crystallin distribution patterns in concentric layers from toad eye lenses

RESEARCH ARTICLE

Crystallin distribution patterns in concentric layers from

toad eye lenses

Jonathan Keenan1, Giuliano Elia2, Michael J. Dunn3, David F. Orr1

and Barbara K. Pierscionek1

1 School of Biomedical Sciences, University of Ulster, Coleraine, UK2 Mass Spectrometry Resource, UCD Conway Institute of Biomolecular and Biomedical Research,

University College Dublin, Dublin, Ireland3 Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research,

School of Medicine and Medical Sciences, University College Dublin, Dublin, Ireland

Received: December 29, 2008

Revised: August 26, 2009

Accepted: September 1, 2009

Protein distribution patterns across eye lenses from the Asiatic toad Bufo gargarizans were

investigated and individual crystallin classes characterised. Special fractionation that follows

the growth mode of the lens was used to yield nine fractions corresponding to layers laid

down at different chronological (developmental) stages. Proportions of soluble and insoluble

crystallins within each fraction were measured by Bradford assay. Water-soluble proteins in

all fractions were separated by size-exclusion HPLC and constituents of each class further

characterised by electrophoresis, RP-HPLC and MS analysis. In outer lens layers, a-crystallin

is the most abundant soluble protein but is not found in soluble proteins in the lens centre.

Water-soluble b-crystallins also decrease from their highest level in the outer lens to negli-

gible mounts in the central lens. The proportion of soluble g-crystallin increases significantly

towards the lens centre where this is the only soluble protein present. Insoluble protein levels

increase significantly towards the lens centre. In B. gargarizans lenses, as with other anurans,

the predominant water-soluble protein class is g-crystallin. No taxon-specific crystallins were

found. The relationship between the protein distribution patterns and the functional prop-

erties of the lens this species is discussed.

Keywords:

Animal proteomics / Bufo gargarizans / Electrophoresis / Eye lens / LC / MS /

Protein distribution patterns.

1 Introduction

The vertebrate eye lens carries out a similar function in all

species but remains a structure of intense interest from an

evolutionary perspective [1, 2]. The lens remains transparent

because of the organization of major structural proteins, the

lens crystallins, which play a crucial role in maintaining the

refractive properties of the lens [1, 3–6]. Active metabolic

pathways in the lens are important in protecting against

post-translational modifications [7].

Although the common crystallins (a, b and g) are present

in all vertebrate lenses, taxon-specific crystallins, of which

d-crystallin is the most extensively studied, exist only in

particular species [2, 8–11]. These crystallins have also been

termed enzyme-crystallins because of their sequence simi-

larity to metabolic enzymes [10].

In amphibian lenses,g-crystallin is the main protein class

[12–14]. Amphibian lens crystallins have been studied to a

much lesser extent than mammalian crystallins. Taxon-

specific crystallins are present in lenses of particular species.

In frog lenses of genera Rana, the taxon-specific protein

r-crystallin has been isolated. This protein is a monomeric

crystallin with a molecular weight around 37 kDa [15–19].

z-crystallin, originally detected in guinea pig eye lenses hasAbbreviations: HMW, high molecular weight; LMW, low mole-

cular weight; MMW, middle molecular weight

Correspondence: Professor Barbara K. Pierscionek, School of

Biomedical Sciences, University of Ulster, Cromore Road,

Coleraine, BT52 1SA, UK

E-mail: [email protected]

Fax: 144-2870-324965

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

5340 Proteomics 2009, 9, 5340–5349DOI 10.1002/pmic.200800986

also been found to be present in Japanese tree-frog (Hylajaponica) lenses [20, 21].

Fewer studies have focused on toad eye lenses. Early studies

using Bufo bufo lenses concentrated for the most part on the

soluble lens protein [14]. de Jorge et al. (1970) found an

accumulation of sodium, magnesium and phosphorus in

cataractous lenses of Bufo marinus icterius toads [22]. More

recently, Roll compared lenses from different amphibian

species including Bufo bufo and several reptilian lenses, find-

ing taxon-specific proteins in only Rana temporaria and

Triturus vulgaris lenses [19]. A study into the major corneal

proteins of the Indian toad (Bufo melanostictus) found taxon-

specific a-enolase/t-crystallin in the lens; this had previously

been seen in turtle, duck, lamprey and crocodile lenses [23–27].

