Functional analyses of two cellular binding domains of bovine lactadherin
Functional analyses of two cellular binding domains of bovine lactadherin
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DOI：
10.1021/bi992221r
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发表时间：
2000-05-23
期刊：
影响因子：
2.9
通讯作者：
Rasmussen, JT
中科院分区：
文献类型：
--
作者：
Andersen, MH;Graversen, H;Rasmussen, JT
牛乳粘附素糖蛋白(以前称为PAS-6/7)由两个EGF样结构域和两个C样结构域组成，在凝血因子V和VIII中发现。牛乳粘着素以RGD依赖的方式与α(V)β(5)整合素结合，也与磷脂，特别是磷脂酰丝氨酸结合。为了定义和表征这些结合，研究了乳胶粘附素与不同类型的哺乳动物细胞之间的相互作用。利用重组形式的牛乳胶粘附素，表达α(V)β(5)整合素受体的人乳腺癌MCF-7细胞被证明与含有乳粘附素的RGD特异性结合，但不与突变的RGE乳粘附素结合。抗α(V)β(5)整合素受体的单抗和合成的含有RGD的多肽可抑制MCF-7细胞与乳胶粘附素的黏附。绿猴肾MA-104细胞也表达α(V)β(3)和α(V)β(5)整合素，显示通过这两种整合素与牛乳粘附素结合。为了研究脂类与乳黏附素的相互作用，分别表达了C1C2结构域和C2结构域的两个片段。这两个片段都以浓度依赖的方式与磷脂酰丝氨酸结合，亲和力类似于天然乳胶粘附素(K-d=1.8 nM)。与C2结构域C末端部分对应的多肽以浓度依赖的方式抑制乳胶粘附素与磷脂的结合，最后证明乳粘附素介导了人工磷脂酰丝氨酸膜与MCF-7细胞的结合。综上所述，这些结果表明，乳粘附素可以通过其N端与整合素受体结合，并通过其C端与磷脂结合，从而起到连接两个表面的作用。
The glycoprotein bovine lactadherin (formerly known as PAS-6/7) comprises two EGF-like domains and two C-like domains found in blood clotting factors V and VIII. Bovine lactadherin binds to alpha(v)beta(5) integrin in an RGD-dependent manner and also to phospholipids, especially phosphatidyl serine. To define and characterize these bindings the interactions between lactadherin and different mammalian cell types were investigated. Using recombinant forms of bovine lactadherin, the human breast carcinomas MCF-7 cells expressing the alpha(v)beta(5) integrin receptor were shown to bind specifically to RGD containing lactadherin but not to a mutated RGE lactadherin. A monoclonal antibody against the alpha(v)beta(5) integrin receptor and a synthetic RGD-containing peptide inhibited the adhesion of MCF-7 cells to lactadherin. Green monkey kidney MA-104 cells, also expressing the alpha(v)beta(3) together with the alpha(v)beta(5) integrin, showed binding to bovine lactadherin via both integrins. To investigate the interaction of lipid with lactadherin two fragments were expressed corresponding to the C1C2 domains and the C2 domain. Both fragments bound to phosphatidyl serine in a concentration-dependent manner with an affinity similar to native lactadherin (K-d = 1.8 nM). A peptide corresponding to the C-terminal part of the C2 domain inhibited the binding of lactadherin to phospholipid in a concentration-dependent manner, and finally it was shown that lactadherin mediates binding between artificial phosphatidyl serine membranes and MCF-7 cells. Taken together these results show that lactadherin can act as link between two surfaces by binding to integrin receptors through its N-terminus and to phospholipids through its C-terminus.