Molecular Vision 2003; 9:110-118 <http://www.molvis.org/molvis/v9/a17/> Received 26 November 2002 | Accepted 14 April 2003 | Published 16 April 2003

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Existence of deamidated αB-crystallin fragments in normal and cataractous human lenses

Om P. Srivastava, Kiran Srivastava
 
 

Department of Physiological Optics, School of Optometry, University of Alabama at Birmingham, Birmingham, AL

Correspondence to: Om P. Srivastava, Department of Physiological Optics, Worrell Building, 924 South 18th Street, University of Alabama at Birmingham, Birmingham, AL, 35294; Phone: (205) 975-7630; FAX: (205) 973-5725; email: Srivastava@icare.opt.uab.edu

Abstract

Purpose: The aims of this study were to characterize lens crystallin fragments having a molecular mass of <10 kDa, isolated by solubilization in trichloroacetic acid, in order to identify cleavage sites in the parent crystallins for their origin and determine post-translational modifications in the fragments.

Methods: The water-soluble (WS) and water-insoluble (WI) protein fractions were isolated from normal human lenses of 60 to 80 year old donors and from age-matched cataractous lenses. Both WS and WI protein fractions were treated with TCA at 60 °C for 2 h and the TCA-soluble fractions were recovered following centrifugation. The preparations were dialyzed against H₂O to remove TCA, concentrated by lyophilization and subjected to two dimensional gel electrophoresis (2D-GE). The spots from 2D-gels were analyzed by western blot analysis, partial N-terminal sequencing, or excised for mass spectrometric analysis.

Results: SDS-PAGE analysis showed that TCA solubilized polypeptides having a molecular mass of <10 kDa from both WS and WI protein fractions of normal and cataractous lenses. Following 2D-GE of TCA-solubilized species from normal lenses, 8 and 5 polypeptides from the WS and WI protein fractions, respectively, were observed. Using similar 2D-GE analysis of TCA solubilized species from cataractous lenses, 9 and 5 polypeptides from WS and WI protein fractions, respectively, were seen. Partial N-terminal sequence analysis showed that the majority of the polypeptides from both WS and WI protein fractions of normal and cataractous lenses were derived from αB-crystallin following cleavage at the D₁₂₉-P₁₃₀ bond. Western blot and partial N-terminal sequence analyses identified three additional 4-kDa αA-crystallin fragments with cleavage at the D₁₃₆-G₁₃₇ bond in the WS proteins from normal lenses. MALDI-TOF mass spectrometric analysis showed that all TCA soluble polypeptides from cataractous lenses, except one from normal lenses, contained residue number 130 to 175 from αB-crystallin. No further truncation occurred at the C-terminal region of the αB-crystallin polypeptides. Following comparison of the isotopic distribution in MALDI-TOF profiles of a tryptic fragment having a mass of 2,014 among the αB-crystallin polypeptides, a gain of one single Dalton was observed. This suggested deamidation of the N₁₄₆ residue in αB-crystallin fragments.

Conclusions: The results show that the N₁₄₆ residue in human αB-crystallin undergoes in vivo deamidation and several fragments containing this modification exist in both WS and WI protein fractions of normal and cataractous human lenses.

Introduction

The lens contains structural proteins (α-, β-, and γ-crystallins), which are long lived and undergo a variety of post-translational modifications. These modifications include glycation (non-enzymatic glycosylation), carbamylation, steroid adduct formation, protein-protein disulfide bond formation, methionine oxidation, racemization, degradation, and deamidation [1]. These modifications, singly or in combination, cause changes in crystallins that lead to their aggregation and cross-linking during aging. The ultimate result of the changes is protein insolubilization due to aggregation and cross-linking and cataract development [1].

Deamidation is the conversion of an asparagine (N) residue to a mixture of isoaspartate and aspartate (D). Similar deamidation of glutamine (Q) to glutamic acid (E) can occur but does so at much lower rate [2]. With storage of proteins at neutral pH, a major modification is deamidation of asparagine residues to isoaspartic acid, which occurs in many proteins [3]. Deamidation has been shown to inhibit functional properties of proteins [4], and can also lead to physiological changes [5,6]. It is also hypothesized that the deamidation of both glutaminyl and aspariginyl residues in proteins may serve as molecular clocks of biological events such as protein turnover, development, and aging [6]. Indeed, the turnover rates of cytochrome c [7,8] and rabbit muscle aldolase reductase [9] were accelerated following deamidation. Similarly, the deamidation of histones [10] and erythrocyte membrane protein 4.1 [11] was linked to cell development and aging. More recently, the deamidation of phenylalanine hydroxylase [12], trisphosphate isomerase [13], ribonuclease [14], and lens crystallins [15] have been reported.

