Diversity in the processing events at the N-terminus of type-V collagen.
Diversity in the processing events at the N-terminus of type-V collagen.
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DOI：
10.1111/j.1432-1033.1994.tb18815.x
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发表时间：
1994-05
期刊：
影响因子：
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通讯作者：
M. Moradi-Améli;J. Rousseau;J. Kleman;M. Champliaud;M. Boutillon;J. Bernillon;J. Wallach;M. Rest-M.
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文献类型：
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作者：
M. Moradi-Améli;J. Rousseau;J. Kleman;M. Champliaud;M. Boutillon;J. Bernillon;J. Wallach;M. Rest-M.
使用抗肽多克隆抗体研究了人胶原V型链的加工，所述抗肽多克隆抗体针对前α 1（V）和前α 2（V）链的N-末端非三螺旋区的肽序列。针对N-末端α 2（V）序列的48-57位产生的抗肽多克隆抗体识别在蛋白酶抑制剂混合物存在下从几种组织中提取的人α 2（V）链的成熟形式，所述人α 2（V）链不经任何蛋白水解处理。它还识别横纹肌肉瘤A204细胞系细胞培养基中分泌的pro-α 2（V）和pN-alpha 2（V）胶原链。来自该细胞系的pN-alpha 2（V）胶原链在电泳过程中与从组织中获得的alpha 2（V）链一起迁移。这表明组织中的α 2（V）链未被完全加工，并以缺乏C-前肽的pN-α 2（V）胶原蛋白链存在。相比之下，针对N-末端α 1（V）人序列的284-299位残基产生的抗肽多克隆抗体不能识别α 1（V）链的成熟形式，而它与pN-α 1（V）胶原蛋白链形式反应。这些结果表明，α 1（V）链在N-末端区域经历了一个加工事件，涉及至少前284个残基的去除。对溴化氰产生的或胰蛋白酶产生的肽进行氨基酸序列分析，这两条电泳带是从组织形式的胶原V中获得的。与预期的一样，对应于完整α 1（V）链的较慢迁移带仅给出对应于α 1（V）链的序列。然而，先前被认为是完整的α 2（V）链的条带除了α 2（V）链之外还给出了α 1（V）链的序列。该结果表明在组织提取物中存在进一步加工形式的α 1（V）链，其与完整的α 2（V）链一起迁移。在进一步的分析中，我们观察到组织形式的胶原V的两条带以1：1的比例出现，而在胃蛋白酶消化以去除非胶原区域后，观察到两条带的α 1（V）/α 2（V）链比例为3：1。这些结果表明α 1（V）链以不同于[α 1（V）]2 α 2（V）的另外的化学计量存在。（400字处截断摘要）
The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibody raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha 2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha 2(V) collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processed and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) human sequence failed to recognize the mature form of the alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain gave, as expected, only sequences corresponding to the alpha 1(V) chain. However, the band previously considered to be the intact alpha 2(V) chain also gave sequences for the alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)]2 alpha 2(V).(ABSTRACT TRUNCATED AT 400 WORDS)