|Molecular Vision 2000;
Received 11 November 1999 | Accepted 4 February 2000 | Published 21 February 2000
Localization of biologically uncommon D-b-aspartate-containing aA-crystallin in human eye lens
Tadashi Shimo-Oka,2 Masayo Ogiso,3 Yuko Momose,3
Toshio Kodama,4 Makoto Kodama,3 Mitsuhiko Akaboshi1
1Research Reactor Institute, Kyoto University Kumatori, Sennan, Osaka 590-0494, Japan; 2Life Science Center, Asahi Techno Glass Corporation, Funabashi 273-0044, Japan; 3Bionic Design, National Institute for Advanced Interdisciplinary Research, Tsukuba, Ibaraki 305-8566, Japan; 4Department of Ophthalmology, Ehime University, School of Medicine, Shitukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0204, Japan
Correspondence to: Noriko Fujii, Ph.D., Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka 590-0494, Japan; Phone: +81-724-51-2496; FAX: +81-724-51-2630; email: nfujii@HL.rri.kyoto-u.ac.jp
Purpose: Previous studies demonstrated that the Asp-151 residue of aA-crystallin from human eye lens is stereoinverted to the biologically uncommon D-isomer and isomerized to the b-aspartyl residue (isoaspartate) with age. To detect the locality of the D-b-Asp-containing peptide in aged human lens, we prepared a highly specific antibody against peptide Gly-Leu-D-b-Asp-Ala-Thr which corresponds to positions 149-153 of human aA-crystallin using peptide Gly-Leu-D-b-Asp-Ala-Thr-Gly-Leu-D-b-Asp-Ala-Thr-Gly-Leu-D-b-Asp-Ala-Thr (designated peptide 3R) as an immunogen.
Methods: Peptide 3R was synthesized with F-moc (9-fluorenylmethoxycarbonyl) solid phase chemistry and then the peptide was immunized in rabbits to generate antibody against peptide 3R. The antibody in rabbit serum was purified by affinity chromatography using peptide 3R and bovine aA-crystallin as ligands. The specificity and titer of antibody were checked by ELISA assay. We synthesized four kinds of peptide T18 (IQTGLDATHAER; corresponding to the amino acid sequences 146-157 in human aA-crystallin) in which Asp-151 residues were normal L-a-Asp, abnormal D-a-Asp, L-b-Asp, and D-b-Asp, respectively. The specificity of antibody was confirmed by ELISA using these peptides and utilized in immunohistochemistry.
Results: The antibody we prepared crossreacted specifically to D-b-Asp-151-containing aA-crystallin. Immunohistochemical staining of human lens with the antibody demonstrated that D-b-Asp-151-containing aA-crystallin was predominantly localized in the core of aged human lens.
Conclusions: The peptide 3R antibody clearly recognized the presence of racemized and isomerized Asp-151 in both protein solution and lens tissue obtained from aged human lens.
a-Crystallin is one of the major proteins in the vertebrate eye lens and its function has been indicated to be maintenance of lens transparency. a-Crystallin exists in the lens fiber cell cytoplasm as a polydisperse aggregate with an average molecular mass of approximately 800 kDa. The aggregate is composed of two kinds of polypeptides, aA- and aB-crystallins, containing 173 and 175 amino acid residues, respectively. The amino acid sequences of aA- and aB-crystallins are approximately 55% homologous [1,2]. Recently, it was demonstrated that a-crystallin is one of the small heat shock proteins  and functions as a molecular chaperone which protects other proteins against denaturation and eventual aggregation . Proteins of the lens have very long half-lives, therefore, various posttranslational modifications such as deamidation [5,6], racemization [7,8], stereoinversion [9-12], isomerization [8-12], truncation [6,10,13-15], phosphorylation [6,16], oxidation [6,8,17], and an increase in intramolecular disulfide bonding [6,18] have been shown to take place during aging. These post-translational modifications may cause the formation of high molecular weight protein aggregates and may lead to the formation of cataract by affecting the close-packing of the crystallins or by reducing the chaperone-like activity of a-crystallins.
