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

TY - JOUR

T1 - Presence of the protein-glycosaminoglycan-protein covalent cross-link in the inter-alpha-inhibitor-related proteinase inhibitor heavy chain 2/bikunin

AU - Enghild, J J

AU - Salvesen, G

AU - Thøgersen, I B

AU - Valnickova, Z

AU - Pizzo, S V

AU - Hefta, S A

PY - 1993

Y1 - 1993

N2 - 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.

AB - 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.

KW - Alpha-Globulins

KW - Amino Acid Sequence

KW - Blood Proteins

KW - Carbohydrate Sequence

KW - Electrophoresis, Polyacrylamide Gel

KW - Glycoproteins

KW - Glycosaminoglycans

KW - Humans

KW - Mass Spectrometry

KW - Membrane Glycoproteins

KW - Molecular Sequence Data

KW - Protease Inhibitors

KW - Trypsin Inhibitor, Kunitz Soybean

M3 - Journal article

C2 - 7682553

SN - 0021-9258

VL - 268

SP - 8711

EP - 8716

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 12

ER -