Recombinant analysis of human alpha 1 (XVI) collagen. Evidence for processing of the N-terminal globular domain.
Recombinant analysis of human alpha 1 (XVI) collagen. Evidence for processing of the N-terminal globular domain.
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人类 α 1 (XVI) 胶原蛋白的重组分析。
DOI：
10.1111/j.1432-1033.1995.tb20245.x
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Timpl,R
中科院分区：
文献类型：
--
作者：
Tillet,E;Mann,K;Nischt,R;Pan,TC;Chu,ML;Timpl,R
人胶原α1（XVI）链的N末端非胶原结构域NC 11是从稳定转染的肾细胞克隆中获得的重组35-kDa蛋白。这种形式在基本切割基序处经历了蛋白水解修剪，表明在体内有类似的释放。结构域NC 11在旋转阴影后显示出球形形状，并且对中性蛋白酶具有抗性。可以产生针对重组NC 11的特异性抗体，并用于分析转染全长α1（XVI）链的其他细胞克隆。代谢标记细胞的洗涤剂提取物的免疫沉淀证明存在具有NC 11表位的二硫键键200-kDa多肽。该材料对胃蛋白酶具有部分抗性，表明形成了具有三螺旋构象的α1（XVI）链同源三聚体。然而，当分泌到培养基中时，这些同源三聚体的相当大比例被降解为可变大小（35-150 kDa）的片段。这些片段中的几个可以在半制备规模上从在中空纤维盒中生长的细胞中获得，并且显示出脯氨酸的大量羟基化，与三螺旋形成一致。Edman降解证明了一些来自胶原蛋白XVI的N末端，一个来自胶原蛋白XVI的C末端位置。这种广泛的降解可以通过NC 11的释放和α1（XVI）链的三螺旋结构域的九个中断中的一些内的进一步裂解来解释。这一过程是否也发生在现场仍有待证明。
The N‐terminal non‐collagenous domain NC11 of the human collagen α1(XVI) chain was obtained as a recombinant 35‐kDa protein from stably transfected kidney cell clones. This form had undergone proteolytic trimming at a basic cleavage motif indicating a similar releasein vivo.Domain NC11 showed a globular shape after rotary shadowing and was resistant to neutral proteases. Specific antibodies could be raised against recombinant NC11 and were used for the analysis of other cell clones transfected with the full‐length α1(XVI) chain. Immunoprecipitation of detergent extracts of metabolically labelled cells demonstrated the presence of disulfide‐bonded 200‐kDa polypeptides possessing NC11 epitopes. This material was partially resistant to pepsin, indicating the formation of α1(XVI) chain homotrimers with a triple‐helical conformation. Yet a substantial proportion of these homotrimers was degraded to fragments of variable size (35–150 kDa) when secreted into the culture medium. Several of these fragments could be obtained on a semi‐preparative scale from cells grown in hollow fiber cassettes and showed substantial hydroxylation of proline, consistent with triple‐helix formation. Edman degradation demonstrated the origin of some from the N‐terminal and of one from a more C‐terminal position of collagen XVI. This extensive degradation may be explained by the release of NC11 and by further cleavages within some of the nine interruptions of the triple‐helical domain of the α1(XVI) chain. Whether this process also occursin situremains to be shown.