Membrane topology of Bves/Pop1A, a cell adhesion molecule that displays dynamic changes in cellular distribution during development
Membrane topology of Bves/Pop1A, a cell adhesion molecule that displays dynamic changes in cellular distribution during development
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DOI：
10.1074/jbc.m301961200
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发表时间：
2003-08-29
影响因子：
4.8
通讯作者：
Backstrom, JR
中科院分区：
文献类型：
--
作者：
Knight, RF;Bader, DM;Backstrom, JR
我们研究了Bves/Pop 1A的膜拓扑结构，以此为基础来剖析Bves/Pop 1A在发育过程中运输的分子基础和功能。Bves在氨基末端含有两个天冬酰胺连接的糖基化位点和三个推定的膜结构域。因此，进行糖基化测定以确定Bves的氨基末端是否被递送到内质网腔中并被糖基化。我们建立了鸡心脏和转染细胞的Bves是糖基化的，这意味着细胞表面分子的氨基末端是细胞外的。利用三种生物化学上不同的方法来确定Bves的羧基末端的取向。首先，在羧基末端内的外源位点处的Bves的糖基化仅在缺乏第三膜结构域的构建体中观察到，这大概逆转了羧基末端的方向。第二，全长Bves与存在于不同亚细胞区室中的可溶性羧基末端Bves构建体的共表达揭示了Bves-Bves相互作用发生在细胞质中。第三，内源性Bves在心外膜细胞表面的免疫反应性显着增强与去污剂。这些结果表明，细胞表面Bves/Pop 1A的膜拓扑结构是由细胞外氨基末端，三个跨膜结构域，和细胞质羧基末端。因此，我们假设羧基末端调节冠状动脉血管发育过程中Bves/Pop 1A的细胞分布。
We investigated the membrane topology of Bves/ Pop1A as a foundation to dissect the molecular basis and function of Bves/ Pop1A trafficking during development. Bves contains two asparagine-linked glycosylation sites within the amino terminus and three putative membrane domains. Therefore, glycosylation assays were performed to determine if the amino terminus of Bves is delivered into the endoplasmic reticulum lumen and glycosylated. We establish that Bves from chick heart and transfected cells is glycosylated, implying that the amino terminus of cell surface molecules is extracellular. Three biochemically distinct approaches were utilized to determine the orientation of the carboxyl terminus of Bves. First, glycosylation of Bves at exogenous sites within the carboxyl terminus was only observed in a construct that lacked the third membrane domain, which presumably reversed the orientation of the carboxyl terminus. Second, co-expression of full-length Bves with soluble, carboxyl-terminal Bves constructs that reside in different subcellular compartments revealed that Bves-Bves interactions occur in the cytoplasm. Third, the immunoreactivity of endogenous Bves at the cell surface of epicardial cells was dramatically enhanced with detergent. These results suggest that the membrane topology of cell surface Bves/ Pop1A is composed of an extracellular amino terminus, three transmembrane domains, and a cytoplasmic carboxyl terminus. We therefore hypothesize that the carboxyl terminus regulates the cellular distribution of Bves/ Pop1A during coronary vessel development.