We found racemization of amino acids in lens protein generated at specific sites of aA- and aB-crystallin which could not be found in other crystallins (our unpublished data). Subsequently, we identified a biologically uncommon D-isomer at Asp-58 and Asp-151 [9,10] in aA-crystallin, and also at Asp-36 and Asp-62 in aB-crystallin,  from aged human lenses. D-Asp-151 of aA-crystallin was found to be present in the lenses of cattle , horse (our unpublished data), rat , and mouse ; however, the D-isomers of Asp-58 in aA-crystallin and Asp-36 and Asp-62 in aB-crystallin were restricted to human lenses. The results showed that the configuration of Asp-151 in aA-crystallin is stereochemically labile. Strikingly, the D isomer to native L ratio (D/L ratio) of Asp-151 of human aA-crystallin was higher than 1.0 [9-12]. Since racemization is defined as a reversible first order reaction, racemization is in equilibrium when the D/L ratio reaches 1.0. Therefore, the reaction which induces D-Asp predominantly (D/L>1.0) is not termed racemization but stereoinversion. There have been no reports of stereoinversion of amino acids in proteins except in human aA-crystallin. D-Asp formation was also accompanied by isomerization from the natural a-Asp to the biologically uncommon b-Asp (isoaspartate) [8-12]. Therefore, four isomers, which are normal L-a-Asp, biologically uncommon L-b-Asp, D-a-Asp, and D-b-Asp are formed in aA-crystallins. Recently, we measured the ratio of the four isomers of Asp-151 in aA-crystallins obtained from human lenses of newborns, and subjects with ages in the 30, 60, and 80 year ranges . The isomers increased with age, and the total amount of three isomers was more than that of normal L-a-Asp in the aA-crystallin of the human lenses of subjects in the 80 year range . In the present study, we prepared polyclonal antibody against the D-b-Asp-151-containing peptide and investigated the localization of the accumulation of D-b-Asp-151 in the lens.
Purification of aA-crystallin from human lens
aA-crystallins were isolated from the water-soluble fraction of human (from an 80 year old and 11 month old for an aged and young sample, respectively), bovine, and calf lens samples as described previously .
The peptide Ile-Gln-Thr-Gly-Leu-Asp-151-Ala-Thr-His-Ala-Glu-Arg (designated T18), corresponding to the amino acid sequence 146-157 in human aA-crystallin, was synthesized in which the Asp-151 residues were L-a-, D-a-, L-b-, and D-b-isomers and were designated as L-a-Asp T18, D-a-Asp T18, L-b-Asp T18, and D-b-Asp T18, respectively. These peptides were synthesized by F-moc (9-fluorenylmethoxycarbonyl) solid phase chemistry. F-moc-L-Asp-(OtBu)-OH, F-moc-D-Asp-(OtBu)-OH, F-moc-L-Asp-OtBu, and F-moc-D-Asp-OtBu were used as building blocks to synthesize L-a-, D-a-, L-b-, and D-b-isomers, respectively. The crude peptide was purified by reverse phase HPLC and the purity (more than 95%) was confirmed by analytical HPLC and amino acid analysis.
Preparation of immunogen
The peptide Gly-Leu-Db-Asp-Ala-Thr-Gly-Leu-Db-Asp-Ala-Thr-Gly-Leu-Db-Asp-Ala-Thr (designated peptide 3R), containing three repeats of position 149-153 of the human aA-crystallin optic isomer, was synthesized. The peptide was coupled to keyhole limpet hemocyanin (KLH) using dimethyl adipimidate.
Preparation of antibody
The peptide-KLH conjugate (mixed with Freund's complete adjuvant) was first injected intracutaneously into rabbits. Booster injections were repeated several times subcutaneously (mixed with Freund's incomplete adjuvant) every 2 weeks after the first injection. The antiserum thus obtained was purified by affinity chromatography using peptide 3R or calf aA-crystallin coupled to Affi-Gel 10 (Bio-Rad Co., Kanagawa, Japan). The purified antibody was dialyzed against phosphate-buffered saline (PBS) and added to 10 mg/ml of bovine serum albumin (BSA) for storage. Specificity and titer of antiserum and the purified antibody were checked by ELISA using a polystyrene microtiter plate coated with peptides or aA-crystallin.
Polystyrene microtiter ELISA plates (Asahi Techno Glass Corp., Funabashi, Japan) were used. Each well was coated with 50 ml of each peptide (20 mg/ml) or aA-crystallin (A280=0.027) in PBS at 4 °C overnight. As a blank, some wells were coated with PBS only. The plates were washed twice with PBS containing 0.1% Tween 20 (Tween-PBS) and coated with PBS containing 10 mg/ml of BSA for 60 min at 25 °C. After the plates were washed twice with Tween-PBS, antiserum diluted 200-, 1000-, 5000-, and 25,000-fold or affinity-purified antibody diluted 10-, 100-, 1000-, and 10,000-fold was added to the wells and incubated for 60 min at 25 °C. Unbound antibodies were washed away by washing the plates five times with Tween-PBS. Bound antibodies were reacted with a goat anti-rabbit immunoglobulin coupled with horseradish peroxidase for 60 min at 25 °C. After washing five times with Tween-PBS, the bound enzymatic activity was measured by the use of tetramethylbenzidine as a substrate. Duplicate determinations were performed for each assay.