Differing results therefore exist regarding the presence of

taxon-specific crystallins in toad lenses of genera Bufo.

Unlike other ocular tissues, the lens continues to grow

throughout life accumulating fibres with little or no protein

turnover. As a result, the lens retains a chronological record

of changes and this renders the lens an ideal tissue for

studying changes that have occurred with development and

ageing. The fractionation technique used in this paper was

originally developed by Pierscionek and Augusteyn to study

chronological changes across human lenses [28]. It has since

been used in studies involving bovine, human, fish and

porcine lenses to examine changes in the protein distribu-

tion patterns with growth and ageing [28–32].

This paper presents an investigation of protein distribu-

tion patterns across the lens of the toad Bufo gargarizans.The crystallin classes have been identified and characterised.

2 Materials and methods

Two lenses were obtained from a 3-year-old Asiatic toad

(B. gargarizans) that had been sacrificed, at the University of

Ulster, for purposes not involved with these investigations.

The lenses were weighed and used immediately after

enucleation. All investigations were carried out at room

temperature unless otherwise stated.

2.1 Lens protein fractionation

The capsule, which envelops the lens, was removed; this

semi-elastic membrane peels off the lens material easily

without any tissue adhesion or loss. Successive concentric

fractions of each lens were isolated through step-wise disso-

lution by gentle agitation in 1 mL of 0.1 M phosphate buffer

(pH 7.3). Using this method, nine extracts were obtained for

each lens in a manner previously described [28–32].

Each extract was then centrifuged at 11 000 g for 20 min

at 41C to separate water-soluble from water-insoluble

protein. The water-insoluble proteins were dissolved in 8 M

urea (which acts to solubilise the water-insoluble fraction)

and protein contents (soluble and insoluble fractions) were

determined by the Bradford assay [33] using BSA as stan-

dard. Measurements of each sample were made in triplicate

and water-soluble and water-insoluble protein contents

expressed as a proportion of total protein in each lens layer.

2.2 Size-exclusion chromatography

Soluble proteins from each lens were separated into high

(HMW), medium (MMW) and low molecular weight (LMW)

fractions by size-exclusion chromatography using a Waters

Biosuite 250 5 mm HR SEC column (7.8� 300 mm) attached

to a Waters HPLC system at a flow rate of 1 mL/min and

detection at 280 nm. 0.1 M phosphate was used as the gel

filtration buffer. All solvents were of HPLC grade. The

column was calibrated using gel filtration standards. At least

two separations were performed on each sample and frac-

tions were collected from each peak. Areas under each peak

were calculated using Waters EmPower software.

2.3 SDS gel electrophoresis

Samples from each chromatographic peak were dried at

room temperature using a Concentrator 5301 (Eppendorf,

Hamburg, Germany). Each preparation was then subjected

to SDS gel electrophoresis by the Laemmli [34] method

using 4–12% NuPAGE Novex Bis-Tris Pre-cast Gels

(10� 10 cm) following the NuPAGE electrophoresis system

protocol (NuPAGE, Invitrogen, Carlsbad, CA, USA). Inso-

luble protein fractions, dissolved in 8 M urea, were also

analysed electrophoretically. Gels were stained with

Coomassie blue (SimplyBlue Safestain; Invitrogen, Carls-

bad, CA, USA).

2.4 2-DE

Soluble protein samples from the outermost lens layer were

also separated by 2-DE. To ensure that sample extracts

contained no contaminants that would interfere with subse-

quent 2-DE, each sample was precipitated in four volumes of

acetone, overnight at –201C and subsequently spun at

14 000 g for 10 min. The resulting pellet was resuspended in a

2-DE compatible lysis buffer (9.5 M Urea; 2% w/v CHAPS;

0.8% v/v Pharmalyte pH 3–10; 1% w/v DTT).

Isoelectric focusing of the sample was carried out on the

IPGphor 3 system (GE Healthcare, Little Chalfont, Bucks,

UK) using IPG strips (24 cm, pH 3–10 NL, Immobiline

Drystrip, GE Healthcare, Little Chalfont, Bucks, UK).