Because of the potential effects of deamidation on protein functions, the detection and quantification of isoaspartate is of great interest [3,16]. Isoaspartic acid is formed from asparagine via generation of a succinimide intermediate (formed by nucleophilic attack of a nitrogen atom at the carboxyl side of the residue on the asparagine side chain). The resulting cyclic intermediate hydrolyzes to form isoaspartic acid or less commonly, aspartic acid [3,10].

Deamidation of asparagine (N) and glutamine (Q) residues have been observed in both bovine and human crystallins. Age-related deamidation of crystallins seems to be one of the major post-translational modifications that are frequently observed [15,17-22]. The deamidation of specific residues was found to increase in the water insoluble protein fraction compared to the water-soluble protein fraction in human lenses [20]. Mass spectrometric analysis showed increased deamidation of human lens α-crystallin with relatively greater deamidation of Q₅₀ compared to Q₆ and Q₁₄₇ in αA-crystallin [21]. This report also showed deamidation of Q₁₀₈ and N₁₄₆ in αB-crystallin. Another study suggested only the deamidation of Q₅₀ in αA-crystallin during aging in human lenses [21]. Furthermore, deamidation of N₁₄₃ in γS-crystallin has been shown to be a cataract-specific but not an age-specific event [22]. Presently, the specific role of deamidation in cataract development is not known.

Deamidation of N has been shown to cause a loss of biological activity in vivo and in vitro in a variety of proteins such as DNAse [23] and recombinant soluble CD4 [24]. However, sometimes deamidation of N does not adversely affect biological activity [25]. A list of the proteins with affected biological activity following deamidation has been compiled by Wright [26]. Taken together, the role of deamidation is unpredictable but it is believed that deamidation may provide a signal for protein degradation, thereby regulating intracellular levels during development and aging [3,6,10]. Because of the relatively greater susceptibility of the C-terminal region compared to the N-terminal region of α-crystallin to degradation, it is possible that deamidation of N and Q residues in the C-terminal region may provide a potential signal for truncation.

The aims of this study were to: (1) Isolate crystallin fragments having a molecular mass of <10 kDa by a special method of TCA solubilization from normal and age-matched cataractous lenses, (2) determine bonds cleaved in vivo in crystallins to produce these fragments, and (3) determine post-translational modifications in the fragments. We report here a selective solubilization of crystallin fragments having a molecular mass of <10 kDa in TCA, identification of the majority of these as αB-crystallin fragments (containing residue numbers 130-175 with a cleavage at the D₁₂₉-P₁₃₀ bond) following separation as individual spots by 2D-GE and further analyses by N-terminal sequencing, western blotting, and mass spectrometric methods. The results show that the N₁₄₆ residue in human αB-crystallin undergoes in vivo deamidation, and several fragments containing this modification exist in both WS and WI protein fractions from normal and cataractous human lenses.

Methods

Materials

Normal human lenses with no apparent opacity were obtained from Dr. Robert Church, Emory University, Atlanta, GA or from the Shared Ocular Tissue Module at the University of Alabama at Birmingham. The lenses were retrieved within 48-72 h post-mortem and stored in medium-199 without phenol red at -20 °C until used. Intact lenses with nuclear cataract were similarly recovered from a local surgeon and stored in medium-199. The prestained and unstained protein molecular weight markers were from Life Technologies (Rockville, MD) and Amersham Biosciences (Piscatway, NJ), respectively. All chemicals for 2D-GE were from either Amersham Biosciences or BioRad (Hercules, CA). Unless indicated otherwise, all other chemicals used in this study were purchased from Sigma (St. Louis, MO) or Fisher (Atlanta, GA).

Isolation and analysis of polypeptides from WS and WI lens protein fractions

Normal human lenses from donors 60-80 years of age and age-matched cataractous lenses were used to isolate WS and WI protein fractions by a procedure previously described [27]. The WS and WI protein fractions contained proteins from epithelium, cortex and nucleus of the lenses. The WI protein fraction was suspended in buffer A (2 ml per lens [27]) and both WS and WI protein fractions were separately treated with 2.5% trichloroacetic acid (TCA, v/v, final concentration) at 60 °C for 2 h. Next, both protein fractions were cooled to room temperature and centrifuged at 25,000x g for 15 min. The TCA soluble preparations were dialyzed against distilled water at 4 °C for 72 h using 3,500 Dalton molecular weight cut off dialysis tubing (Spectrum, Rancho Dominguez, CA), and concentrated by lyophilization to dryness. The TCA soluble preparations from WS and WI protein fractions were individually dissolved in resolubilization buffer (5 M urea, 2 M thiourea, 2% 3-[C3-cholamidoproyl] dimethyl-ammonio-1-propansulfonat (CHAPS), 2% caprylyl sulfobetaine 3-10, 2 mM tri-butyl phosphine, 40 mM Tris, pH 8.0). Each preparation was subjected to 2D-gel electrophoresis (IEF in the first dimension and SDS-PAGE in the second dimension). IEF separation was carried out using Immobiline Dry Strips (pH range of 3-10) and utilizing the manufacturer's method (Amersham Biosciences). SDS-PAGE in the second dimension was performed using 15% polyacrylamide gels [28].