Human eyeballs were obtained from a 2-year-old child with retinoblastoma (young eye) and a 60-year-old donor within 1 h after death (aged eye) with informed consent of the families of these patients. There were no clinical abnormalities in these lenses. Enucleated eyes were fixed in 10% formaldehyde in phosphate buffer (pH 7.5) for five days at 4 °C. After fixation, the eyeballs were embedded in paraffin wax and sectioned with a microtome. Sections were deparaffinized with xylene and rehydrated through a graded alcohol series. After rinsing in PBS (8.1 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4), the sections were incubated for 1 h in blocking solution (PBS containing 0.5% BSA and 1/300 volume of protein A [Wako Pure Chemical Industries, Osaka, Japan]) to block non-specific antibody-binding sites on the sections. The sections were washed several times with PBS and incubated with the anti-peptide 3R antibody at a dilution of 1:300 (for aged eye) or 1:100 (for young eye) in PBS containing 0.5% BSA for 1 h. Control for nonspecific secondary binding was incubated without the anti-peptide 3R antibody. After washing several times with PBS, the sections were incubated with peroxidase-conjugated protein A (Bio-Rad) at a dilution of 1:300 (for aged eye) or 1:100 (for young eye) in PBS containing 0.5% BSA for 1 h. Peroxidase activity was detected with DAB substrate solution (0.02% diaminobenzidine [Wako Pure Chemical Industries, Ltd.], 0.005% H2O2, 0.01 M Tris-HCl, pH 7.5) for 30 min. The stained sections were dehydrated through a graded alcohol series, permanently mounted and analyzed by light microscopy. All incubation, washing and staining procedures were performed at room temperature.
Characterization of anti-peptide 3R antibody
To obtain antibody which specifically recognizes aA-crystallin in which the Asp-151 residue is a D-b-isomer, we designed an immunogen peptide (peptide 3R) composed of three repeats of Gly-Leu-D-b-Asp-Ala-Thr corresponding to positions 149-153 of the human aA crystallin optic isomer. ELISA was carried out to evaluate the specificity and titer of the anti-peptide 3R antibody. First, the specificity and titer of the antiserum were assayed using aA-crystallin proteins as antigens (Figure 1A). The antiserum reacted most strongly with human aA-crystallin derived from an aged person (80-year-old subject). The antiserum also reacted with both bovine aA-crystallin and human aA-crystallin from the 11-month-old subject, but reactivity to calf aA-crystallin was negligible. For immunostaining studies, the antiserum was purified by affinity chromatography using bovine aA-crystallin or peptide 3R coupled to Affi-Gel 10 (Bio-Rad). Specificities are shown in Figure 1B,C. Both bovine aA-crystallin-purified antibody and peptide 3R-purified antibody had very similar specificities, with strong reactivity to aA-crystallin derived from both aged human and aged bovine lenses. the findings that the bovine aA-crystallin-purified antibody reacted to aA-crystallin derived from the aged human lens and that the antiserum did not recognize calf aA-crystallin (Figure 1A), which does not contain the D-b-Asp-151 residue (our unpublished data), strongly suggested that the antibody specifically recognizes the D-b-Asp-151-containing peptide in both human and bovine aA-crystallin. This assumption was supported by the results that the peptide 3R-purified antibody reacted very strongly with the D-b-Asp T18 peptide and very weakly with the L-b-Asp T18 and D-a-Asp-T18 peptides, but scarcely reacted with the L-a-Asp T18 peptide, in which the only difference was the configuration of the Asp-151 residue (Figure 1D). From these results, we concluded that the antibody we produced is highly specific to aA-crystallin in which the Asp-151 residue is a D-b-isomer. The reactivity of the antibody to aA-crystallin derived from the young human can be explained by the fact that the aA-crystallin contains small amounts of D-b-Asp-151 residue [9,12].
Immunohistochemical staining of human eyes (60 years and 2 years old) was performed with the purified anti-peptide 3R antibody, which has specific reactivity with D-b-Asp-151-containing peptides. The intense immunoreactivity of D-b-Asp-151-containing peptides was specifically localized in the core of the aged lens (Figure 2A), while in the young lens, immunostaining was not observed, even though 3-fold concentrations of antibody were used for staining (Figure 2B).
