Zhao M, Yoneda M, Ohashi Y, Kurono S, Iwata H, Ohnuki Y, Kimata K
Institute for Molecular Science of Medicine, Aichi Medical University, Japan.
J Biol Chem. 1995 Nov 3;270(44):26657-63. doi: 10.1074/jbc.270.44.26657.
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-alpha-trypsin inhibitor (ITI) (Huang, L., Yoneda, M., and Kimata, K. (1993) J. Biol. Chem. 268, 26725-26730). In this study, the SHAP.HA complex was isolated from pathological synovial fluid from human arthritis patients. The SHAP.HA complex was digested with thermolysin, 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 SHAP.HA 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-alpha-trypsin inhibitor (HC1 and HC2), and heavy chain 3 of pre-alpha-trypsin inhibitor (HC3), respectively, and a heptapeptide from HC1. Mass spectrometric analyses suggested that the C-terminal 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.
我们先前发现，血清来源的85-kDa蛋白（SHAPs，血清来源的透明质酸相关蛋白）与培养的成纤维细胞合成的透明质酸（HA）紧密结合。随后鉴定出SHAPs为α-胰蛋白酶抑制剂（ITI）的重链（Huang, L., Yoneda, M., and Kimata, K. (1993) J. Biol. Chem. 268, 26725 - 26730）。在本研究中，从人类关节炎患者的病理性滑液中分离出SHAP.HA复合物。用嗜热菌蛋白酶消化SHAP.HA复合物，然后进行CsCl梯度离心。将由此获得的含HA片段进一步用软骨素酶AC II消化，并进行TSK凝胶高效液相色谱（HPLC）分析。含肽-HA二糖的级分（SHAP.HA结合区域）通过反相HPLC进一步纯化。通过蛋白质测序和质谱分析（电喷雾电离质谱和碰撞诱导解离-MS/MS）分析主要峰。通过与报道的人类ITI家族的C末端序列进行比较，发现这些肽分别对应于α-胰蛋白酶抑制剂重链1和2（HC1和HC2）以及前α-胰蛋白酶抑制剂重链3（HC3）的C末端衍生的四肽，以及来自HC1的七肽。质谱分析表明，每条重链的C末端天冬氨酸被酯化为HA链内部N-乙酰葡糖胺的C6-羟基。本报告首次证明了蛋白质与HA的共价结合。