Miscellaneous methods

For western blot analysis, the 2D-GE separated species were electrophoretically transferred to a PVDF membrane [29], immunoreacted with either anti-human αA-crystallin peptide (N-terminal residues 1-9) or anti-human αA-crystallin peptide (C-terminal residues 165-173) antibodies, and anti-rabbit IgG-peroxidase-conjugated secondary antibody was used to visualize immunoreactive spots. The two antibodies were raised in our laboratory using a procedure previously described [30]. Partial N-terminal sequencing of the desired spots was performed at the core facility of University of Alabama at Birmingham. For this purpose, the polypeptides, after their transfer to a PVDF membrane, were briefly stained with Coomassie blue and individual spots excised and used for sequencing. The sequences of desired fragments were matched with that of published sequences of human lens crystallins. For sequence searching, the on-line search engine (via SEQSRCH) using the database from the Protein Research Foundation (Japan) was used. For mass spectrometric analyses, the protein spots were excised from a 2D-gel, washed with doubly deionized water, and destained after treating with ammonium bicarbonate and acetonitrile. A trypsin solution (12 ng/μl) was added and the preparation was resuspended in 25 mM ammonium bicarbonate, pH 7.8. The samples were digested with trypsin at 37 °C overnight, and next day, they were analyzed by MALDI-TOF method (Pespective Biosciences, Model Voyager-DE2 PRO). The MALDI-analysis and ES-MS/MS sequencing (Micromass QTOF-2) were performed at the University of Alabama at Birmingham (UAB) mass spectrometry core facility. The matrix assisted laser desorption ionization-time of flight (MALDI-TOF) identity of proteins was established by using the NCBInr database of Matrix Science. Protein concentration was determined by a modified method of Lowry using the Pierce protein determination kit.

Results

Solubilization of polypeptides from human lenses and 2D-gel electrophoretic analysis

The WS and WI protein fractions from normal and cataractous human lenses were treated with 2.5% TCA, and the TCA-solubilized polypeptides from the two fractions were separated by 2D-GE as described in Methods. From normal lenses, approximately 2.8% of the total WS proteins, and 13.8% of the total WI proteins were TCA-solubilized. Similarly, from the cataractous lenses, 6% of the total WS proteins and 13.8% of WI proteins were TCA solubilized. SDS-PAGE analysis of TCA-soluble fractions recovered from WS and WI protein fractions of individual normal and cataractous lenses showed that the majority of the solubilized polypeptides had a molecular mass of <10 kDa (Figure 1, lanes 1 to 7). For this analysis, proteins from individual lenses were identically solubilized in TCA, dialyzed at 4 °C, freeze dried, suspended in identical volumes, and identical aliquots of from each preparation were used. In Figure 1, lane 2 contained TCA-solubilized polypeptides from WS proteins of a cataractous lens, and lanes 3 and 4 contained TCA-solubilized proteins from WS proteins of individual normal lenses. Similarly, lane 5 contained TCA solubilized polypeptides from WI proteins of a cataractous lens, and lanes 6 and 7, TCA-solubilized polypeptides from WI proteins from individual normal lenses. Because identical quantities of either WS or WI proteins from normal and cataractous lenses were used during the TCA solubilization and the final TCA soluble polypeptides were suspended in identical volumes, the SDS-PAGE profile in Figure 1 indicates the relative amounts of each TCA-solubilized species. It was noted that TCA soluble polypeptides having a molecular mass of <10 kDa were absent in the TCA insoluble (precipitated) fractions of both normal and cataractous lenses (Figure 1, lanes 9-13). However, two species of 25 and 43 kDa were occasionally seen in the TCA soluble fractions. These were later identified as contaminants from the TCA precipitated proteins. This conclusion was based on SDS-PAGE analyses of at least five individual TCA soluble fractions isolated from WS and WI proteins of either normal or cataractous lenses.

