Wong E F, Brar S K, Sesaki H, Yang C, Siu C H
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
J Biol Chem. 1996 Jul 5;271(27):16399-408. doi: 10.1074/jbc.271.27.16399.
Dictyostelium discoideum expresses EDTA-sensitive cell-cell adhesion sites soon after the initiation of development, and a Ca2+-binding protein of Mr 24,000 (designated DdCAD-1) has been implicated in this type of adhesiveness. We have previously purified DdCAD-1 to homogeneity and characterized its cell binding activity (Brar, S. K., and Siu, C.-H. (1993) J. Biol. Chem. 268, 24902-24909). In this report, we describe the cloning of DdCAD-1 cDNAs. DNA sequencing revealed a single open reading frame coding for a polypeptide containing 213 amino acids. The identity of the cDNA was confirmed by amino acid sequences of two cyanogen bromide peptides. The deduced amino acid sequence of DdCAD-1 exhibits a relatively high degree of sequence similarity with members of the cadherin family and protein S of Myxococcus xanthus. Unlike the other cadherins, the carboxyl-terminal region of DdCAD-1 contains a Ca2+-binding motif. Although analyses of the sequence suggest that the polypeptide lacks a signal peptide sequence and a transmembrane domain, immunofluorescence microscopy demonstrates the association of DdCAD-1 with the ecto-surface of the plasma membrane. To investigate the structure/function relationships of DdCAD-1, glutathione S-transferase fusion proteins containing different DdCAD-1 fragments were expressed and assayed for their 45Ca2+ and cell binding activities. These studies revealed that the cell binding activity is dependent on the amino-terminal segment and not the carboxyl-terminal Ca2+-binding domain and showed additional Ca2+-binding site(s) within the amino-terminal segment.
盘基网柄菌在发育开始后不久就会表达对乙二胺四乙酸（EDTA）敏感的细胞间黏附位点，一种分子量为24000的钙结合蛋白（命名为DdCAD-1）与这种黏附性有关。我们之前已将DdCAD-1纯化至同质，并对其细胞结合活性进行了表征（布拉尔，S.K.，和萧，C.-H.（1993年）《生物化学杂志》268，24902 - 24909）。在本报告中，我们描述了DdCAD-1 cDNA的克隆。DNA测序揭示了一个单一的开放阅读框，编码一个含有213个氨基酸的多肽。通过两个溴化氰肽的氨基酸序列证实了该cDNA的身份。DdCAD-1推导的氨基酸序列与钙黏蛋白家族成员和黄色黏球菌的蛋白S表现出相对较高程度的序列相似性。与其他钙黏蛋白不同，DdCAD-1的羧基末端区域包含一个钙结合基序。尽管对序列的分析表明该多肽缺乏信号肽序列和跨膜结构域，但免疫荧光显微镜显示DdCAD-1与质膜的外表面相关联。为了研究DdCAD-1的结构/功能关系，表达了含有不同DdCAD-1片段的谷胱甘肽S-转移酶融合蛋白，并检测了它们的45Ca2+结合活性和细胞结合活性。这些研究表明，细胞结合活性取决于氨基末端片段，而非羧基末端的钙结合结构域，并在氨基末端片段内显示出额外的钙结合位点。
