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

J J Enghild, G Salvesen, I B Thøgersen, Z Valnickova, S V Pizzo, S A Hefta

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    Abstract

    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.
    Original languageEnglish
    JournalJournal of Biological Chemistry
    Volume268
    Issue12
    Pages (from-to)8711-6
    Number of pages5
    ISSN0021-9258
    Publication statusPublished - 1993

    Keywords

    • Alpha-Globulins
    • Amino Acid Sequence
    • Blood Proteins
    • Carbohydrate Sequence
    • Electrophoresis, Polyacrylamide Gel
    • Glycoproteins
    • Glycosaminoglycans
    • Humans
    • Mass Spectrometry
    • Membrane Glycoproteins
    • Molecular Sequence Data
    • Protease Inhibitors
    • Trypsin Inhibitor, Kunitz Soybean

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