11s storage globulin from pumpkin seeds: regularities of proteolysis by papain

11S globulin (legumin) and 7S globulin (vicilin) are

the storage proteins most characteristic of plant seeds [1].

To date, extensive information describing primary and

higher order structures of 11S and 7S globulins [2] and

their evolutionary pathway of descent from bacterial

oxalate decarboxylases [3] is available. Common principles

of structural organization of the storage globulin subunits

can be suggested on the basis of comparison of their highly

conserved amino acid sequences and tertiary structures.

The subunits of 11S globulins consist of disulfide�

bonded N�terminal (α�chain) and C�terminal (β�chain)

domains [2]. The N� and C�terminal domains are homolo�

gous and structurally equivalent. A β�barrel of eight antipar�

allel β�strands BCDEFGHI conjoined with additional

antiparallel β�strands A′, A, and J, J′ forms the structural

basis of the domains (Fig. 1). In the α�chain structure, addi�

tional antiparallel β�strands E′ and F′ are formed between

strands E and F. The sequence between strands J and J′forms a group of α�helices – h1, h2, and h3. The sequence

between strands A and B forms the additional α�helix h0.

The mature hexameric molecule of 11S globulins is

formed by combination of subunit trimers. The interac�

tions between the α� and β�chain α�helices h1�h3 from

subunits neighboring in the trimers are essential for for�

mation of the quaternary structure of 11S globulin [4, 5].

The regularities of storage globulin degradation,

which is an essential part of their function, have been less

studied. The degradation of storage globulins in vivo dur�

ing seed germination and in vitro follows two radically dis�

tinct mechanisms, namely limited and cooperative pro�

teolyses [3]. The limited (“zipper” [6]) proteolysis is

restricted to cleavage of peptide bonds in the protein sub�

strate structure that are specifically susceptible to proteo�

lytic attack. Degradation by the cooperative mechanism

(one�by�one [6]) depends on the conformational state of

the protein [7] and consists of deep one�by�one proteo�

lysis of the substrate molecules [3, 6].

Depending on their conformation, native storage

globulin molecules might be either susceptible or insus�

ceptible to cooperative proteolysis. For instance, the

packing density of soybean 7S globulin tertiary structure

is relatively low. Therefore, its degradation follows the

cooperative mechanism irrespective of the limited pro�

teolysis that also occurs independently, in parallel [8]. In

contrast, the native common bean 7S globulin with a rel�

atively high packing density of its molecule [8] is inacces�

ISSN 0006�2979, Biochemistry (Moscow), 2014, Vol. 79, No. 8, pp. 820�825. © Pleiades Publishing, Ltd., 2014.

Published in Russian in Biokhimiya, 2014, Vol. 79, No. 8, pp. 1024�1030.

820

* To whom correspondence should be addressed.

11S Storage Globulin from Pumpkin Seeds:Regularities of Proteolysis by Papain

A. S. Rudakova, S. V. Rudakov, I. A. Kakhovskaya, and A. D. Shutov*

State University of Moldova, Mateevici str. 60, Kishinev, MD�2009 Moldova; fax: 37�322�244�248;

E�mail: rud�[email protected]; [email protected]; [email protected]; [email protected]

Received April 11, 2014

Revision received May 14, 2014

Abstract—Limited proteolysis of the α� and β�chains and deep cleavage of the αβ�subunits by the cooperative (one�by�one)

mechanism was observed in the course of papain hydrolysis of cucurbitin, an 11S storage globulin from seeds of the pump�

kin Cucurbita maxima. An independent analysis of the kinetics of the limited and cooperative proteolyses revealed that the

reaction occurs in two successive steps. In the first step, limited proteolysis consisting of detachments of short terminal pep�

tides from the α� and β�chains was observed. The cooperative proteolysis, which occurs as a pseudo�first order reaction,

started at the second step. Therefore, the limited proteolysis at the first step plays a regulatory role, impacting the rate of

deep degradation of cucurbitin molecules by the cooperative mechanism. Structural alterations of cucurbitin induced by

limited proteolysis are suggested to generate its susceptibility to cooperative proteolysis. These alterations are tentatively dis�

cussed on the basis of the tertiary structure of the cucurbitin subunit pdb|2EVX in comparison with previously obtained data

on features of degradation of soybean 11S globulin hydrolyzed by papain.

