The putative chloride channel hCLCA2 has a single C-terminal transmembrane segment
The putative chloride channel hCLCA2 has a single C-terminal transmembrane segment
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DOI：
10.1074/jbc.m605919200
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发表时间：
2006-10-06
影响因子：
4.8
通讯作者：
Pauli, Bendicht U.
中科院分区：
文献类型：
--
作者：
Elble, Randolph C.;Walia, Vijay;Pauli, Bendicht U.
钙激活氯离子通道 (CLCA) 蛋白首先被描述为可以被钙激活的质膜 Cl-通道家族。遗传和电生理学研究支持了这一观点。人 CLCA2 蛋白表达为 943 个氨基酸的前体，其 N 端信号序列被去除，然后在氨基酸位置 680 附近进行内部切割。早期对跨膜几何形状的研究表明需要五次膜通过。然而，通过最近衍生的简单模块化架构研究工具算法进行的分析预测，C 端 22 个氨基酸疏水片段构成唯一的跨膜通道。为了解决这个问题，我们提出了一种针对 hCLCA2 的抗体，并研究了该蛋白质的合成、定位、成熟和拓扑结构。细胞表面生物素化和糖苷内切酶 H 分析显示，转染后 48 小时，128 kDa 前体局限于内质网，而细胞表面有成熟糖基化的 141 kDa 前体。 72 小时时，在细胞表面检测到 109 kDa N 端和 35 kDa C 端裂解产物，但在内质网中未检测到。然而，令人惊讶的是，109-kDa 产物自发地脱落到介质中或通过酸洗去除，而前体和 35-kDa 产物则被膜保留。另外两个 CLCA 家族成员 bCLCA2 和 hCLCA1 也表现出 N 端产物的优先释放。将 hCLCA2 C 末端疏水片段转移至分泌形式的绿色荧光蛋白足以将该蛋白靶向质膜。总之，这些数据表明 hCLCA2 大部分位于细胞外，仅具有单个跨膜片段，后面跟着一个短的细胞质尾，并且其本身不太可能形成通道。
Calcium-activated chloride channel (CLCA) proteins were first described as a family of plasma membrane Cl- channels that could be activated by calcium. Genetic and electrophysiological studies have supported this view. The human CLCA2 protein is expressed as a 943-amino-acid precursor whose N-terminal signal sequence is removed followed by internal cleavage near amino acid position 680. Earlier investigations of transmembrane geometry suggested five membrane passes. However, analysis by the more recently derived simple modular architecture research tool algorithm predicts that a C-terminal 22-amino-acid hydrophobic segment comprises the only transmembrane pass. To resolve this question, we raised an antibody against hCLCA2 and investigated the synthesis, localization, maturation, and topology of the protein. Cell surface biotinylation and endoglycosidase H analysis revealed a 128-kDa precursor confined to the endoplasmic reticulum and a maturely glycosylated 141-kDa precursor at the cell surface by 48 h post-transfection. By 72 h, 109-kDa N-terminal and 35-kDa C-terminal cleavage products were detected at the cell surface but not in the endoplasmic reticulum. Surprisingly, however, the 109-kDa product was spontaneously shed into the medium or removed by acid washes, whereas the precursor and 35-kDa product were retained by the membrane. Two other CLCA family members, bCLCA2 and hCLCA1, also demonstrated preferential release of the N-terminal product. Transfer of the hCLCA2 C-terminal hydrophobic segment to a secreted form of green fluorescent protein was sufficient to target that protein to the plasma membrane. Together, these data indicate that hCLCA2 is mostly extracellular with only a single transmembrane segment followed by a short cytoplasmic tail and is itself unlikely to form a channel.