Structure-function Studies On A Cancer Cell-specific Cell Adhesion Protein

多肽通常在mRNA翻译后被切割。典型的例子是信号肽，它是蛋白质分泌或将蛋白质靶向到各种膜所必需的。还有称为“前肽”的肽，其功能通常是未知的。我们研究了血管性血友病因子原多肽（pp-vWF,-100kDa）的生理功能，并从分子角度阐明了pp-vWF与胶原蛋白的相互作用。在本研究中，我们扩展了这一研究，发现pp-vWF被纳入细胞外基质，并作为细胞粘附蛋白。我们最初的观察表明，它特异性地介导了黑色素瘤细胞的粘附。利用我们的抗pp-vWF单克隆抗体和蛋白酶消化技术，我们发现负责细胞粘附的结构域位于中央8-kDa区域。利用基因工程和合成肽技术，我们已经将结构域缩小到15个氨基酸片段Asp395-Gln409。以该肽为配体，设计亲和层析，分离出细胞粘附受体，鉴定为alpha4beta1 integrin， vla4。将该整合素cDNA转染动物细胞，并检测细胞对pp-vWF的粘附能力，进一步证实了这一点。VLA-4不仅存在于黑色素瘤细胞中，也存在于淋巴细胞等多种血细胞中。我们确实证实了淋巴细胞Molt-3与pp-vWF的粘附。总之，我们推测pp-vWF不仅参与黑色素瘤的转移，还参与淋巴细胞对炎症部位的粘附。

Very often polypeptides are cleaved after translation from mRNA.Typical examples are signal peptides, which are necessary for protein secretion or targeting proteins to various membranes. There are also peptides called "propeptides", whose functions are usually not known. We had previously investigated physiological functions of propolypeptide of von Willebrand factor (pp-vWF,-100kDa), and elucidated the interaction between pp-vWF and collagen in molecular terms. In the present investigation we have extended this study and found that pp-vWF is incorporated into extra cellular matrix, and that it serves as a cell adhesion protein. Our initial observation suggested that it specifically mediated the adhesion of melanoma cells. Using our monoclonal antibodies against pp-vWF and technique of protease digestion, we found that the domain responsible for the cell adhesion resided in the central 8-kDa region. Using genetic engineering and synthetic peptide techniques we have narrowed down the domain to 15 amino acid segment Asp395-Gln409. Using this peptide as the ligand, we have devised affinity chromatography and isolated the receptor for the cell adhesion, which was identified as alpha4beta1 integrin, VLA-4. This point was further confirmed by transfection of the cDNA of this integrin into animal cells and testing the cell adhesion to pp-vWF.VLA-4 is found not only in melanoma cells but also various blood cells such as lymphocytes. We indeed confirmed the adhesion of lymphocyte Molt-3 to pp-vWF.In conclusion, we speculate that pp-vWF is involved not only in metastasis of melanoma but also in adhesion of lymphocytes to the site of inflammation.