A serum-derived hyaluronan-associated protein (SHAP) is the heavy chain of the inter alpha-trypsin inhibitor.
We showed previously that hyaluronan (HA) synthesized by cultured fibroblasts firmly bound 85-kDa proteins. The proteins were derived from serum used for the culture and appeared to be covalently linked to HA (Yoneda, M., Suzuki, S., and Kimata, K. (1990) J. Biol. Chem. 265, 5247-5257). In these regards, we named this molecule SHAP (serum-derived HA associated proteins). Incubation of serum with exogenous HA under physiological conditions enabled us to prepare SHAP.HA complex without cell cultivation. The complex thus obtained from bovine or human serum was served for the characterization of SHAP. Digestion with HA-lyase and subsequent separation on SDS-polyacrylamide gel electrophoresis yielded two components, X and Y. Because of the block of their NH2 termini, peptides were obtained by the digestion of X and Y with V8 protease, separated on SDS-polyacryl-amide gel electrophoresis and then subjected to the analysis. Peptides from X and Y showed a high degree of sequence similarity to the two heavy chains, HC2 and HC1, of human inter-alpha-trypsin inhibitor (ITI), respectively (over 80% with bovine SHAP and essentially 100% with human SHAP). Cross-reactivity with antibodies against ITI supported the findings. Direct digestion of the complex with V8 protease and the subsequent purification of the HA-resistant fragment complex were performed to identify the HA-binding domains. NH2-terminal sequences of the fragments suggested the participation of the COOH-terminal half of ITI with an amphipathic alpha helix structure in the HA binding.
Huang
L
L
Institute for Molecular Science of Medicine, Aichi Medical University, Japan.
Yoneda
M
M
Kimata
K
K
D000510:Alpha-Globulins / Q000737:chemistry* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D001769:Blood; D002417:Cattle; D002478:Cells, Cultured; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017386:Sequence Homology, Amino Acid
D000510:Alpha-Globulins; C011877:inter-alpha-inhibitor; D006820:Hyaluronic Acid