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Function of the formation of SHAP (serum-derived hyaluronan associated protein) -HA complex

SHAP（血清透明质酸相关蛋白）-HA 复合物形成的功能

基本信息

批准号：
06680594
负责人：
YONEDA Masahiko
金额：
$ 1.41万
依托单位：
AICHI MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

We previously showed that hyaluronan (HA) synthesized by cultured fibloblasts firmly bound serum-derived 85kDa proteins (SHAPs, serum-derived hyaluronan associated proteins). SHAPs were identified with the heavy chains of inter alpha-trypsin inhibitor (ITI) (J.Biol.Chem.268,2672526730,1993). ITI consist of three genetically different peptides, a light chain (bikunin) and two heavy chains (HC1 and HC2). ITI doesn't bind to HA in any assay experiments. We appeared that SHAPs bind covalently to HA.We subjected SHAP-HA complex to limited proteolysis and hyaluronidase-digestion to obtain fragments of the linkage regions. The fragments were analyzed with protein sequencer and electrospray ionization mass spectrometry. The C-terminal Asp of each heavy chain was esterified with 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.The reaction of formation requires one of the serum factors (enzymes). It is intersting that the formation of the SHAP-HA complex from HA and ITI is accompanied by the release of bikunin.The highly metastatic subclone of mouse mammary carcinoma (FM3A P15A) with a high activity in HA synthesis has a large distribution of pericellular HA (HA rich-matrix). Additions of purified ITI have no effect on a size of HA rich-matrix of P15A cultures. Both ITI and the partially purified enzyme added to cultures made it enlarge. It means that the formation of the SHAP-HA complex increases a volume of HA richmatrix. We studied a distribution HA and SHAP in tumor tissues in vivo by immunostaining. We had results that the accumulation of both HA and SHAP in tumor and bikunin localize around tumor. The formation of the SHAP-HA complex on the surface of cells may regulate a construction and size of extracellular matrix and a release of bikunin around cells.

我们先前表明，由培养的成纤维细胞合成的透明质酸（HA）牢固地结合血清衍生的85 kDa蛋白（SHAP，血清衍生的透明质酸相关蛋白）。SHAPs经α-胰蛋白酶间抑制剂（ITI）重链鉴定（J.Biol.Chem.268，2672526730，1993）。ITI由三种遗传上不同的肽组成，一条轻链（bikunin）和两条重链（HC 1和HC 2）。在任何测定实验中，ITI不与HA结合。我们将SHAP-HA复合物进行有限的蛋白水解和透明质酸酶消化，以获得连接区域的片段。用蛋白质测序仪和电喷雾质谱仪对片段进行分析。每个重链的C-末端Asp与HA链的内部N-乙酰葡糖胺的C6羟基酯化。本报告首次证明了蛋白质与HA的共价结合。形成反应需要血清因子（酶）之一。HA和ITI形成SHAP-HA复合物的过程中伴随着bikunin的释放，具有高HA合成活性的小鼠乳腺癌高转移性亚克隆（FM 3A P15 A）具有大量的细胞周HA分布（HA富集基质）。添加纯化的ITI对P15 A培养物的富含HA的基质的大小没有影响。ITI和加入到培养物中的部分纯化的酶都使其扩大。这意味着SHAP-HA复合物的形成增加了HA富集基质的体积。我们通过免疫组化染色研究了HA和SHAP在体内肿瘤组织中的分布。我们的结果是HA和SHAP在肿瘤中积聚，bikunin定位于肿瘤周围。SHAP-HA复合物在细胞表面的形成可以调节细胞外基质的结构和大小以及细胞周围bikunin的释放。