DOI: 10.1134/S0006297914080100

Key words: Cucurbita maxima, seed storage globulin, limited proteolysis, papain, kinetics

PROTEOLYSIS OF PUMPKIN 11S GLOBULIN BY PAPAIN 821

BIOCHEMISTRY (Moscow) Vol. 79 No. 8 2014

sible to cooperative proteolysis under the action of differ�

ent proteinases [9]. In this case, the onset of the cooper�

ative proteolysis is delayed and starts only after comple�

tion of limited proteolysis that generates a certain change

in the 7S globulin tertiary structure [9]. A similar pattern

of proteolysis has been shown to occur during papain

hydrolysis of soybean 11S globulin [5].

In this report, the relationships between limited and

cooperative proteolyses during the hydrolysis of cucur�

bitin, the 11S globulin from pumpkin seeds, were studied.

Papain has been used here as a model enzyme because

homologous papain�like proteinases are responsible for

mobilization of storage globulins in germinating seeds

[10].

Fig. 1. The α�chain structure of pumpkin 11S globulin cucurbitin pdb|2EVX. a) Ribbon diagram of the tertiary structure. Disordered regions

1�4 are shown by dashed lines. The dark part of the diagram corresponds to the C�terminal sequence region that is presumably detached dur�

ing limited proteolysis due to cleavage of the disordered region 3 between residues E191 and E204. Cysteine residue Cys103 is involved in for�

mation of a disulfide bond between the α� and β�chains. b) Structurally aligned amino acid sequences of cucurbitin 2EVX and glycinin 1OD5

α�chains. Numbers in square brackets correspond to the number of residues in cucurbitin and glycinin disordered regions. Residues of

enhanced (64�75%) accessibilities to the solvent are printed in bold. The glycinin C�terminal sequence shown in italics is detached during

papain hydrolysis [5].

b

a

822 RUDAKOVA et al.


MATERIALS AND METHODS

The defatted meal from Cucurbita maxima cotyle�

dons was washed with water (1 : 70 w/v) to eliminate pro�

tein and non�protein contaminations. The pellet was

extracted (1 : 10 w/v) with 0.5 M NaCl in buffer A

(0.02 M Tris�HCl, pH 8.0, 0.02% NaN3, 1 mM EDTA).

Cucurbitin was salted out from the extract with the addi�

tion of ammonium sulfate up to 40% saturation. The pre�

cipitate was dissolved in 3.0 M NaCl in buffer A, and the

solution was loaded onto a column of phenyl�Sepharose

CL�4B (Pharmacia Biotech, Sweden) equilibrated with

3.0 M NaCl in buffer A. After removal of the non�

adsorbed fraction, the cucurbitin was eluted with 2.0 M

NaCl in buffer A.

For the cucurbitin digestion, the reaction mixture

containing 5 mg/ml substrate and 50 μg/ml papain

(Sigma Life Science, USA) in buffer B (32.25 mM

Na2HPO4, 4.75 mM citric acid, pH 6.8, 0.417 M NaCl,

0.02% NaN3, 1 mM EDTA, 10 mM 2�mercaptoethanol)

was incubated for 14 h at 30°C. To follow the time course

of the digestion, aliquots of the incubation mixture were

sampled from time to time and the reaction was stopped

by addition of trichloroacetic acid (TCA) to final concen�

tration 5% (w/v).

