Presence of the protein-glycosaminoglycan-protein covalent cross-link in the inter-alpha-inhibitor-related proteinase inhibitor heavy chain 2/bikunin

• Published in: The Journal of biological chemistry 1993-04, Vol.268 (12), p.8711-8716
• Main Authors: ENGHILD, J. J, SALVESEN, G, THØGERSEN, I. B, VALNICKOVA, Z, PIZZO, S. V, HEFTA, S. A
• Format: Article
• Language: English
• Subjects: Alpha-Globulins - metabolism; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Blood Proteins - metabolism; Carbohydrate Sequence; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glycoproteins - blood; Glycoproteins - chemistry; Glycoproteins - metabolism; Glycosaminoglycans - metabolism; Humans; Hydrolases; Mass Spectrometry; Membrane Glycoproteins; Molecular Sequence Data; Protease Inhibitors - blood; Protease Inhibitors - chemistry; Protease Inhibitors - metabolism; Trypsin Inhibitor, Kunitz Soybean
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)52933-1

HC2/bikunin is a human plasma proteinase inhibitor composed of two polypeptide 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. We show that the cross-link between the two protein chains is sensitive to chondroitin sulfate-degrading enzymes and to 50 mM NaOH, properties shared by the protein-glycosaminoglycan-protein cross-link found in the related pre-alpha-inhibitor (Enghild, J. J., Salvesen, G., Hefta, S., Thøgersen, I. B., Rutherfurd, S., and Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751). Biochemical and mass spectrometric analysis of the peptides containing the cross-link indicate that it is mediated by a chondroitin-4-sulfate chain that originates from a typical O-glycosidic link to Ser10 of bikunin. The COOH-terminal Asp648 residue of heavy chain 2 is esterified via the alpha-carbon to C-6 of an internal N-acetylgalactosamine of the chondroitin-4-sulfate chain. This suggests that the protein-glycosaminoglycan-protein cross-link that assembles the chains of pre-alpha-inhibitor is identical to that which assembles HC2/bikunin, and is probably a characteristic of the bikunin proteins.