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Study on the SHAP-hyaluronoan (HA) complex as a functional entity of HA in the process of inflammation.

SHAP-透明质酸（HA）复合物作为HA在炎症过程中的功能实体的研究。

基本信息

批准号：
14380298
负责人：
KIMATA Koji
金额：
$ 8.77万
依托单位：
INSTITUTE FOR MOLECULAR SCIENCE OF MEDICINE, AICHI MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380298/
关键词：
inflammation hyaluronan SHAP trypsin inhibitor CD44 cumulas-oocyte complex ovulation cell adhesion

项目摘要

We have shown that HA in the inflammatory tissues with influx of blood usually contain covalently bound proteins that we named SHAP and fount to correspond to the heavy chain subunits of inter a-trypsin inhibitor (ITI) circulating in blood. Therefore, we hypothesized that the SHAP-HA complex is a functional entity of HA involved in inflammation. Here, we demonstrate evidences obtained from the following experiments shown below, and our final goal is to find some ways to regulate inflammations through the study on the regulation of the SNAP-HA complex formation.1) Electron microscopic observation and biochemical analysis of the complex purified from the synovial fluid obtained from rheumatoid arthritic patients have revealed that the HA of-Mr 1,000,000 in the patient fluid bound about 5 SHAPs and is polymerized via SNAP.2) SHAP-HA complex-knockout mice obtained by homologous recombination of the genes for molecules involved in the ITI synthesis show not only a marked decrease in the ovu … More lation of the cumulus-oocyte complex (COC) which is caused by the defective formation of the COC, due to the absence of the SNAP-HA complex in the COC matrix, but also the impaired fertilization which is likely due to the abnormality of the zona pellucida judging from the IVF (in vitro fertilization) and ISCI (intra-cytoplasmic spermatozoa injection) experiments.3) Comparison of cell adhesion and activation of CD44-positive inflammatory lymphocyte cells, HUT78 to the SHAP-HA complex substrata with those to HA alone has revealed that the cell adhesion to the complex is about 100 times more efficient than that to HA and SHAP bound to HA mediates such a high capacity of the HA-CD44 interaction.4) We have developed the highly sensitive assay method for the activity of the SHAP-HA complex formation and found the presence of the activity in human sera and conditioned media of some liver cells. In collaboration with the Kaketsu Institute Corporation we successfully purified the fraction with the high activity and, based upon the informations of the peptide analysis, have obtained the cDNA of the candidate molecule. We are now on the way to the definitive answer.5) In order to clarify in vivo role of the SNAP-HA complex in inflammation, we induced type II collagen-induced arthritis and dextran sulfate-induced colitis in the knockout mice, and have found that the disease conditions tented to be significantly reduced, compared to those in wild mice. However, this seemed not the case for ConA-induced hepatitis, which suggests some involvement of the deficient formation of ITI itself in the hepatitis. Less

我们已经表明，在炎症组织中的HA与血液流入通常含有共价结合的蛋白质，我们命名为SHAP，并发现对应于在血液中循环的间α-胰蛋白酶抑制剂（ITI）的重链亚基。因此，我们假设SHAP-HA复合物是HA参与炎症的功能实体。在此，我们证明了从下述实验中获得的证据，我们的最终目标是通过研究SNAP-HA复合物形成的调节来找到一些调节炎症的方法。1）从类风湿性关节炎患者的滑液中纯化的复合物的电镜观察和生化分析显示，-Mr 1，000的HA，2）通过同源重组ITI合成相关分子的基因获得SHAP-HA复合物敲除小鼠，不仅显示出卵子中SHAP-HA复合物的表达显著降低，而且显示出与ITI合成相关分子的表达显著降低。 ...更多信息卵丘-卵母细胞复合体（COC）的形成，这是由于COC基质中缺少SNAP-HA复合体，而且从IVF判断，可能是由于透明膜异常导致受精障碍（体外受精）和ISCI（胞质内精子注射）实验。3）比较CD 44阳性炎性淋巴细胞的细胞粘附和活化，HUT 78对SHAP-HA复合物基质的粘附与对单独HA的粘附相比，揭示了细胞对复合物的粘附比对HA的粘附效率高约100倍，并且与HA结合的SHAP介导了HA-CD 44相互作用的这种高能力。我们已经建立了高灵敏度的SHAP-HA复合物形成活性的测定方法，并在人血清和某些肝细胞的条件培养基中发现了该活性的存在。我们与Kaketsu Institute Corporation合作，成功地纯化了具有高活性的级分，并根据肽分析的信息，获得了候选分子的cDNA。5）为了阐明SNAP-HA复合物在炎症中的体内作用，我们在敲除小鼠中诱导II型胶原诱导的关节炎和硫酸葡聚糖诱导的结肠炎，并且发现与野生小鼠相比，疾病状况显著减少。然而，这似乎不是ConA诱导的肝炎的情况下，这表明一些参与ITI本身的缺陷形成的肝炎。少