SDS�PAGE was carried out in 15% (w/v) gels using

the buffer system of Laemmli [11]. Prior to electrophore�

sis, the residual TCA in precipitated protein samples was

removed by washing with acetone, the pellets were dried

and dissolved in sample buffer with or without 2�mercap�

toethanol under standard conditions. Molecular mass

markers from Fermentas Life Sciences (Lithuania) were

used. Mature glycinin polypeptides of molecular masses

known from their sequences [4] were used as additional

markers. The electrophoregrams were scanned

(ImageScanner III; GE Healthcare, GB) and analyzed

quantitatively using Phoretix 1D Gel Analysis v.5.10 soft�

ware. The relative molar amounts of polypeptides detect�

ed by SDS�PAGE in the intact cucurbitin and in the resid�

ual protein after proteolysis were calculated using their

molecular masses and relative weight concentrations.

The accessibility of a given amino acid residue (X) in

the cucurbitin tertiary structure, defined as the percent�

age of its surface accessibility to the solvent in the extend�

ed pentapeptide GGXGG, was calculated using the pro�

gram DeepView/Swiss�PdbViewer v.3.7. This program

was also used for structural alignments and contouring of

ribbon diagrams.

For analysis of proteolysis kinetics, residual TCA�

insoluble protein was determined by a dye�binding

method [12]. The number�average molecular masses of

the intact cucurbitin subunits and of those of the residual

protein samples in the course of the reaction were calcu�

lated using the molecular masses of polypeptides separat�

ed by SDS�PAGE and their relative weight amounts.

Concentrations of the residual protein Pt and its molecu�

lar masses Mt were expressed as percentages of the initial

values P0 and M0.

The proteolysis kinetics was analyzed following a

previously described strategy [5]. During mixed�type pro�

teolysis, when limited and cooperative proteolyses occur

either in parallel independently from each other or suc�

cessively, the input of the limited proteolysis in the total

decline of the protein weight concentration of the

hydrolysate (P0 → Pt) is equal to M%0 – M%t. Hence, the

equation P%t + (M%0 – M%t) = f(t) exclusively describes

the kinetics of the cooperative proteolysis, which occurs

as a pseudo�first order reaction [13, 14]. Accordingly, the

decline of the relative molecular mass value of the protein

substrate (M%0 → M%t) exclusively describes the kinetics

of the limited proteolysis.

RESULTS AND DISCUSSION

According to the SDS�PAGE data, the subunits of

the intact cucurbitin consist of a group of similarly sized

α�chains α (apparent molecular mass 34.8 kDa) and a

minor α�chain α′ (24.0 kDa) and three β�chains (22.4,

21.5, and 20.8 kDa). Thus, the oligomeric cucurbitin

molecule is formed by a combination of at least three

kinds of subunits (Fig. 2, lane 1). Only a single amino acid

sequence of cucurbitin subunits is available (pdb|2EVX of

α� and β�chain molecular masses 31.5 and 20.9 kDa,

respectively). It seems likely that the apparent molecular

mass of the α�chains α shown above is overestimated,

which is characteristic of other seed 11S globulins [5].

Therefore, the molecular mass value of similarly sized α�

chains α, which has been used in all further calculations,

was taken equal to that of the cucurbitin 2EVX subunit.

Proteolysis of cucurbitin started with formation of a

transient α�chain fragment Ft (29.9 kDa) and fragment

F2 of 20.0 kDa (Fig. 2, lane 2). During further limited

proteolysis finalized by the formation of cucurbitin�P

(lane 5), the relative amounts of fragment F2 and the β�

chain β3 are increased, and the α�chain minor fragment

F1 (23.3 kDa) is formed. Relatively low�molecular�mass

(less than 20 kDa) TCA�insoluble products are not

detectable in the cucurbitin�P preparations. Therefore, it

was suggested that the limited proteolysis of cucurbitin

polypeptides is restricted to their N� and/or C�terminal

truncation.

SDS�PAGE in the absence of 2�mercaptoethanol

(Fig. 2, lanes 6 and 7) revealed bands of disulfide�bonded

α� and β�chains (intact cucurbitin) and the products of

their limited proteolysis (cucurbitin�P).

During the first step of the proteolysis (up to 2 h), the

decline of protein mass concentration P exceeds the

decrease of its molecular mass M but only slightly (Fig.

3a, curves 1 and 2, respectively). Thus, no significant

qualitative evidence of the occurrence of cooperative pro�

teolysis is observed at the first step.



