N-LINKED AND O-LINKED KERATAN SULFATE ON THE HYALURONAN-BINDING REGION OF AGGRECAN FROM MATURE AND IMMATURE BOVINE CARTILAGE

In the hyaluronan binding region (HABR) peptide of aggrecan, there is a marked increase in the level of keratan sulfate (KS) during aging. T o determine the sites of RS attachment, KS containing peptides were pr epared from HABRs from immature and mature bovine articular cartilage by digestion with trypsin or papain followed by carbohydrate analysis and peptide sequencing. KS is attached to Thr(42) within loop A in mat ure, but not in immature, HABR. Within loop B KS is N-linked to Asn(22 0) in both HABRs, but in the immature HABR the chains are shorter. Asn (314) in loop B' of mature HABR is substituted either with a KS chain or with an oligosaccharide of the complex type. In immature HABR this site does not carry KS. In the interglobular domain, 2 threonine resid ues within the sequence TIQTVT are substituted in both calf and steer, and in steer further substitution occurs within the sequence NITEGEA, which contains a major catabolic cleavage site (Sandy, J., Neame, P. J., Boynton, R., and Flannery, C. R. (1991) J. Biol. Chem. 266, 8683-8 685). The extreme polydispersity of mature HABR was investigated by pr eparing four subfractions of increasing molecular size which had essen tially the same protein core, i.e. Val(1)-Arg(367) or Val(1)-Arg(375). The smaller species lacked the KS chains attached to loop A. These re sults show that KS substitution occurs within each of the disulfide-bo nded loops of the HABR, that the KS may be either N- or O-linked, and that variations in the addition of KS are responsible for the polydisp ersity of mature HABR.