Proteoglycans analyzed by composite gel electrophoresis and immunoblotting.

Abstract

Proteoglycans are the protein products of diverse genes posttranslationally modified with highly negatively charged side chains, commonly known as glycosami-noglycans. The latter consist of repeating disaccharides capable of forming polymers of varying size, which, depending on the specific disaccharide composition, are known as the chondroitin sulfates, the iduronate-containing dermatan sulfates, keratan sul-fate, and heparan sulfate. Some of the more complex proteoglycans, such as aggrecan, are decorated by additional nonglycosaminoglycan oligosaccharides (). Aggrecan contains additionally a hyaluronan-binding domain that in the extracellular matrix facilitates formation of large noncovalently-bound complexes containing one hyaluronan molecule and numerous aggrecans (). Analysis of proteoglycans by standard SDS-PAGE is complicated by the presence of the negatively charged side chains, which prevent the linerarization of molecules usually achieved by treatment with a mercaptoethanol and SDS. Consequently, in SDS-PAGE most proteoglycans with multiple glycosaminoglycan side chains, if they enter the gel at all, migrate as broad bands and appear as smears due to size heterogeneity and large electroendosmotic effects particularly when the acrylamide composition of the PAGE gel is held to a minimum.