Type XIII collagen is identified as a plasma membrane protein
Type XIII collagen is identified as a plasma membrane protein
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DOI：
10.1074/jbc.273.25.15590
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发表时间：
1998-06-19
影响因子：
4.8
通讯作者：
Pihlajaniemi, T
中科院分区：
文献类型：
--
作者：
Hagg, P;Rehn, M;Pihlajaniemi, T
通过cDNA克隆确定了小鼠XIII型胶原蛋白链的完整初级结构。将小鼠氨基酸序列与先前确定的人氨基酸序列进行比较，显示出90%的高同源性。令人惊讶的是，小鼠的cdna在5'方向上比先前鉴定的人类克隆延伸得更远。5'序列包含一个新的帧内ATG密码子，用于翻译起始，导致n端非胶原结构域延长了81个残基。这些n端序列缺乏典型的信号序列，但包括一个高度疏水的片段，显然满足跨膜结构域的标准。因此，序列数据出人意料地表明，XIII型胶原可能位于质膜上，具有较短的胞质内n端部分和较长的胶原细胞外部分。这些序列数据促使我们产生针对XIII型胶原的抗肽抗体，以研究该蛋白及其亚细胞位置。人肿瘤HT-1080细胞提取液的Western blotting显示条带大于180kda。这些似乎代表了二硫键多聚多肽形式，在还原成85-95 kda带后分解，可能代表了单片段型13型胶原链的剪接形式的混合物。这些链被证明包含预测的n端延伸，因此也包含假定的跨膜段。从表面标记的HT-1080细胞中对生物素化的XIII型胶原进行免疫沉淀、亚细胞分离和免疫荧光染色，证明XIII型胶原分子确实位于这些细胞的质膜中。
The complete primary structure of the mouse type XIII collagen chain was determined by cDNA cloning. Comparison of the mouse amino acid sequences with the previously determined human sequences revealed a high identity of 90%. Surprisingly, the mouse cDNAs extended further in the 5' direction than the previously identified human clones. The 5' sequences contained a new in-frame ATG codon for translation initiation which resulted in elongation of the N-terminal noncollagenous domain by 81 residues. These N-terminal sequences lack a typical signal sequence but include a highly hydrophobic segment that clearly fulfills the criteria for a transmembrane domain. The sequence data thus unexpectedly suggested that type XIII collagen may be located on the plasma membrane, with a short cytosolic N-terminal portion and a long collagenous extracellular portion.These sequence data prompted us to generate antipeptide antibodies against type XIII collagen in order to study the protein and its subcellular location. Western blotting of human tumor HT-1080 cell extract revealed bands of over 180 kDa. These appeared to represent disulfide-bonded multimeric polypeptide forms that resolved upon reduction into 85-95-kDa bands that are likely to represent a mixture of splice forms of monomelic type XIII collagen chains. These chains were shown to contain the predicted N-terminal extension and thus also the putative transmembrane segment. Immunoprecipitation of biotinylated type XIII collagen from surface-labeled HT-1080 cells, subcellular fractionation, and immunofluorescence staining were used to demonstrate that type XIII collagen molecules are indeed located in the plasma membranes of these cells.