NONENZYMATIC GLYCATION OF TYPE-IV COLLAGEN AND MATRIX METALLOPROTEINASE SUSCEPTIBILITY

The glomerular basement membrane (GBM) is damaged in diabetes through complex mechanisms that are not fully understood. Prominent among them is nonenzymatic protein glycation leading to the formation of so-call ed advanced glycation end products (AGEs). We examined the effects of in vitro glycation of intact collagen type IV in bovine lens capsule ( LBM) and kidney glomerular (GBM) basement membranes on their susceptib ility to matrix metalloproteinases, using stromelysin 1 (MMP-3) and ge latinase B (MMP-9). Sites of cleavage of unmodified LBM collagen were located in the triple helical region. In vitro glycation by glucose se verely inhibited the release of soluble collagen cleavage peptides by MMP-3 and MMP-9. The distribution of AGEs within the three domains of collagen IV (7S, triple helical, and noncollagenous NC1) were compared for LBM glycation using AGE fluorescence, pentosidine quantitation, a nd immunoreactivity towards anti-AGE antibodies that recognize the AGE carboxymethyllysine (CML). Marked asymmetry was observed, with the fl exible triple helical domain having the most pentosidine and fluoresce nt AGEs but the least CML. The in vivo relevance of these findings is supported by preliminary studies of AGE distribution in renal basement membrane (RBM) collagen IV domains from human kidneys of two insulin- dependent diabetics and one normal subject. Pentosidine and fluorescen t AGE distributions of diabetic RBM were similar to LBM, but the CML A GE in diabetic kidney was less in the triple helical domain than in NC 1. Our results support the hypothesis that nonenzymatic glycation of c ollagen IV contributes to the thickening of basement membranes, a hall mark of diabetic nephropathy.