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

癌细胞特异性细胞粘附蛋白的结构功能研究

• 批准号: 06454644
• 负责人: SAITO Yuji
• 金额: $ 1.66万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454644/
• 关键词: propolypeptide; von Willebrand factor; cell adhesion; integrin; melanoma; VLA-4; フォンビルブラント因子; α4β1; プロペプチド

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.

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各种膜所必需的。也有称为“前肽”的肽，其功能通常是未知的。本实验室前期研究了血管性血友病因子多肽原（pp-vWF，~ 100 kDa）的生理功能，并从分子水平阐明了pp-vWF与胶原的相互作用。在本研究中，我们扩展了这项研究，发现pp-vWF被纳入细胞外基质，它作为一种细胞粘附蛋白。我们的初步观察表明，它特异性介导的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Asp 395-Gln 409。利用该肽作为配体，我们设计了亲和层析并分离了细胞粘附的受体，其被鉴定为α 4 β 1整联蛋白，VLA-4。这一点通过将该整联蛋白的cDNA转染到动物细胞中并测试细胞对pp-vWF的粘附性而得到进一步证实。VLA-4不仅存在于黑素瘤细胞中，而且存在于各种血细胞如淋巴细胞中。我们确实证实了淋巴细胞Molt-3对pp-vWF的粘附。总之，我们推测pp-vWF不仅参与黑色素瘤的转移，而且还参与淋巴细胞对炎症部位的粘附。

项目成果

Takagi et al.: "Identification of Factor XIIIa-Reactive Glutaminyl Residues in the Propolypeptide of Bovine von Willebrand factor" European Journal of Biochemistry. 232. 773-777 (1995)

Takagi 等人：“牛血管性血友病因子原多肽中因子 XIIIa 反应性谷氨酰胺残基的鉴定”《欧洲生物化学杂志》。

T.Isobe: "Propolypeptide of von Billebrand factor is a novel ligand for VLA-4 integrin" Journal of Biological Chemistry. (印刷中).

T. Isobe：“von Billebrand 因子的前多肽是 VLA-4 整合素的新型配体”《生物化学杂志》（正在出版）。

J.Takagi, T.Iosbe, Y.Takada and Y.Saito: "Structural interlock between ligand binding site and stalk like region of beta1 integrin revealed by a monoclonal anitibody recognizing conformation" J.Biochem. (Tokyo). (in press).

J.Takagi、T.Iosbe、Y.Takada 和 Y.Saito：“单克隆抗体识别构象揭示了配体结合位点和 β1 整联蛋白茎状区域之间的结构联锁”J.Biochem。

J.Takagi, Y.Sudo, T.Saito & Y.Saito: "beta1 integrin mediated adhesion of melanoma cells to the propolypetide of von Willebrand factor" Eur.J.Biochem.222. 861-867 (1994)

J.高木、Y.须藤、T.斋藤

斉藤佑尚: "インテグリンリガンドスーパーファミリー" 生体の科学. 46. 533-536 (1995)

Yutaka Saito：“整合素配体超家族”生物科学。 46. 533-536 (1995)