Approximately 100mg of sample was added to the rehydra-

tion buffer (8 M Urea, 0.5% w/v CHAPS, Bromophenol blue

(trace), 0.5% w/v DTT, 0.2% v/v pharmalyte) to give a final

volume of 450 mL. The sample was loaded by passive in-gel

rehydration overnight at room temperature [35]. The

proteins were separated by isoelectric focusing at 0.05 mA/

IPG strip for 75 000 Vh at 201C. Prior to separation by SDS-

PAGE the IPG strips were equilibrated for 15 min in equi-

libration buffer (6 M Urea; 50 mM Tris-HCl, pH 8.8 30% v/v

Proteomics 2009, 9, 5340–5349 5341


glycerol; 2% w/v SDS), which contained 10% w/v DTT. This

was followed by equilibration for 15 min in equilibration

buffer containing 25% w/v iodoacetamide. The strips were

loaded onto 15% polyacrylamide gels (1 mm� 24 cm� 22

cm) and the second dimension SDS-PAGE was carried out

overnight using a Protean plus Dodeca system (Bio-Rad,

Hemel Hempstead, Herts, UK) at 151C and 1 W/gel. Spots

were visualised using a silver stain (PlusOne Silver stain, GE

Healthcare, Little Chalfont, Bucks, UK).

2.5 MS analysis

Selected spots revealed by 2-DE were excised for tryptic

digestion, according to the protocol described by Shevchenko

et al. [36]. The resulting tryptic peptides were desalted and

concentrated using ZipTips (Eppendorf) according to the

manufacturer’s instructions. The lyophilized peptides were

dissolved in matrix buffer (70% ACN, 0.1% TFA in MilliQ

water), mixed with 3 mg/mL CHCA and spotted onto a 384-

well MALDI target plate (Applied Biosystems, Foster City,

CA). MALDI-TOF/TOF analysis was carried out with a 4800

MALDI-TOF/TOF Analyzer (Applied Biosystems). MS spec-

tra were acquired by 2000 laser shots from an Nd:YAG laser

operating at 355 nm and 200 Hz. After measuring all samples

in the MS mode, a maximum of 15 precursors per spot were

selected for subsequent fragmentation by CID. The resulting

spectra were processed and analysed using the Global Protein

Server (GPS Explorer) workstation (Applied Biosystems),

which uses internal MASCOT (Matrix Sciences) software for

matching MS and MS/MS data against databases of in silicodigested proteins. The data obtained were screened against a

non-redundant database of all known proteins downloaded

from the Expasy homepage (http://www.expasy.org). The

following analysis settings were used for the identification of

peptides and proteins: (i) precursor tolerance: 30 ppm, (ii)

MS/MS fragment tolerance: 0.2 Da, (iii) maximum missed

cleavages: 2 and (iv) variable modifications: oxidation of

methionine. Protein identifications were considered correct

calls when the confidence interval (CI) was greater than 95%.

2.6 Reverse-phase HPLC

Selected pooled samples of each size-exclusion peak (HMW,

MMW and LMW peaks) were subjected to RP-HPLC frac-

tionation using a 4.6� 150 mm, 5mm analytical Phenomenex

C-8(2) column (Hichrom, Reading, Berks, UK) eluted at a

flow rate of 2 mL/min with a gradient of TFA/water (0.1:99.9,

v/v) to TFA/water/ACN (0.1:19.9:80.0, v/v/v) over 80 min

using a Waters separation system. The column eluent was

monitored simultaneously at 214 and 280 nm and fractions

(1 mL) were collected and numbered at 1 min intervals.

2.7 Molecular mass determination

MALDI TOF-MS was performed on all peaks using an

Applied Biosystems Voyager instrument with delayed

extraction and autosampler (Applied Biosystems, CA, USA).