To identify various polypeptides species present in the TCA solubilized fractions, each fraction was subjected to 2D-GE and the gel spots were used either for western blotting, partial N-terminal sequencing, or mass spectrometric analyses. On 2D-GE, a total of thirteen TCA-soluble spots (8 spots from the WS protein fraction Figure 2A and 5 spots from the WI protein fraction Figure 2B) from normal lenses were observed. A similar 2D-GE of cataractous lenses showed a total of 14 spots (9 spots from the WS protein fractions and 5 spots from the WI protein fractions, Figure 3).

Western blot and N-terminal sequence analyses of the TCA-soluble polypeptides isolated from WS proteins from normal lenses

Partial N-terminal sequence analysis showed that the majority of the polypeptides (spots number 1-5 from WS proteins and spots number 9-13 from WI proteins, Figure 2) from normal lenses were derived from the αB-crystallin following cleavage at the D₁₂₉-P₁₃₀ bond (Table 1). This suggested a repeated cleavage of this bond to produce several polypeptides with approximately identical molecular mass but different charges, as was evident from the 2D-GE separation. Spots number 7 and 8 from the WS protein fraction (Figure 2) originated from γS-crystallin with cleavage at G₉₀-G₉₁ (Table 1). Because of relatively poor staining of certain spots, western blot analysis was used to identify them. On such an analysis with anti-αA N-terminal (residue numbers 1-9) and anti-αA-C-terminal (residue numbers 165-173) antibodies, three spots of 4 kDa exhibited immunoreaction with the anti-αA-C-terminal (residue numbers 165-173) antibody (Figure 4). Following partial N-terminal sequence analysis, all three spots showed an identical N-terminal sequence of GMLTF, suggesting a cleavage at D₁₃₆-G₁₃₇ in αA-crystallin. Therefore, the three αA-crystallin fragments having an identical molecular mass but differing in charges existed in the WS protein fraction of human lenses.

To determine whether charge differences in the αB-crystallin fragments were due to additional truncation at the C-terminal region (between residue numbers 130-175), each 2D-gel separated polypeptide was trypsin-digested and analyzed by MALDI-TOF. A typical MALDI profile of spot numbers 1-5 from the WS TCA soluble fractions and spot numbers 9, 11, 12, and 13 from the WI TCA soluble fractions of normal lenses is shown in Figure 5.

The known sequence of human lens αB-crystallin from residues number 130-175 is; DPLTITSSLSSDGVLTVNGPRKQVSGPERTIPITREEKPAVTAAPKK. In the MALD-TOF profile of Figure 5, the tryptic fragment peaks having a mass of 2,014 (representing the N-terminal sequence of residues number 130-150 having a sequence of; DPLTITSSLSDGVLTVNGPR) and 1,823 (representing C-terminal residues number 157-174, having a sequence of; TIPITREEKPAVTAAPK) were present.

Assuming that the bond with the last two C-terminal residues of K₁₇₄-K₁₇₅ was cleaved by trypsin in the above tryptic fragment having a mass of 1,823, the MALDI data suggested the presence of intact C-termini in the αB-crystallin fragments. A tryptic fragment having a mass of 1,141 representing the sequence EEKPAVVTAAPK (residues number 164-174) of αB-crystallin was also observed in all of the αB-crystallin fragments except spot number 10, suggesting that spot number 10 of αB-crystallin was truncated at the C-terminal region.

A similar MALDI-TOF mass spectrometric analysis of TCA soluble polypeptides from cataractous lenses (spots number 1-7, Figure 3A), isolated from the WS protein fraction, and spots number 10-12 isolated from WI protein fractions (Figure 3B) showed peaks having a mass of 2,014 (representing the N-terminal sequence of residues number 130-150) and 1,823 (representing the C-terminal residues number 157-174, results not shown). The results showed that these were fragments of αB-crystallin having an intact C-terminus. The identity of spots number 8 and 9 (isolated from the WS protein fraction) and spots number 12 and 13 (isolated the from WI protein fraction) of cataractous lenses are, at present, unknown.

Determination of deamidation in αB-crystallin fragments

From the above partial N-terminal sequence and mass spectrometric data, it was clear that the majority of the αB-crystallin fragments contained residues number 130-175, and yet demonstrated charge differences. To examine whether this could be due to post-translational modifications, three tryptic fragments from spot number 1, isolated from the WS protein fraction of normal lenses (Figure 2) were subjected to ES-MS/MS analysis using Micromass QTOF-2 (Figure 6). The first fragment having a mass of 899.48 was doubly charged and had a sequence of KQVSGPER (residues number 150-157 from αB-crystallin) with no modified amino acids (Figure 6A). The second tryptic fragment having a mass of 1,139.62 from MS/MS analysis was doubly charged and had the sequence of EEKPAVTAAPK (residues number 164-174 from αB-crystallin), and exhibited no modified amino acids. Similarly, the third tryptic fragment having a mass of 2,014 was doubly charged during MS/MS analysis, and had a sequence of PLTITTSSLSSDGVLTVNGPR (residues number 130-174 from αB-crystallin) with no amino acid modification.

