Human placental calreticulin - Characterization of domain structure and post-translational modifications
Human placental calreticulin - Characterization of domain structure and post-translational modifications
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DOI：
10.1046/j.1432-1327.2001.02138.x
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发表时间：
2001-05-01
期刊：
影响因子：
--
通讯作者：
Houen, G
中科院分区：
文献类型：
--
作者：
Hojrup, P;Roepstorff, P;Houen, G
用质谱结合蛋白酶切分析了人胎盘钙网蛋白的结构域组成和翻译后修饰。胰蛋白酶、糜蛋白酶、弹性蛋白酶、金黄色葡萄球菌V8蛋白酶或蛋白酶K的长期治疗均导致钙网蛋白分子量减少6- 7-kDa。发现该降低是由于残基340周围区域中的裂解。此外，次要片段产生的二次切割接近N-末端观察，但没有稳定的片段的中间尺寸被发现。这些结果表明，钙网蛋白的C-域是容易被蛋白水解切割和N-和P-域形成蛋白水解稳定的紧密association。前两个半胱氨酸之间的二硫桥被映射在N-结构域中，第三个半胱氨酸被发现处于还原形式。没有发现糖基化或磷酸化形式的翻译后修饰。分离出了一种缺乏C-末端六肽的钙网蛋白修饰形式，其中包括KDEL内质网滞留序列子。这种截短可能是钙网蛋白从内质网逃逸的机制。
The domain organization and the post-translational modifications of human placenta calreticulin were analysed by MS in combination with proteolytic digestion. Prolonged treatment with trypsin, chymotrypsin, elastase, Staphylococcus aureus V8 protease, or proteinase K all led to a 6- to 7-kDa decrease in the molecular mass of calreticulin. The decrease was found to be due to cleavages in the region around residue 340. In addition, minor fragments resulting from secondary cleavages close to the N-terminus were observed, but no stable fragments of intermediate size were found. These results show that the C-domain of calreticulin is susceptible to proteolytic cleavage and that the N- and P-domains form a proteolytically stable tight association. A disulfide bridge between the first two cysteines was mapped in the N-domain, and the third cysteine was found in the reduced form. No post-translational modifications in the form of glycosylation or phosphorylation were found. A modified form of calreticulin lacking the C-terminal hexapeptide including the KDEL endoplasmic reticulum retention sequon was isolated. Such a truncation may point to a mechanism that allows escape of calreticulin from the endoplasmic reticulum.