Evidence for the Covalent Binding of SHAP, Heavy Chains of Inter-α-Trypsin Inhibitor, to Hyaluronan

Ming Zhao, Masahiko Yoneda, Yoko Ohashi, Sadamu Kurono, Hisashi Iwata, Youji Ohnuki, Koji Kimata
1995 Journal of Biological Chemistry  
We previously showed that serum-derived 85-kDa proteins (SHAPs, serum-derived hyaluronan associated proteins) are firmly bound to hyaluronan (HA) synthesized by cultured fibroblasts. SHAPs were then identified to be the heavy chains of inter-␣-trypsin inhibitor (ITI) (Huang, L., Yoneda, M., and Kimata, K. (1993) J. Biol. Chem. 268, 26725-26730). In this study, the SHAP⅐HA complex was isolated from pathological synovial fluid from human arthritis patients. The SHAP⅐HA complex was digested with
more » ... ermolysin, followed by CsCl gradient centrifugation. The HA-containing fragments thus obtained were further digested with chondroitinase AC II and subjected to TSK gel high performance liquid chromatography (HPLC). Peptide-HA disaccharide-containing fractions (the SHAP⅐HA binding regions) were further purified by reverse phase HPLC. Major peaks were analyzed by protein sequencing and mass spectrometry (electrospray ionization mass spectrometry and collision induced dissociation-MS/MS). By comparison with the reported C-terminal sequences of the human ITI family, the peptides were found to correspond to tetrapeptides derived from the C termini of heavy chains 1 of and 2 of inter-␣-trypsin inhibitor (HC1 and HC2), and heavy chain 3 of pre-␣-trypsin inhibitor (HC3), respectively, and a heptapeptide from HC1. Mass spectrometric analyses suggested that the Cterminal Asp of each heavy chain was esterified to the C6-hydroxyl group of an internal N-acetylglucosamine of HA chain. This report is the first demonstration to give evidence for the covalent binding of proteins to HA.
doi:10.1074/jbc.270.44.26657 pmid:7592891 fatcat:py2b4h2x7nbipfocf5dnkowo2q