PRESENCE OF THE PROTEIN-GLYCOSAMINOGLYCAN-PROTEIN COVALENT CROSS-LINKIN THE INTER-ALPHA-INHIBITOR-RELATED PROTEINASE-INHIBITOR HEAVY CHAIN-2 BIKUNIN

HC2/bikunin is a human plasma proteinase inhibitor composed of two pol ypeptide chains that resist dissociation under reducing conditions in SDS-polyacrylamide gel electrophoresis. This observation suggests that a nondisulfide cross-link is responsible for the association of these two polypeptide chains. In this study, we have utilized a variety of techniques to investigate the structural basis for this observation. W e show that the cross-link between the two protein chains is sensitive to chondroitin sulfate-degrading enzymes and to 50 mM NaOH, propertie s shared by the protein-glycosaminoglycan-protein cross-link found in the related pre-alpha-inhibitor (Enghild, J. J., Salvesen, G., Hefta, S., Thogersen, I. B., Rutherfurd, S., and Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751). Biochemical and mass spectrometric analysis of t he peptides containing the cross-link indicate that it is mediated by a chondroitin-4-sulfate chain that originates from a typical O-glycosi dic link to Ser10 of bikunin. The COOH-terminal Asp648 residue of heav y chain 2 is esterified via the alpha-carbon to C-6 of an internal N-a cetylgalactosamine of the chondroitin-4-sulfate chain. This suggests t hat the protein-glycosaminoglycan-protein cross-link that assembles th e chains of pre-alpha-inhibitor is identical to that which assembles H C2/bikunin, and is probably a characteristic of the bikunin proteins.