Samples were prepared by mixing a 1mL aliquot with 1mL of

matrix solution. The matrix solution was made by saturating

a water–ACN (50:50), 0.05% TFA solution with sinapinic

acid. Samples of 1 mL were spotted into wells of the MALDI

sample plate and allowed to air-dry before being placed in

the mass spectrometer. All peptides were analysed in the

linear, positive ion mode by delayed extraction using an

accelerating voltage of 20 kV, unless otherwise noted, giving

0.05% mass accuracy. The limit for detection using this

system was 10 fmol. External calibration was achieved using

a standard ‘‘calibration 2’’ mixture from PE Biosystems.

Mass spectra were obtained on a PerSeptive Voyager-DE

STR MALDI-TOF (Applied Biosystems).

3 Results

Lens wet weights were 0.1334 and 0.1402 g with total protein

contents representing approximately 30 and 32% of the wet

AU

0.00

0.20

0.40

0.60

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0

AU

0.00

0.05

0.10

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0

AU

0.00

0.02

0.04

0.06

AU

0.00

0.02

0.04

0.06

0.08

0.10

669 136 12kDa36 16

Layer 1

HMW MMW LMW

Layer 3

Layer 6

Layer 9

Figure 1. Typical size-exclusion HPLC elution profiles of the

water-soluble proteins from selected layers of B. gargarizans

lens. The layers are numbered consecutively from the lens

periphery inwards. Numbers above the chromatograms indicate

positions at which various molecular weight standards eluted.

From lens periphery to centre, HPLC profiles show a decrease in

HMW and MMW proteins and an increase in the proportions of

LMW proteins.

5342 J. Keenan et al. Proteomics 2009, 9, 5340–5349


weights. HPLC elution profiles obtained from selected toad

lens layers are shown in Fig. 1. In outer lens layers the

chromatographs demonstrated a high proportion of HMW

proteins and a low proportion of LMW proteins. The HMW,

MMW and LMW proteins eluted as sets of multiple rather

than single peaks. With progression into the central layers

of the lens, the peaks coalesced to single peaks and there

was a decrease in the proportion of the HMW and MMW

protein and a concomitant increase in the proportion of

LMW proteins. In the innermost lens layers, there was a

single LMW protein peak.

The proportions of each class of proteins (HMW, MMW

and LMW), obtained by calculating the area under each

peak, are plotted in Fig. 2 against the cumulative weight of

total soluble protein from lens periphery to centre. The

HMW proteins represented around 52% of the total soluble

protein in outer lens layers decreasing to less than 1% in the

central layers (Fig. 2A). The total MMW protein also

decreased towards the lens centre from around 24% of the

total soluble protein in the outer layers to less than 1% in

the central layers (Fig. 2B). The LMW proteins increased

considerably from around 22% of the total soluble protein in

the outer layers to around 98% in the central region

(Fig. 2C). There was an increase in insoluble proteins from

around 2% of the total protein in the outer layers to around

63% in the inner layers (Fig. 2D).

Constituents of individual peaks from an outer layer were

further characterised electrophoretically. The peaks sepa-

rated by size-exclusion HPLC were subjected to 1-DE (Fig. 3)

to broadly identify HPLC peak components; 2-DE (Fig. 4)

was used for more specific differentiation of subunits. The

LMW group of proteins were found to consist mainly of

g-crystallins with bands around the 19 000–21 000 Da range

(Figs. 3A and B). Some bands at molecular weights

consistent with b-crystallins were also visible in outer lens

layer fractions (Fig. 3A, lane 6) and in inner layers the

constituents of the single HPLC peak were g-crystallins

(Fig. 3B). Fractions from MMW peaks (Fig. 3A, lanes 3–5)

were dominated by bands between 23 000–32 000 Da

consistent with b-crystallins; a band around 26 000 Da was

the most intense. In lane 5, a faint band was also evident

around 12 000 Da. (MMW fractions from every layer are not

shown). Fractions from the HMW peaks showed bands with

molecular weights consistent with subunits of both a-crys-

tallin (19 000–20 000 Da) and b-crystallin (23 000–32 000 Da)

(Fig. 3A, lanes 1–2). The initial HMW peak was dominated

by a-crystallins with bands consistent with b-crystallins

appearing very faintly. b-crystallins were more apparent in

the second HMW peak. (HMW fractions from every layer

are not shown). The main constituents of the water-inso-

luble fractions were composed of subunits in the

19 000–28 000 Da range; faint bands of larger subunits were

less apparent (Fig. 3C).

