Presence of the protein-glycosaminoglycan-protein covalent cross-link in the inter-alpha-inhibitor-related proteinase inhibitor heavy chain 2/bikunin.
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.
Enghild
J J
JJ
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.
Salvesen
G
G
Thøgersen
I B
IB
Valnickova
Z
Z
Pizzo
S V
SV
Hefta
S A
SA
D000510:Alpha-Globulins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000378:metabolism; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D014360:Trypsin Inhibitor, Kunitz Soybean
D000510:Alpha-Globulins; D001798:Blood Proteins; D006023:Glycoproteins; D006025:Glycosaminoglycans; D008562:Membrane Glycoproteins; D011480:Protease Inhibitors; C109148:SPINT2 protein, human; C011877:inter-alpha-inhibitor; D014360:Trypsin Inhibitor, Kunitz Soybean