Glycation of lens membrane intrinsic proteins.
Changes occurring at the membrane are believed to be the decisive factors in the initiation of diabetic cataract. During diabetic hyperglycemia lens crystallins were shown to undergo glycation. Several studies indicated that glycation brings about protein conformational changes thus implicated in cataractogenesis. Since the membrane proteins are the first targets for glycation, in this study we measured the glycation of alkali washed urea-insoluble membrane proteins from control and diabetic rats by two different methods, phenyl-boronate affinity chromatography and [3H]NaBH4 reduction, and confirmed by amino acid analysis. There was a significant increase in the glycation of membrane proteins in diabetic cataract lenses when compared to controls. It appears that lysine is the major site of glycation. Concomitant to early glycation, there was an increase in non-tryptophan fluorescence (Ex: 350 nm/Em: 440 nm) in the diabetic lens membrane proteins suggesting the presence of advanced glycation mediated protein cross-links. In order to identify whether the major membrane intrinsic protein, MIP26, undergoes glycation, we isolated MIP26 along with its degradatory product MIP22 as one peak on molecular sieve HPLC. HPLC isolated MIP26/MIP22 was further separated on SDS-PAGE followed by slicing and counting. This analysis revealed that MIP26 and MIP22 were more or less equally glycated in controls, however, in diabetic rats glycation of MIP22 was glycated slightly higher than MIP26. Moreover, the proportion of MIP22 increased by about 2-fold in diabetic lenses compared to controls. Thus it appears that major glycation sites are still retained in MIP22 in diabetic rat lenses. In vitro glycation studies with bovine lens membranes were also done using 14C glucose, followed by SDS-PAGE and autoradiography. The major protein glycated in vitro also seems to be MIP26. Interestingly, MIP22 was less glycated than MIP26 in vitro.
Swamy
M S
MS
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912.
Abraham
E C
EC
D000818:Animals; D020346:Aquaporins; D001345:Autoradiography; D001894:Borohydrides; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000378:metabolism*; D006031:Glycosylation; D007908:Lens, Crystalline / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D013311:Streptozocin
D020346:Aquaporins; D001894:Borohydrides; D005136:Eye Proteins; D008562:Membrane Glycoproteins; C030328:aquaporin 0; D013311:Streptozocin; C025364:sodium borohydride