Soluble lens proteins from the outermost layer, isolated

by 2-DE, are shown in Fig. 4. Peptides corresponding to a, band g-crystallins within the same molecular weight range as

in Fig. 3A were detected. Gel spots (numbered 1–30 in

Fig. 4) were selected to cover the complete range of spots

detected and identified by MALDI-TOF/TOF following

digestion with trypsin. Results from database searches are

presented in Table 1. The vast majority of spots analysed

demonstrated highest sequence identity with amphibian

crystallins, mainly from the lens of bullfrog (Rana catesbei-ana). Spots 1–14 corresponded to b-crystallins with bA1-1,

bA2, bB2 and bB3-crystallins identified. Spots 15–18 were

identified as g-crystallins. Spots 19–21 corresponded to

R. catesbeiana aB-crystallin and spot 22 to aA-crystallin

(R. catesbeiana). Peptides at lower molecular weights (Spots

23–30), likely to represent degradation products, were found

to be derived from both a and b-crystallins.

Intact molecular masses of the constituents of size-

exclusion HPLC fractions were also subjected to RP-HPLC

followed by MALDI-TOF MS. RP-HPLC of the HMW size-

exclusion fraction yielded three separate peaks, the masses

of which were found to be 19 606, 20 029 and 23 565 Da

(Figs. 5A and B). These are thought to correspond to aA-

crystallin, aB-crystallin and bB2-crystallin subunits, respec-

tively. As the latter is the main subunit extracted from the

0

20

40

60

80

100

0 10 20 30 40 50

Distance from Epithelium (cumulative mg protein)

% H

MW

Pro

tein

0

20

40

60

80

100

0 10 20 30 40 50


% M

MW

Pro

tein

0

20

40

60

80

100

0 10 20 30 40 50


% L

MW

Pro

tein

0

20

40

60

80

100

0 10 20 30 40 50


% In

solu

ble

Pro

tein

A B C D

Figure 2. Protein distribution patterns in B. gargarizans lenses. The proportions of (A) HMW proteins; (B) MMW proteins and (C) LMW

proteins plotted as a function of the cumulative amount of soluble protein from the lens periphery to centre. (D) The proportion of

insoluble protein plotted as a function of the cumulative amount of total protein from the lens periphery to centre. With progression

towards the lens centre there is a decrease in the HMW and MMW proteins and a concurrent increase in LMW and insoluble proteins.

Proteomics 2009, 9, 5340–5349 5343


pooled MMW peaks, a separate MALDI-TOF profile of this

subunit from the pooled HMW peaks is not shown.

RP-HPLC results from the MMW proteins produced two

peaks. When subjected to MALDI-TOF MS, the first of these

peaks gave a molecular mass of 23 599 Da, which is likely to

correspond to bB2-crystallin (Fig. 5C) (The peak appears to

be split; 23 599 Da refers to the higher peak). The finding

that two peaks, corresponding to molecular masses of 23 565

and 23 599 Da, from HMW and MMW fractions, respec-

tively, are both identified as bB2 crystallin, may indicate the

presence of age-related post-translational modifications in

this lens. Fractions from the second RP-HPLC peak

produced a MALDI-TOF profile similar to that obtained

from HMW fractions with peaks at molecular masses 19 600

and 20 041 Da (data not shown).

RP-HPLC of pooled LMW fractions produced two main

peaks with molecular masses of 21 173 and 20 048 Da

(Figs. 5D and E). These masses are likely to correspond to

g-crystallin subunits.

4 Discussion

In recent years, interest in amphibian research has

increased with the discovery of various antimicrobial

peptides in the skin and stomach of many amphibian

species [37, 38]. In the stomach of B. gargarizans one such

antimicrobial peptide with activity against Gram-positive

bacteria, Gram-negative bacteria and fungi has been isolated

[39].

Amphibians are of considerable interest to scientists as

they mark the evolutionary junction between aquatic and

terrestrial life. An important characteristic of anurans is that

as they grow they undergo metamorphosis which includes

ocular changes such as modifications to lenticular shape and

position to compensate for the living environmental change

from aquatic to terrestrial life [40].