The cooperative proteolysis of a changeless rate con�

stant started at the second step (Fig. 3a, curve 3).

Extrapolation of the linear part of curve 3 to zero time led

to P value that exceeds 100%. The fact of this excess can

be regarded as a qualitative indication that the rate con�

stant of the cooperative proteolysis depends on structural

alterations of the cucurbitin molecule generated by limit�

ed proteolysis at the first step. It should be taken into con�

Fig. 2. SDS�PAGE of intact cucurbitin and products of its proteolysis in the presence (lanes 1�5) and in the absence (lanes 6 and 7) of 2�mer�

captoethanol. Masses of molecular markers (kDa) are shown on the left side. Numbers below correspond to the duration of reaction (h).

1 2 3 4 5 6 7

0 0.5 1 2 14 0 14

Fig. 3. Proteolysis of cucurbitin by papain. a) Proteolysis kinetics. Curves: 1) weight concentration P of the residual (TCA insoluble) protein;

2) its number�average molecular mass M. P and M are expressed as percentage of the initial values; 3) the plot (P% + [100% – M%])

describes the kinetics of exclusively cooperative (one�by�one) proteolysis (see above). The ordinate axis is logarithmic. b) Relative molar

amounts (%) of polypeptide components in the intact cucurbitin subunits and in high�molecular�mass products of their proteolysis: 1) the

sum of β�chains β1 and β2; 2) β�chain β3; 3) sum of α�chains α and α′, and fragments Ft and F1; 4) fragment F2; 5) sum of β�chains β1,

β2, and β3; 6) sum of α�chains α and α′ and fragments Ft, F1, and F2.

a b

Time, h

824 RUDAKOVA et al.


sideration that the limited proteolysis deflects from ideal�

ity. Hence, low amounts of structurally altered cucurbitin

molecules as a substrate for the cooperative proteolysis

should appear before completion of the first stage of the

reaction.

Similar dependence of the rate of the cooperative

proteolysis on structural alterations of the protein sub�

strate induced by limited proteolysis was observed previ�

ously when the proteolysis of soybean 11S globulin by

papain was studied [5]. In contrast, cooperative proteoly�

sis of oat 11S globulin by papain starts from the very

beginning of the reaction irrespective of limited proteoly�

sis that occurs in parallel [3].

Limited proteolysis detectable by a continuous

decrease in the molecular mass of the residual protein

occurs at the second step (Fig. 3a, curve 2) under change�

less rate constant of the cooperative process. Thus, struc�

tural consequences of the limited proteolysis at the sec�

ond step seem to be inessential.

To follow the time course of cucurbitin�P formation,

the relative molar amounts (and thus the relative number

of particles) of the polypeptides detected by SDS�PAGE

under reducing conditions were plotted vs. the reaction

time (Fig. 3b). The decrease in summed molar amounts

of the β�chains β1 and β2 was found to accompany the

increase in molar amounts of the β�chain β3 (curves 1

and 2, respectively). It seems likely that during cucur�

bitin�P formation some of the β1 (22.4 kDa) and β2

(21.5 kDa) polypeptides are truncated, generating β3�

like (20.8 kDa) bands. Similarly, the decrease in summed

molar amounts of the α�chains α and α′ together with

obvious products of their proteolysis Ft and F1 was found

to reflect the increase in the molar amounts of fragment

F2 (curves 3 and 4, respectively).

The summed molar amounts of the β�chains β1�β3

(curve 5) is constant (~50%) up to 2 h of the reaction.

Hence, the fragment F2 that appeared just at the first step

of the reaction (Fig. 2, 0.5�2 h) is formed due to the

hydrolysis of α�chains. The apparent increase in the

summed molar amounts of polypeptides, which form the

bands β1, β2, and β3, and the simultaneous equivalent

decrease in the summed molar amounts of the α�chain

fragments during the second step of the reaction suggest

the formation of an additional α�chain fragment F2′(20.8 kDa), which co�migrates with the band of the β�

chain β3 in SDS�PAGE.