We next determined the potential deamidation of N and E residues in αB-crystallin fragments. The αB-crystallin fragment contained one N (at position 146) and four E residues (at positions 155, 156, 164, and 165). The tryptic fragments having a molecular mass of 1,140 (amino acid sequence; EEKPAVTAAPK), 2,014 (amino acid sequence; PLTITSSLSSDGVLTVNGPRK), and 2,175 (amino acid sequence; PLTITSSLSSDGVLTVNGPRK) were examined for their isotopic mass distribution. When N and Q are deamidated to D and E, respectively, a change (gain) of only one mass unit occurs with each deamidation. The isotopic peak distribution in MALDI profiles of tryptic fragments having a mass of 1,140 and 2,014 from spot no. 1 (Figure 7A,B) and spot no. 2 (Figure 7C,D), isolated from WS proteins of normal lenses (Figure 2A), were compared. The isotopic distribution of peak 1,140 remained almost identical in spots number 1 and 2 (compare Figure 7A,C) whereas the 2,014 peak disappeared in spot number 2 (compare Figure 7B,D). This suggested that the N residue in the fragment having a mass of 2,014 was deamidated to D by gaining one mass unit. A similar isotopic peak analysis in the MALDI profiles of other αB-crystallin fragments (from normal and cataractous lenses) showed an identical shift of one mass unit and disappearance of the peak having a mass of 2,014 (results not shown). The data strongly suggested that the N₁₄₆ residue was deamidated in all of the αB-crystallin polypeptides from normal lenses except for spot number 1. In contrast, the deamidation of N₁₄₆ was observed in all of the αB-crystallin polypeptides isolated from cataractous lenses.

Discussion

The major finding of this report are that WS and WI protein fractions of normal and cataractous lenses contained; (1) αB-crystallin fragments (residues number 130-175) with cleavage at the D₁₂₉-P₁₃₀ bond, (2) αA- and γS-crystallin fragments with cleavage sites at D₁₃₆-G₁₃₇ and G₉₀-G₉₁, respectively, and (3) the N₁₄₆ residue of αB-crystallin fragments demonstrated deamidation. The association of the deamidated αB-crystallin fragments with the WI protein fractions suggested aggregation and cross-linking during aging and cataractogenesis. While both nondeamidated and deamidated N₁₄₆ αB-crystallin fragments were present in normal lenses, cataractous lenses contained only the deamidated αB-crystallin fragments. Presently, it is difficult to distinguish whether deamidation in αB-crystallin fragments occurred following their insolubilization or the deamidation caused their insolubilization. However, the data did show deamidation of N₁₄₆ in all of the αB-crystallin fragments from cataractous lenses, but not in all such fragments from aging lenses. Therefore, this deamidiation could be a cataract-specific change like other deamidation previously reported in human cataractous lenses [15,22].

SDS-PAGE and 2D-GE analyses showed that polypeptides having a molecular mass <10 kDa were selectively solubilized in TCA from both WS and WI protein fractions while the majority of lens crystallins were precipitated (Figure 1, Figure 2, and Figure 3). Additional MALDI-TOF analysis of crystallin fragments from WS and WI protein fractions of normal and cataractous human lenses showed the presence of αB-crystallin fragments having a molecular mass <10 kDa (unpublished results). Therefore, the αB-crystallin fragments that were selectively solubilized in TCA existed in vivo.

Based on previously published reports, the TCA solubilization method was chosen to isolate polypeptides having a molecular mass of <10 kDa. Previously, the TCA solubilization method has been employed to isolate and purify low molecular weight proteinase inhibitors [31], and it was also used in our laboratory to selectively solubilize a 5.5 kDa α-crystallin fragment from WS and WI protein fractions from bovine lenses [32,33]. Further, a relatively greater level of TCA solubilization of the 5.5 kDa species from bovine lenses occurred at an elevated temperature (up to 60 °C) compared to lower temperatures [33]. A previous study used immunology to identify a bovine lens 5.5 kDa polypeptide as an α-crystallin fragment [31]. The data presented in this report showed selective TCA solubilization yielded αB-crystallin fragments composed of residue numbers 130-175, produced by cleavage at the D₁₂₉-P₁₃₀ bond (Figure 2 and Figure 3). This identification was possible only because the 2D-GE resolved a mixture of the TCA solubilized polypeptides into individual spots (13 spots from WS and WI proteins of normal lenses, and 14 spots from WS and WI proteins of cataractous lenses).

