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

我们已经证明，在血液流入的炎症组织中，HA通常含有共价结合蛋白，我们称之为Shap和Funt，与血液中循环的α-胰蛋白酶间抑制物(ITI)的重链亚基相对应。因此，我们假设Shap-HA复合体是HA参与炎症反应的一个功能实体。1)从类风湿关节炎患者滑液中提纯的复合体的电子显微镜观察和生化分析表明，患者滑液中的-mR-1,000,000的HA结合了大约5个形状，并通过SNAP聚合。2)通过同源重组参与ITI合成的分子的基因获得Shap-HA复合体-敲除小鼠，结果不仅显示Ovu…显著减少卵丘-卵母细胞复合体(COC)由于COC基质中缺少SNAP-HA复合体而导致的卵丘-卵母细胞复合体(COC)的缺陷，以及受精障碍，从IVF(体外受精)和ISCI(胞浆内精子注射)实验来看，很可能是由于透明带的异常。3)CD44阳性的炎性淋巴细胞的细胞黏附和激活的比较，HUT78与Shap-HA复合底物的粘附率约为HA的100倍，而与HA结合的Shap介导了如此高容量的HA-CD44相互作用。4)我们建立了高灵敏的Shap-HA复合体形成活性的测定方法，并在人血清和一些肝细胞的条件培养液中发现了该活性。在Kaketsu Institute Corporation的合作下，我们成功地纯化了活性较高的部分，并根据多肽分析的信息获得了候选分子的cDNA.5)为了阐明SNAP-HA复合体在体内炎症中的作用，我们在基因敲除的小鼠中诱导了II型胶原诱导的关节炎和葡聚糖诱导的结肠炎，并发现与野生小鼠相比，疾病状况明显减轻。然而，这似乎不是ConA诱导的肝炎的情况，这表明ITI本身的缺陷形成参与了肝炎的某些过程。较少

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