Proteoglycans isolated from dissociative extracts of differently aged human articular cartilage: characterization of naturally occurring hyaluronan-binding fragments of aggrecan

Abstract

Proteoglycans extracted with 4 M guanidinium chloride from young (mean 20 years) or old (mean 79 years) macroscopically normal human articular cartilage were separated by density gradient centrifugation and Q-Sepharose chromatography and characterized by gradient gel SDS/PAGE and immunodetection before and after removal of glycosaminoglycan chains. The extracts contained two large populations of aggrecan, a population of small N-terminal aggrecan fragments, as well as decorin, biglycan and fibromodulin. The distribution of all these species in density gradient fractions has been determined. The large aggrecan populations comprised four different chondroitin sulphate-bearing core proteins while the population of smaller fragments comprised eight different components. The two smallest fragments (35 and 42 kDa), identified as the first globular domain of aggrecan (N-terminal) (G1) and containing no glycosaminoglycan, were detected only in extracts of old cartilage. A 55 and a 70 kDa fragment of G1 were present in both keratan sulphate-containing and non-keratan sulphate-containing forms. Four other fragments, each containing keratan sulphate epitopes, were identified and these contained either G1 epitopes (one 95 kDa species), or G1 and G2 epitopes (three species). These results have suggested that proteolytic processing at the N-terminus is more extensive than has previously been recognized and raises the possibility that more than one proteinase may be involved in aggrecan degradation in vivo. With the exception of the two smallest G1 fragments, the repertoire of proteoglycan fragments found in young and old human articular cartilage is essentially the same, although the relative abudnance of various species differed. The older tissue contains a larger proportion of C-terminally truncated aggrecan fragments and a significantly decreased content of decorin and biglycan.