotl hemoglobin: cDNA-derived amino acid sequences of two α globins and a β globin from an adult Ambystoma mexicanum. Comparative Biochemistry and Physiology, Part B 142 (2005) 258–268.

2. Wood et al (1982). Control of hemoglobin function in the salamander, Ambystoma tigrinum. Molecular Physiology. 2: 263-272.

3. Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32(5):1792-1797.

4. Edgar, R.C. (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics.

5. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 Oct;25(13):1605-12.

6. N. Eswar, M. A. Marti-Renom, B. Webb, M. S. Madhusudhan, D. Eramian, M. Shen, U. Pieper, A. Sali. Comparative Protein Structure Modeling With MODELLER. Current Protocols in Bioinformatics, John Wiley & Sons, Inc., Supplement 15, 5.6.1-5.6.30, 2006.

7. M.A. Marti-Renom, A. Stuart, A. Fiser, R. Sánchez, F. Melo, A. Sali. Comparative protein structure modeling of genes and genomes. Annu. Rev. Biophys. Biomol. Struct. 29, 291-325, 2000.

8. A. Sali & T.L. Blundell. Comparative protein modeling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815, 1993.

9. A. Fiser, R.K. Do, & A. Sali. Modeling of loops in protein structures, Protein Science 9. 1753-1773, 2000.

10. Shindyalov, Ilya N., and Philip E. Bourne. "Protein Structure Alignment by Incremental Combinatorial Extension (CE) of the Optimal Path." PubMed. 1998: n. pag. NCBI.

11. Riggs, AF. The Bohr Effect. 1988. Ann. Rev. Phys. 50:181-204.