Differences exist between the optics of mammalian and

amphibian eyes [41]. While frogs are thought to be emme-

tropic (no refractive error) in air, studies have shown toads

to be either emmetropic or myopic (short-sighted) once the

small eye artefact is taken into account [42–47]. Toads judge

distance using monocular and binocular cues [48, 49] and

Bufo bufo species are extremely reliant on visual rather than

on olfactory cues when catching prey [50, 51]. The lens

contributes refractive power to the eye and the magnitude of

this power depends on the lens shape as well as the material

property of refractive index. The refractive index of the eye

lens is not constant but follows an increasing gradient into

the lens centre [41]. The shape and magnitude of this

gradient is determined by the concentration and distribution

of the lens proteins. The contribution each protein class

makes to the refractive index varies depending on the

specific refractive increment [52]. The g-crystallin class of

proteins has the highest refractive increment of all the

crystallin classes and lenses with high proportions of

g-crystallin have steep refractive index gradients with high

index magnitudes [41].

The Bufo bufo group of toads which inhabit the eastern

escarpment of the Tibetan plateau includes species

B. gargarizans, Bufo andrews, Bufo minshanicus and Bufotibetanus which are believed to derive from a common

ancestor [53–55]. Since the sequences of all toad lens crys-

tallins are unknown, crystallins in this study were identified

based on sequence homology comparison to known

sequences of amphibian crystallins.

1 2 3 4 5 6 7 8 91417

28

Approx MW (kDa)

1 2 3 4 5 6 7

1417

28

38

Approx MW (kDa)

Approx MW (kDa)

1 2

62

49

38

28

17

A

B

C

Figure 3. Representative SDS gel electrophoresis results from (A)

the water-soluble protein fraction of B. gargarizans Layer 1.

Lanes 1–2 5 HMW peaks; lanes 3–5 5 MMW peaks; lanes

6–7 5 LMW peaks; constituents of the first HMW peak corre-

sponds to a-crystallins, constituents of MMW peaks to b-crys-

tallins and constituents of LMW peaks to g-crystallins. (B) The

LMW protein from concentric layers of B. gargarizans. The layers

are numbered consecutively (1–9) from the lens periphery

inwards; constituents of LMW peaks corresponds solely to

g-crystallins in all layers. (C) The water-insoluble protein fraction

of toad lens. Lane 1 5 outermost lens layer; lane 2 5 innermost

lens layer. The main constituents of water-insoluble protein

fractions are a-crystallins with bands corresponding to other

crystallin classes visible.



There are differences between mammalian and amphi-

bian lens crystallin patterns with g-crystallins representing

the major soluble protein in amphibian lenses [12–14,

56–59]. As seen with other anurans, this study has shown

that when the soluble lens proteins from the adult toad,

B. gargarizans, are separated, the common vertebrate lens

crystallins a, b and g-crystallins are present. It is possible

that the chromatograph integration method used may have

overestimated the proportion of a-crystallins present and

underestimated that of b-crystallins since the areas classified

as HMW proteins included some b-crystallins; MMW and

LMW proteins appeared to be more homogeneous. This

study concurs with previous work, that the major crystallin

class in the amphibian lens is g-crystallin. This study

is the first to show the distribution of this protein across

the lens and to demonstrate that this is the only crystallin

class present in the B. gargarizans lens centre, at a level of

98% of total soluble protein. This has significant implica-

tions for the optics of the toad lens and the function

of its eye as g-crystallin is associated with a high refractive

power [41].

The fractionation procedure in this paper followed the

growth mode of the lens, and hence, unlike previous work,

was able to show the protein patterns in layers laid down at

different life stages rather than averaged out across the

whole lens. The presence of high MW proteins in outer lens

layers has been reported in other species [4, 32]. This form

of fractionation has not been used for amphibian lenses;

cortical and nuclear separations have been made and the

presence of higher MW species was reported only in the

cortical regions that correspond to outer layers in this work.

