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

J Biol Chem. 1993 Apr 25;268(12):8711-6.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

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

Substances

  • Alpha-Globulins
  • Blood Proteins
  • Glycoproteins
  • Glycosaminoglycans
  • Membrane Glycoproteins
  • Protease Inhibitors
  • SPINT2 protein, human
  • inter-alpha-inhibitor
  • Trypsin Inhibitor, Kunitz Soybean