Glycation induced crosslinking of link proteins, in vivo and in vitro

Objective. Reducing sugars have the ability to crosslink proteins through c reation of advanced glycosylated end products (AGE). In this study, we dete rmined the ability of AGE to induce crosslinking of link proteins and aggre can proteoglycans. Methods. Aggrecan proteoglycans and link proteins were purified from adult human articular cartilage and from young bovine nasal cartilage for in vivo and in vitro studies, respectively, In vitro studies concerned incubation of aggrecan aggregates or link proteins with ribose under physiological con ditions. After 30 days, aggregates were centrifuged dissociatively to obtai n aggrecan monomers and link proteins. Aggrecan monomers were analyzed by i mmunoblot assay. Incubated link proteins were analyzed by SDS-PAGE and Seph acryl-200 column chromatography, Results. After extensive purification, adult human cartilage aggrecan conti nued to show the presence of link protein antigens by immunoblot analysis. Immunoblot analysis of purified aggrecan derived from ribose-treated aggreg ates also showed the presence of link protein antigens. Ribose treatment of link protein lead to polymerization that was confirmed by Sephacryl-200. Conclusions. These studies suggest that human link proteins tend to become crosslinked to aggrecan in adult cartilage. A likely cause of the crosslink ing is formation of AGE due to reducing sugars. (C) 2000 Academic Press.