Polansky and Bennett (1970) reported that adult bullfrog

lens nuclear crystallins were similar to those of a whole

tadpole lens [60]. The fractionation technique used in this

study enables lens fibres from concentric layers to be

examined more quantitatively and indicates which proteins

are preferentially synthesized at different times in the life of

the lens. Inner lens fractions contain proteins laid down in

embryonic life and these are chiefly g-crystallins. This

agrees with previous anuran work that has found g-crystallin

to be the earliest detectable protein in embryonic amphibian

lenses [56, 57, 59]. McDevitt et al. (1969) suggested that

g-crystallins were indicative of lens fibre differentiation in

the normal developing anuran lens [57].

Previous findings of high proportions of g-crystallin in

embryonic and adult fish lenses coupled with findings of

low levels of g-crystallin in bird lenses, have led to sugges-

tions that amphibian lenses occupy a phylogenetic niche

between fish and avian lenses and that g-crystallin was the

‘‘primitive’’ vertebrate crystallin [12, 61–64].

The study of Roll et al. [19] on whole Bufo bufo lenses

found electrophoresis bands within a similar molecular

weight range as this study. Additional peptides, such as a

12 kDa band, that were seen in this study, may have formed

as a result of post-translational modifications, causing

protein species to aggregate [65–68]. The lenses used in this

study were from an older toad than those in the study of Roll

et al. [19].

The constituents of the HMW proteins from size-exclu-

sion HPLC were found to be mainly a-crystallin with the

presence of some b-crystallins. This is consistent with

observations from previous studies which found that it was

more difficult to separate the a-crystallin from the b-crys-

tallin fraction in amphibian lenses compared to other

mammals or reptiles [32, 69].

MS analysis of the 2-D gel showed spots corresponding to

aA, aB, bA1-1, bA2, bB1, bB2, bB3, gII, gM1-2 and gM3-

crystallin (Table 1); several spots were matched to more than

one protein species. This is likely to be a consequence of

internal sequence homology and conservation across species

for each of the main crystallin classes. This is particularly

evident for the b-crystallins such as bA1-1 and bB2-crystallin

which were each detected in six spots (Fig. 4; Table 1). For

the majority of peptides analysed, the highest amino acid

coverage was seen with bullfrog lenses (R. catesbeiana).

Further work is needed to examine the extent of sequence

similarity between crystallins from R. catesbeiana and

B. gargarizans and to determine whether phylogenetically

55

29

2420

14.2

6.5

45

kDa

pH 3 pH 10

24

5

6

7

8

9

10 11

12

13

14 16

17

18 19 21

22

23

26

28 29 30

1

315

20

24

25 27

Figure 4. 2-DE results from the water-

soluble protein fraction of B. gargar-

izans Layer 1. Spots selected for MS

analysis by MALDI-TOF/TOF after

in-gel digestion with trypsin are

numbered 1–30. Spots were found to

correspond to a, b and g-crystallins as

detailed in Table 1.

Proteomics 2009, 9, 5340–5349 5345


R. catesbeiana lens crystallins are most similar to B. gargar-izans crystallins.

The results of this study agree with previous work that

showed proteins such as r- or z-crystallin are not expressed

taxon-specifically in Bufo bufo lenses [19]. The presence of

a-enolase has been reported in the lens of B. melanostictus [27];

the results of this study show that this protein was not found

in the lens of B. gargarizans. It is not known why a-enolase/t-

Table 1. MS analysis results from Bufo gargarizans lens listing the major proteins identified in selected 2-D gel spots.

Spot Accessionno.

Protein Peptidesmatched

ProteinpI

Proteinscore

Protein scoreCI %

Total ionscore

Total ionCI %

ProteinMW

1 Q91315 bA1-1-crystallinR. catesbeiana

5 6.16 119 100 90 100 23 221

2 P19141 bB3-crystallin B. taurus 5 6.36 189 100 160 100 24 3133 Q91315 bA1-1-crystallin

R. catesbeiana5 6.16 273 100 247 100 23 221

4 Q91315 bA1-1-crystallinR. catesbeiana

5 6.16 158 100 132 100 23 221

5 P19141 bB3-crystallin B. taurus 5 6.36 116 100 88 100 24 3136 P55164 bA2-crystallin G. gallus 5 6.23 77 99.198 51 98.498 22 989.17 Q91315 bA1-1-crystallin