The TCA soluble polypeptides from the WS and WI protein fractions of cataractous lenses showed a somewhat higher molecular size (Figure 3) compared to those exhibited by the TCA solubilized polypeptides from normal lenses (Figure 2). This difference may be due to the use of different sets of protein molecular weight markers in the analysis (the markers used for the analysis of normal lenses had a molecular size range of 2.9 to 43.7 kDa whereas the markers used for the analysis of cataractous lenses had a molecular size range of 8.1 to 16.9 kDa). The markers having a range of 8.1 to 16.9 kDa may not be accurate because of two reasons; (1) as shown in Figure 1, the TCA soluble preparations from normal and cataractous lenses on SDS-PAGE analysis using the 2.9 to 43.7 kDa range standard showed polypeptides that had an almost identical molecular mass of 3 to 4 kDa. This figure also shows that the molecular mass of TCA-soluble polypeptides from WS proteins in lane 2 (cataractous) and lanes 3 and 4 (normal) were identical, and similarly, the molecular mass of polypeptides isolated from WI proteins in lane 5 (cataractous) and lane 6 (normal) were identical, and (2) as described later, the partial N-terminal sequencing and MALD-TOF mass spectrometric analyses showed that the majority of the TCA soluble polypeptides from both normal and cataractous lenses were αB-crystallin fragments containing residues number 130 to 175.

The partial N-terminal sequence analysis showed that the majority of the TCA soluble polypeptides were αB-crystallin fragments having a cleavage site at the D₁₂₉-P₁₃₀ bond. Although addition data from our laboratory suggest in vivo cleavage of this bond, the cleavage may also occur during TCA treatment because the D-P bond is susceptible to cleavage during acid treatment [34]. However, the presence of two γs-crystallin fragments with cleavage at the G₉₀-G₉₁ bond, and αA-crystallin fragments with cleavage at the D₁₃₆-G₁₃₇ bond suggested in vivo cleavage to generated the αB-crystallin fragments. This is supported by the MALDI-TOF analysis of 2D-GE-separated crystallin fragments (molecular mass of <10 kDa) of WS and WI protein fractions, which identified the presence of C-terminal intact αB crystallin fragments (unpublished results). The MALDI-TOF mass spectrometric results also showed that the C-terminal region in the αB-crystallin fragments were intact, they contained residue number 130-175. Therefore, the observed charge differences in the αB-crystallin fragments during 2-D-GE must be due to certain post-translational modifications. One such modification was identified as the deamidation of N₁₄₆ in these fragments. As stated above, the deamidation of N to D results in an increase in one single Dalton mass, which is detectable during isotopic peak analysis of tryptic fragments during MALDI-TOF analysis. Comparison of the isotopic distribution of a tryptic fragment with mass of 2,014 (which contained the single N₁₄₆ residue in the αB-crystallin fragment) of polypeptide spot number 1 with other peptides of identical mass from different spots (isolated from WS or WI proteins of normal and cataractous lenses), the latter spots showed a gain of one Dalton, suggesting deamidation of N₁₄₆ (Figure 7). It is well established that carbon in nature mainly exists as C¹² but 1% also exists as C¹³. While determining the mass by MALDI-TOF, one always finds this 1% of the C¹³ carbon and this gives the additional minor peaks observed in Figure 7. The additional peaks are therefore of the C¹³ isotopic forms, which are observed after amplification. Among many known factors that affect deamidation of a particular N residue in a protein, one is the sequence consideration [3,10]. The highest frequency of this process is observed in proteins having an N-G sequence. Indeed, among proteins in which deamidation has been established and the site characterized, G has been most common residue (N+1). The N+1 residue in the αB-crystallin fragment is also a G residue (see sequence αB-crystallin fragment in Results). This again suggests in vivo deamidation of N₁₄₆ in the fragments.

