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

本文研究了α-胰蛋白酶抑制剂（ITI）与透明质酸（HA）经酯交换反应形成的共价结合物SHAP （ITI重链）-HA复合物的生理功能和血清酶活性；参与反应的因子。通过预先建立的纯化方法，利用一系列亲和柱从血清组分库中获得候选蛋白，鉴定为FHR-1（因子h相关蛋白-1）。然而，FHR-1 cdna转染的肝癌HLF细胞的中等部分没有显示出任何活性。同时，我们通过抗体亲和柱发现，转染了TSG-6 （tnf α-刺激基因6产物）cDNA的培养基在去除TSG-6后仍保持了显著增加的活性，说明酶因子不可能是TSG-6本身，而可能是由TSG-6在细胞中诱导产生的。因此，我们现在正处于对其进行净化和鉴定的过程中。我们之前开发了bikunin （ITI短链）敲除小鼠系统，以研究未形成复合物的shape - ha复合物的体内功能。我们发现敲除小鼠对d -半乳糖胺/E诱导的急性肝炎具有抗性。在大肠杆菌脂多糖（LPS）中，CD44-HA依赖于中性粒细胞与活毛细血管窦细胞表面的相互作用，参与了对LPS刺激的反应。然后，我们通过培养的白细胞发现，SHAP-HA复合物激活CD44-HA相互作用依赖的细胞粘附超过100次，这是SHAP-HA复合物参与炎症细胞反应的决定性证据。我们还发现，在肝脏疾病、关节软骨损伤、妊娠、卵巢癌等患者的血清样本中，SHAP-HA复合物水平与疾病进展之间存在良好的关系，提示SHAP-HA复合物在这些疾病中的功能意义，以及该复合物作为疾病标志物的高可靠性。少

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