Early glycation products produce pentosidine cross-links on native proteins - Novel mechanism of pentosidine formation and propagation of glycation

Bovine lens alpha -crystallin was immobilized on EAH-Sepharose gel and glyc ated using D-ribose. Incubation with 500 and 100 mM D-ribose for 2 and 15 d ays produced short-term glycated (STGP gel) and long-term glycated proteins (LTGP gel). Both STGP and LTGP gels produced oxygen free radicals. Hydroxy l radical production was twice that in STGP gel compared with the LTGP gel. Incubation with the glycated gels produced pentosidine in a mixture of N-a lpha -acetylarginine + N-alpha -acetyllysine, bovine lens proteins (BLP), a nd lysozyme; the amounts measured with STGP gel were higher than those with LTGP gel. Reactive oxygen species scavengers decreased the formation of pe ntosidine. Pentosidine was also formed in BLP when incubated with water-ins oluble proteins extracted from aged or brunescent human lenses. Early glyca ted proteins from aged or diabetic lenses were bound to a boronate affinity column, the protein-containing gel was incubated with BLP, and pentosidine was measured in the incubation mixtures. With this method we found that di abetic lens proteins produced more pentosidine on BLP than did aged lens pr oteins. Further investigation indicates that two and three carbon carbohydr ates possibly formed from oxidative cleavage of early glycation products ar e involved in pentosidine formation. Based on our findings, we propose a no vel pathway for pentosidine formation on native proteins from glycated prot eins.