Crystallins of the eye lens are long-lived proteins because they hardly turn over. Such proteins are constantly subjected to posttranslational modifications such as nonenzymatic glycosylation (glycation). Diabetic patients are four to six times more likely to develop cataract at a younger age than the normal population and glycation of lens crystallins could play a significant role in diabetic cataractogenesis. Glycation proceeds primarily in two stages, formation of Schiff-base and Amadori products or early glycation and formation of advanced glycation end products that are fluorescent and having protein cross-linking properties. The long-range goal of the proposal is to study the role of glycation in diabetic cataractogenesis. The immediate goals are to identify the amino acid sites of different crystallins that are being glycated and to determine the rates or extents of glycation of such sites during the progression of diabetes and cataract in streptozotocin-diabetic rats, in aging and diabetic humans, and during in vitro glycation of rat and human crystallins with various sugars and sugar phosphates. Such information is essential to understand why glycation would induce protein conformational changes, increased reactivity of thiols and enhanced thiol oxidation. To achieve these goals modern techniques in protein chemistry will be applied which are: molecular sieve HPLC for separation of [3H]NaBH4 reacted native crystallins including HMW aggregates, reverse-phase HPLC for separation of crystallin subunit polypeptides and tryptic and chymotryptic peptides, Affigel 601 affinity chromatography for purification of glycated peptides, determination of amino acid composition by a HPLC/post-column derivatization method and amino acid sequencing by a manual microsequencing method. For identification of the crystallins in the in vitro and in vivo generated soluble or insoluble fractions, in addition to classical chemical characterization, immunologic identification will be carried out with monoclonal antibodies that will be produced against various rat and human crystallins. With the aid of inhibitors of early glycation and advanced glycation such as acetylsalicylic acid (aspirin) and aminoguanidine it will be shown whether early glycation and advanced glycation play any role in diabetic cataractogenesis. Moreover, with aspirin treatment it will be shown that glycation of which crystallins are inhibited in vitro (in rat and human crystallins) and in vivo (only in rat crystallins) and whether the mechanism involves acetylation of specific amino acid residues.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY007394-10A1
Application #
2019694
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1987-09-30
Project End
2000-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
10
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Medical College of Georgia (MCG)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Augusta
State
GA
Country
United States
Zip Code
30912
Thampi, Prajitha; Zarina, Shamshad; Abraham, Edathara C (2002) alpha-Crystallin chaperone function in diabetic rat and human lenses. Mol Cell Biochem 229:113-8
Bera, Sibes; Abraham, Edathara C (2002) The alphaA-crystallin R116C mutant has a higher affinity for forming heteroaggregates with alphaB-crystallin. Biochemistry 41:297-305
Shroff, N P; Bera, S; Cherian-Shaw, M et al. (2001) Substituted hydrophobic and hydrophilic residues at methionine-68 influence the chaperone-like function of alphaB-crystallin. Mol Cell Biochem 220:127-33
Shroff, N P; Cherian-Shaw, M; Bera, S et al. (2000) Mutation of R116C results in highly oligomerized alpha A-crystallin with modified structure and defective chaperone-like function. Biochemistry 39:1420-6
Zarina, S; Zhao, H R; Abraham, E C (2000) Advanced glycation end products in human senile and diabetic cataractous lenses. Mol Cell Biochem 210:29-34
Swamy-Mruthinti, S; Shaw, S M; Zhao, H R et al. (1999) Evidence of a glycemic threshold for the development of cataracts in diabetic rats. Curr Eye Res 18:423-9
Cherian-Shaw, M; Smith, J B; Jiang, X Y et al. (1999) Intrapolypeptide disulfides in human alphaA-crystallin and their effect on chaperone-like function. Mol Cell Biochem 199:163-7
Zhao, H R; Nagaraj, R H; Abraham, E C (1997) The role of alpha- and epsilon-amino groups in the glycation-mediated cross-linking of gammaB-crystallin. Study of three site-directed mutants. J Biol Chem 272:14465-9
Matsumoto, K; Ikeda, K; Horiuchi, S et al. (1997) Immunochemical evidence for increased formation of advanced glycation end products and inhibition by aminoguanidine in diabetic rat lenses. Biochem Biophys Res Commun 241:352-4
Cherian, M; Smith, J B; Jiang, X Y et al. (1997) Influence of protein-glutathione mixed disulfide on the chaperone-like function of alpha-crystallin. J Biol Chem 272:29099-103

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