R. catesbeiana5 6.16 299 100 273 100 23 221

8 Q91318 bB2-crystallinR. catesbeiana

3 6.75 91 99.969 78 99.997 23 634.5


9 6.75 383 100 316 100 23 634.5

10 P55164 bA2-crystallin G. gallus 4 6.23 66 90.361 46 95.563 22 989.111 Q91315 bA1-1-crystallin

R. catesbeiana5 6.16 281 100 254 100 23 221


13 6.75 475 100 360 100 23 634.5


12 6.75 494 100 390 100 23 634.5

14 P55164 bA2-crystallin G. gallus 3 6.23 67 92.165 53 99.387 22 989.115 Q91321 gM1-2-crystallin

R. catesbeiana2 6.3 83 99.808 75 99.997 20 836.8

16 P28022 gM3-crystallin C. carpio 4 7.51 60 58.883 50 98.975 20 935.317 Q91321 gM1-2-crystallin

R. catesbeiana4 6.3 58 41.92 40 91.292 20 836.8

18 P02531 gII-crystallinR. temporaria

3 7.08 74 98.437 63 99.962 20 146.5

19 Q91312 aB-crystallinR. catesbeiana

5 6.31 66 89.431 36 68.029 20 066.2


3 6.31 75 98.7 63 99.952 20 066.2


3 6.31 117 100 99 100 20 066.2

22 Q91311 aA-crystallinR. catesbeiana

5 5.87 188 100 161 100 19 886.7

23 Q91311 aA-crystallinR. catesbeiana

6 5.87 180 100 141 100 19 886.7

24 P02477 aA-crystallin P. phocoena 4 5.78 187 100 166 100 19 755.825 Q91315 bA1-1-crystallin

R. catesbeiana3 6.16 86 99.904 74 99.993 23 221

26 P02524 bB3-crystallinR. norvegicus

3 7.1 93 99.981 80 99.998 24 319.1


4 6.75 95 99.987 76 99.995 23 634.5


8 6.75 302 100 246 100 23 634.5


3 6.31 105 99.999 87 100 20 066.2

30 P02482 aA-crystallinA. jamaicensis

4 5.78 185 100 164 100 19 794.8



crystallin is present in lenses of some species of Bufo but not

others and what specific function this protein performs. One

characteristic of enzyme-crystallins is that the majority have

subunits with molecular weights between 35–37 kDa [70]. In

this study a faint band eluting with the g-crystallins around

38 kDa (Fig. 3A) was isolated by 1-DE. Sequence analysis (not

shown) confirmed that this band was derived from b-crystal-

lins rather than r- or z-crystallin with 61% identity to

R. catesbeiana bB2-crystallin. No spots corresponding to this

band were found in the 2-D gel. b-crystallin bands at these

higher molecular masses have been noted previously [32].

Taxon-specific crystallins have been found in amphibian

species R. catesbeiana, R. temporaria, Rana nigromaculata,

Rana rugosa, H. japonica, T. vulgaris and B. melanostictus[15, 18, 19, 21, 27]. Further studies are required to investi-

gate which amphibian species have taxon-specific crystal-

lins, why the lenses of only certain species contain these

crystallins, what their evolutionary origins may be and what

function they perform in the eye lens.

Funding from the Department of Employment andLearning (DEL), the College of Optometrists and the VisionRecognised Research Group of Northern Ireland’s Research andDevelopment Office is gratefully acknowledged (http://www.visionrrg.com). Research in M.J.D.’s laboratory is funded by

Science Foundation Ireland under Grant No. 04/RPI/B499.Access to and use of the UCD Conway Mass SpectrometryResource instrumentation is gratefully acknowledged. Gratitudeis expressed to Kasper Pedersen, Caitriona Scaife, GwenManning and Kieran Wynne for assistance in the preparation of2-D gels and MS analysis.

The authors have declared no conflict of interest.

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14000 19500 25000 30500 36000

% In

ten

sity

10

20

30

40

0

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20048

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Mass (m/z)

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Figure 5. MALDI-TOF mass spectra from RP-

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crystallin distribution patterns in concentric layers from toad eye lenses

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