The deamidation of N₁₄₆ in αB-crystallin may be significant for many reasons. First, deamidation has been hypothesized to alter protein stability due to an increase in charge [10], and increased deamidation of crystallins does occur during aging and cataractogenesis [15,21,22]. Because several factors (age-related post-translational modifications such as degradation, deamidation, oxidation, mixed disulfide formation, and glycation [1]) could reduce the chaperone activity of α-crystallin, the deamidation N₁₄₆ would destabilize the αB-crystallin structure and affect chaperone activity. Second, because the chaperone activity of α-crystallin is dependent on interactions of αA- and αB-crystallin subunits in an oligomer [35], deamidation may affect interactions of these subunits. Indeed, on deamidation of N₂₀₄ in βB1-crystallin, the less compact dimers were observed, suggesting its affect on oligomerization properties [36]. Third, studies of age-related human nuclear cataracts showed that αB-crystallin became part of the colored proteins, and therefore participated in the cataractogenic process [35]. The deamidation of αB-crystallin may affect its stability and therefore may result in post-translational modifications leading to a build up of colored crystallins and enhanced cataractogenesis. Fourth, It has been postulated that deamidation may provide a signal for protein degradation, and thereby regulates their intracellular levels [3,10]. Because the C-terminus of α-crystallin is more susceptible to proteolysis than its N-terminal region (the C-terminus is cleaved in bovine lens αA- and αB-crystallins [αA-crystallin at 1-168, αA-crystallin at 1-151, αA-crystallin at 1-101, and αB-crystallin at 1-170]) [17,37], the deamidation of N₁₄₆ in αB-crystallin might play a role in this susceptibility. Because a truncation of the C-terminal region could lead to a loss of chaperone activity in α-crystallin [38], a correlation between the proteolytic susceptibility of the C-terminal region of N₁₄₆-deamidated αB-crystallin and a loss in chaperone activity might exist.

As stated above, several TCA solubilized αB-crystallin fragments exist that differ in their charges in spite of having an identical amino acid sequence. Because the deamidation of N₁₄₆ was the only post-translational modification identified in the polypeptides, it partly explains the charge difference. Therefore, additional unknown modifications in the polypeptide must exist and are yet to be identified. Indeed, anomalous tryptic peptide peaks of αB-crystallin polypeptides whose mass could not be explained in a typical MALDI profile (represented in Figure 5) were seen. These fragments are presently under investigation to identify additional modifications to explain the charge differences among the αB-crystallin fragments.

Acknowledgements

This work was supported by grants from National Eye Institute (EY-06400 and EY-03039), and the Retirement Research Foundation, Inc. The authors express their sincere appreciation to Ms. Martha Robbins for her help during the preparation of the manuscript. Ms. Janelle Thompson, a summer student under the McNair Scholar program initiated part of the work presented.

References

1. Harding J. Cataract: biochemistry, epidemiology, and pharmacology. New York: Chapman & Hall; 1991.

2. Robinson AB, Rudd CJ. Deamidation of glutaminyl and asparaginyl residues in peptides and proteins. Curr Top Cell Regul 1974; 8:274-95.

3. Robinson NE. Protein deamidation. Proc Natl Acad Sci U S A 2002; 99:5283-8.

4. Kim E, Lowenson JD, MacLaren DC, Clarke S, Young SG. Deficiency of protein-repair enzyme results in accumulation of altered proteins, retardation of growth and fatal seizures in mice. Proc Natl Acad Sci U S A 1997; 94:6132-7.

5. Liu DT. Deamidation: a source of microheterogeneity in pharmaceutical proteins. Trends Biotechnol 1992; 10:364-9.

6. Robinson NE, Robinson AB. Molecular clocks. Proc Natl Acad Sci U S A 2001; 98:944-9.

7. Flatmark T, Sletten K. Multiple forms of cytochrome c in the rat. Precursor-product relationship between the main component Cy I and minor components Cy II and Cy 3 in vivo. J Biol Chem 1968; 243:1623-9.

8. Robinson AB, McKerrow JH, Legaz M. Sequence dependent deamidation rates for model peptides of cytochrome C. Int J Pept Protein Res 1974; 6:31-5.

9. Midelfort CF, Mehler AH. Deamidation in vivo of an asparagine residue of rabbit muscle aldolase. Proc Natl Acad Sci U S A 1972; 69:1816-9.

10. Lindner H, Sarg B, Hoertnagl B, Helliger W. The microheterogeneity of the mammalian H1(0) histone. Evidence for an age-dependent deamidation. J Biol Chem 1998; 273:1324-30.

11. Inaba M, Gupta KC, Kuwabara M, Takahashi T, Benz EJ Jr, Maede Y. Deamidation of human erythrocyte protein 4.1: possible role in aging. Blood 1992; 79:3355-61.

12. Solstad T, Flatmark T. Microheterogeneity of recombinant human phenylalanine hydroxylase as a result of nonenzymatic deamidations of labile amide containing amino acids. Effect on catalytic and stability properties. Eur J Biochem 2000; 267:6302-10.

13. Sun AQ, Yuksel KU, Gracy RW. Terminal marking of triosephosphate isomerase: consequences of deamidation. Arch Biochem Biophys 1995; 322:361-8.

14. Capasso S, Salvadori S. Effect of three-dimensional structure on deamidation reaction of ribonuclease A. J Pept Res 1999; 54:377-82.