We have reported that the configuration of the Asp-151 residue of human aA-crystallin converted to a D-b-Asp isomer during aging [9,12]. To detect the locality of D-b-Asp-containing aA-crystallin in the lens, we prepared an antibody which reacts specifically with D-b-Asp-containing peptides. Antiserum to peptide 3R reacted with aA-crystallins derived from aged bovine lenses and those of 80-year-old and 11-month-old humans but did not react to calf aA-crystallin (Figure 1A). Our previous data indicated large amounts of D-b-Asp-151 in human aA-crystallin from aged subjects, about 42% of the total Asp-151, and also that aA-crystallin from young humans contains slight amounts (about 5% of the total Asp-151) of the D-b-Asp-151 residue [9,12]. As shown in Figure 1D, the purified antibody specifically recognized the D-b-Asp-containing peptide, indicating that D-b-Asp-151 residues of human aA-crystallins from both young and aged subjects were detected with high sensitivity. In bovine lens, the D/L ratio of Asp-151 was reported to be about 0.5, although the linkage of Asp-151 was not analyzed . Our present result suggests that the racemized Asp-151 of bovine aA-crystallin must have b-linkage because the antibody recognized only the D-b-Asp-containing peptides. On the other hand, calf aA-crystallin did not contain the D-b-Asp-151 residue (our unpublished data), therefore, the antibody did not react with calf aA-crystallin.
The sequences of amino acids surrounding Asp-151 in human and bovine aA-crystallin resemble each other. In order to prepare an antibody which recognizes the D-b-Asp-containing peptide, we used three repeats of Gly-Leu-D-b-Asp-Ala-Thr which corresponded to residues 149-153 of human aA-crystallin. The sequence of residues 149-153 of bovine aA-crystallin is Gly-Val-Asp-Ala-Gly and the substitution of Val for Leu at the 150 residue would not change the property of the sequence, although the effect of the substitution of Thr to Gly is not known. Therefore, anti-peptide 3R antibody recognized the D-b-Asp-containing peptide specifically in both human and bovine aA-crystallin. The difference of reactivity of anti-peptide 3R between aged human and bovine aA-crystallin may depend on the slight difference in the sequence of 149-153 of aA-crystallins obtained from human and bovine lenses. Regardless, the antibody we prepared has high specificity to D-b-Asp-containing peptides. The similarity of the structure surrounding the D-b-Asp-151 was also shown by the results that the antibody against three repeats of Gly-Val-D-b-Asp-Ala corresponding to positions 149-152 of the bovine aA-crystallin optic isomer recognized aA-crystallin derived from aged human lens (data not shown). As we reported previously, D-Asp formation was accompanied by isomerization from the natural a-Asp to the biologically uncommon b-Asp (isoaspartate) [8-12]. D-Asp formation may occur via a succinimide intermediate. When the carbonyl group of the side chain of the L-a-aspartyl residue is attacked by the nitrogen of the amino acid residue following the Asp residue, L-succinimide is formed by intramolecular cyclization. L-succinimide is converted to D-succinimide. The D- and L-succinimide are hydrolyzed at either side of their two carbonyl groups, yielding D-b-Asp, D-a-Asp, L-b-Asp, and L-a-Asp residues, respectively. Therefore, the normal L-a-Asp residue converts to L-b-Asp, D-b-Asp, and D-a-Asp-isomers simultaneously [8-12,21]. The probability that the succinimide opens to L-b-Asp or a-Asp is not known, although it prefers to open to b-Asp rather than to a-Asp in small peptides [22-24] and human aA-crystallin [9-12]. The antibody would be useful to distinguish b- and a-Asp.
The result of immunohistochemistry demonstrated that the core of the lens from the 60-year-old subject contained D-b-Asp-containing aA-crystallin, whereas this was not observed in the lens from the 2-year-old subject. The non-staining could be attributed to a sparse and diffuse distribution of D-b-Asp-151-containing aA-crystallin in young human lens. The sensitivity of ELISA is extremely high compared with that of immunohistochemistry, which also could explain why D-b-Asp could be detected in even young human aA-crystallin by ELISA but not by immunohistochemistry.
The localization of D-b-Asp-151-containing aA crystallin in the core of the aged human lens is consistent with a previous report that showed that the racemization rate of Asp in the water insoluble fraction was higher than that in the water soluble fraction . It is well known that the water insoluble fraction is increased and that an increase of insoluble protein is more marked in cataracts. The protein in the core of the lens is old protein, therefore, D-b-Asp-151-containing aA crystallin is localized in the core of aged lens.
This work was supported in part by Grants-in-Aid for Scientific Research 10309005 from the Ministry of Education, Science, Sports, and Culture of Japan.
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