Studies on the formation mechanism and functions of the covalently bound complex of hyaluronan with SHAP, the functional molecular entity of hyaluronan

透明质酸与透明质酸功能分子实体SHAP共价结合复合物的形成机制和功能研究

• 批准号: 17370041
• 负责人: KIMATA Koji
• 金额: $ 9.96万
• 依托单位: 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: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17370041/
• 关键词: hyaluronan; SHAP; inflammation; inter-α-trypsin inhibitor; complex-forming enzyme factor; hepatitis; TSG-6; compliment; インターα-トリプシンインピビター; ビクニン; サイトカイン; インタ-α-トリプシンインヒビター; factor H-related protein 1; TSG6

The covalently bound complex formed from inter-α-trypsin inhibitor (ITI) and hyaluronan (HA) by transesterification reaction, the SHAP (ITI heavy chain)-HA complex was investigated with regard to its physiological functions and serum enz; yme factors involved in the reaction. The candidate protein obtained from one of the library of serum fractions by the beforehand establisehd purification methods using a series of affinity columns was identified FHR-1 (factor H-related protein-1). However, the medium fraction of FHR-1 cDNA-transfected hepatocarcinoma HLF cells did not show any of the activity. Meanwhile, we found that the medium of the transfectants with cDNA of TSG-6 (TNFα-stimulated gene 6 product) had the dramatically increased activity that was yet retained after removal of TSG-6 by passing through the antibody affinity column, suggesting that the enzyme factors could not be TSG-6 itself and be induced with TSG-6 in the cells. Thus, we are now in the processes to the purification … More and identification of the factors. We previously developed the bikunin (ITI short chain)-knockout mouse system in order to investigate in vivo functions of the SHAP-HA complex where the complex was not formed. We found that the knockout mouse was resistant to the induction of acute hepatitis by the administration of D-galactosamine/E.coli lipopolysaccharide (LPS) in which the CD44-HA interaction-dependent attachment of neutrophils to live capillary sinusoidal cell surfaces was involved in response to the LPS stimuli. We then found using culthred leukocytes that the SHAP-HA complex activates the CD44-HA interaction-dependent cell adhesion more than 100 times, which is a decisive evidence for the involvement of the SHAP-HA complex in inflammatory cell response. We also found the good relationship between the SHAP-HA complex levels and the disease progression in the serum samples from patients of liver diseases, articular cartilage injury, gestoses, ovarian cancer etc, suggesting functional significances of the SHAP-HA complex in those diseases and the high reliability of the complex as a disease marker. Less

本文研究了α-胰蛋白酶抑制剂（ITI）与透明质酸（HA）通过酯交换反应形成的共价结合复合物SHAP（ITI重链）-HA复合物的生理功能和参与反应的血清酶因子。通过预先建立的使用一系列亲和柱的纯化方法从血清组分库中获得的候选蛋白被鉴定为FHR-1（H因子相关蛋白-1）。然而，FHR-1 cDNA转染的肝癌HLF细胞的培养基级分没有显示出任何活性。同时，我们还发现含有TSG-6（TNFα-stimulated gene 6产物）cDNA的转染子的培养液，其酶活性在通过抗体亲和柱去除TSG-6后仍有明显的提高，说明这些酶因子不是TSG-6本身，而是在细胞内被TSG-6诱导产生的。因此，我们现在正处于净化过程中 ...更多信息 和因素的识别。我们以前开发了bikunin（ITI短链）-敲除小鼠系统，以研究在体内的SHAP-HA复合物的功能，其中复合物没有形成。我们发现，基因敲除小鼠对D-半乳糖胺/大肠杆菌脂多糖（LPS）诱导的急性肝炎具有抗性，其中中性粒细胞对活毛细血管窦细胞表面的CD 44-HA相互作用依赖性附着参与了对LPS刺激的响应。然后，我们发现使用培养的白细胞，SHAP-HA复合物激活CD 44-HA相互作用依赖性细胞粘附超过100倍，这是SHAP-HA复合物参与炎症细胞应答的决定性证据。在肝病、关节软骨损伤、妊娠、卵巢癌等疾病患者血清中，SHAP-HA复合物的表达水平与疾病进展有良好的相关性，提示SHAP-HA复合物在这些疾病中的功能意义以及作为疾病标志物的可靠性。少

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