15. Takemoto L, Boyle D. Increased deamidation of asparagine during human senile cataractogenesis. Mol Vis 2000; 6:164-8 <http://www.molvis.org/molvis/v6/a22/>.

16. Robinson NE, Robinson AB. Prediction of protein deamidation rates from primary and three-dimensional structure. Proc Natl Acad Sci U S A 2001; 98:4367-72.

17. Van Kleef SM, Willems-Thijssen W, Hoenders HJ. Intracellular degradation and deamidation of alpha-crystallin subunits. Eur J Biochem 1976; 66:477-83.

18. Groenen PJ, van Dongen MJ, Voorter CE, Bloemendal H, de Jong WW. Age-dependent deamidation of alpha B-crystallin. FEBS Lett 1993; 322:69-72.

19. Miesbauer LR, Zhou X, Yang Z, Yang Z, Sun Y, Smith DL, Smith JB. Post-translational modifications of water-soluble human lens crystallins from young adults. J Biol Chem 1994; 269:12494-502.

20. Lund AL, Smith JB, Smith DL. Modifications of the water-insoluble human lens alpha-crystallins. Exp Eye Res 1996; 63:661-72.

21. Takemoto L, Boyle D. Specific glutamine and asparagine residues of gamma-S crystallin are resistant to in vivo deamidation. J Biol Chem 2000; 275:26109-12.

22. Takemoto L, Boyle D. Deamidation of specific glutamine residues from alpha-A crystallin during aging of the human lens. Biochemistry 1998; 37:13681-5.

23. Cacia J, Quan CP, Vasser M, Sliwkowski MB, Frenz J. Protein sorting by high-performance liquid chromatography. I. Biomimetic interaction chromatography of recombinant human deoxyribonuclease I on polyionic stationary phases. J Chromatogr 1993; 634:229-39.

24. Teshima G, Porter J, Yim K, Ling V, Guzzetta A. Deamidation of soluble CD4 at asparagine-52 results in reduced binding capacity for HIV-1 envelop glycoprotein gp120. Biochemistry 1991; 30:3916-22.

25. Becker GW, Tackitt PM, Bromer WW, Lefeber DS, Riggin RM. Isolation and characterization of sulfoxide and desamido derivative of biosynthetic human growth hormone. Biotechnol Appl Biochem 1988; 10:326-37.

26. Wright T. Amino acid abundance and sequence data: clues to biological significance of non-enzymatic asparagine and glutamine deamidation in proteins. In: Aswad DW, editor. Deamidation and isoaspartate formation in peptides and proteins. Boca Raton: CRC Press; 1995. p. 229-251.

27. Srivastava OP, McEntire JE, Srivastava K. Identification of a 9 kDa gamma-crystallin fragment in human lenses. Exp Eye Res 1992; 54:893-90.

28. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-5.

29. Towbin H, Stahelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979; 79:4350-4.

30. Srivastava OP, Srivastava K, Silney C. Covalent modification at the C-terminal end of a 9 kDa gamma D-crystallin fragment in human lenses. Exp Eye Res 1994; 58:595-603.

31. Laskowski M Jr, Kato I. Protein inhibitors of proteinases. Annu Rev Biochem 1980; 49:593-626.

32. Srivastava OP, Ortwerth BJ. Purification and properties of a protein from bovine lens which inhibits trypsin and two endogenous lens proteinases. Exp Eye Res 1983; 36:363-79.

33. Srivastava OP, Ortwerth BJ. Age-related and distributional changes in the trypsin inhibitor activity of bovine lens. Exp Eye Res 1983; 36:695-709.

34. Cook LA, Schey KL, Wilcox MD, Dingus J, Hildebrandt JD. Heterogeneous processing of a G protein gamma-subunit at a site critical for protein and membrane interactions. Biochemistry 1998; 37:12280-6.

35. Truscott RJ, Chen YC, Shaw DC. Evidence for the participation of alpha B-crystallin in human age-related nuclear cataract. Int J Biol Macromol 1998; 22:321-30.

36. Lampi KJ, Oxford JT, Bachinger HP, Shearer TR, David LL, Kapfer DM. Deamidation of human beta B1 alters the elongated structure of the dimer. Exp Eye Res 2001; 72:279-88.

37. van Kleef FS, Nijzink-Maas MJ, Hoenders HJ. Intracellular degradation of alpha-crystallin. Fractionation and characterization of degraded alpha A-chains. Eur J Biochem 1974; 48:563-70.

38. Takemoto L, Emmons T, Horwitz J. The C-terminal region of alpha-crystallin: involvement in protection against heat-induced denaturation. Biochem J 1993; 294:435-8.