Structure of type IV collagen-binding protein from mushroom Hypsizigus marmoreus and its inhibitory activity for cell adhesion

阐明癌细胞与细胞外基质成分相互作用的机制，对于了解肿瘤的转移机制和设计预防肿瘤转移的方法具有重要意义。癌细胞与内皮细胞基底膜的粘连。膜在转移过程中尤为重要，抑制这一步骤将有效地抑制肿瘤转移。在寻找防止癌细胞与基底膜成分纤维连接蛋白和胶原粘连的化合物过程中，我们发现某些蘑菇提取物可以抑制小鼠Lewis肺癌细胞与IV型胶原的粘连。本研究用亲和层析的方法研究了从食用菌中分离得到的23 kDa IV型胶原结合蛋白(HM-23)的性质。结果发现，HM-23对3LL细胞与IV型胶原的黏附有抑制作用。部分氨基酸序列d…进一步的测定表明，HM-23与调节胶原纤维形成的纤维调节蛋白具有同源性，并且HM-23是迄今未知的蛋白质。溴化氰降解HM-23产生了两个约110kDA的多肽，表明蛋氨酸是分子的中心。这些多肽的氨基酸序列分析表明，该蛋氨酸残基是三个已确定序列中的一个。用抗HM-23抗体检测HM-23与细胞外基质蛋白的相互作用。结果表明，HM-23与I型胶原、IV型胶原和明胶结合，与纤维连接蛋白结合较弱，与层粘连蛋白无结合。除了这种与动物细胞外基质蛋白相互作用的蘑菇蛋白外，我们还发现米糠中含有纤维连接蛋白结合蛋白。这些关于非动物蛋白与动物细胞外基质蛋白相互作用的发现将开启广泛的生物学和细胞生物学兴趣，从而为导致癌症转移的问题提供答案。较少

It is very important to elucidate the mechanism of interection between cancer cells and extracellular matrix components for understanding the cancer metastasis and designing the method useful for its prevention. The adhesion of cancer cells to the endothelial basement. membrane is especially important in the metastatic process, and the inhibition of this step would be effective to inhibit cancer metastasis. During search for the compounds which prevent the adhesion of cancer cells to the basement membrane components, fibronectin and collagen, we have found that the extracts from certain mushrooms can inhibit the adhesion of mouse Lewis lung carcinoma cells to type IV collagen. In the present study, we examined the characteristics of the 23kDa type IV collagen-binding protein (HM-23) which was isolated from mushroom Hypsizigus marmoreus by affinity chromatography. As a result, we found that HM-23 inhibited the adhesion of 3LL cells to type IV collagen. The partial amino acid sequences d … More etermined has revealed that HM-23 has homology to fibromodulin, which regulates the formation of collagen fibrils, and that HM-23 is hitherto unknown protein. Cyanogen bromide degradation of HM-23 resulted in generation of two poly-peptides with about 110kDA,suggesting that methionine is present at the center of the molecule. The amino acid sequencing of these polypeptides suggested that this methionine residue is the one contained in one of the three determined sequences. Anti-HM-23 antibodies were used to examine the interaction between HM-23 and extracellular matrix proteins by application of the enzyme-linked immunoadsorbent assay. The results have indicated that HM-23 binds to type I collagen, type IV collagen and gelatin, and to fibronectin weakly, but not to laminin at all. In addition to this mushroom protein exhibiting interaction with animal extracellular matrix proteins, we have also found that rice bran contains fibronectin-binding proteins.These findings on the interaction between non-animal proteins and animal extracellular matrix proteins would open a wide range of biological and cell biological interests which will lead to provide the answers for the problems inculuding cancer metastasis. Less