The summary information on primary and higher

order structures of seed storage 11S globulins [2] (includ�

ing cucurbitin subunit 2EVX, Fig. 1) as well as on the data

on the regularities of their proteolysis [3] allow us to char�

acterize in first approximation those alterations of pri�

mary and tertiary structure of cucurbitin that impart sus�

ceptibility of its molecules for the cooperative proteolysis.

Cysteine residues located inside conserved 11S glob�

ulin sequence regions (between the strands E and E′ in α�

chains (Fig. 1) and inside the extreme β�chain N�termi�

nus) are responsible for formation of the inter�domain

disulfide bond. During limited proteolysis of 11S globu�

lins (including cucurbitin, Fig. 2) this bond is retained

[3]. Hence, the decline in the molecular masses of the β�

chains β1 and β2 is due to truncation of their C�terminal

region that is usually disordered (irregular) in 11S globu�

lins [2]. Therefore, the detachment of this region during

limited proteolysis cannot be accompanied by any essen�

tial change in cucurbitin structure. This might explain at

least partially the invariant cooperative proteolysis rate

constant irrespective of limited proteolysis continued at

the second step (Fig. 3a, curves 2 and 3).

Four disordered variable regions are characteristic of

11S globulin α�chains [2] (Fig. 1a): 1) an N�terminal

region specifically extended in cucurbitin structure (Fig.

1b); 2) a loop between strands E′ and F′; 3) a loop

between the β�barrel and α�helices; and 4) a hyper�vari�

able C�terminal extension (the inter�domain linker). The

α�chain disordered regions formed during 11S globulin

evolution [3] served as initial targets for proteolytic attack

during seed germination and in vitro [15]. In 11S globulin

structures (including cucurbitin, Fig. 1b), the disordered

regions are bordered with amino acid residues of

enhanced accessibility to the solvent, which coincides

with enhanced susceptibility to limited proteolysis [9].

During papain hydrolysis of glycinin, the soybean

11S globulin, cleavage of the disordered region between

β�barrel and α�helices brings about the removal from the

final product of the limited proteolysis the entire C�ter�

minal region of α�chains including the inter�domain

linker together with α�helices and strand J′ [5] (Fig. 1b).

The residual sequences of glycinin α�chains, which cor�

respond to a “denudated” β�barrel containing non�

cleaved E′F′ loop, form fragments of molecular masses

20.1�21.9 kDa. Similar fragments (F2, 20.0 kDa, and

presumed fragment F2′, 20.8 kDa) are basic cucurbitin�P

polypeptides (Fig. 2). As with soybean glycinin, cucur�

bitin acquires susceptibility to cooperative proteolysis

after achieving a certain level of the limited proteolysis.

Therefore, it can be suggested that the limited proteolysis

of the structurally highly related glycinin and cucurbitin

α�chains follow similar scenarios, i.e. the detachment of

C�terminal helical domain (Fig. 1a). This detachment

results in either loosening of the subunit interactions

inside the trimers [5] or more significant alterations of the

11S globulin conformational state.

Controlled degradation of storage globulins in ger�

minating seeds is an integral part of their function. Rapid

but limited mobilization of the extended disordered

regions in 11S and 7S globulin polypeptide chains direct�

ly susceptible to proteolytic attack reflects the initial level

of degradation control. This is characteristic of both

angiosperms and gymnosperms in accordance with all the

available data. A slower but deeper degradation of storage

globulin molecules by the cooperative mechanism may

occur regardless of limited proteolysis as was shown to



occur in vitro for oat 11S globulin [3] and soybean 7S

globulin [8].

A higher level of degradation control demonstrated

for common bean 7S globulin [9] and 11S globulins from

soybeans [5] as well as from pumpkin seeds (this report) is

determined by the individual characteristics of the native

globulin structure of these species that provide its insus�

ceptibility to the cooperative proteolysis. In these cases,

massive storage globulin mobilization starts only after

achievement of a relatively deep level of preceding limit�

ed proteolysis.

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11s storage globulin from pumpkin seeds: regularities of proteolysis by papain

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