Fragmentation of proteins in cartilage treated with interleukin-1 -: Specific cleavage of type IX collagen by matrix metalloproteinase 13 releases the NC4 domain
Fragmentation of proteins in cartilage treated with interleukin-1 -: Specific cleavage of type IX collagen by matrix metalloproteinase 13 releases the NC4 domain
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DOI：
10.1074/jbc.m702491200
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发表时间：
2007-12-21
影响因子：
4.8
通讯作者：
Heinegard, Dick
中科院分区：
文献类型：
--
作者：
Danfelter, Mikael;Onnerfjord, Patrik;Heinegard, Dick
用白细胞介素-1（IL-1）诱导的牛鼻软骨降解来研究16天内组织中的分解代谢事件。通过二维电泳（等电聚焦和SDS-PAGE）对培养基进行分级。通过肽质量指纹图谱鉴定组分揭示了在第12天和第16天释放的代表IX型胶原α 1链的NC 4结构域的片段。针对NC 4结构域的近N-末端表位的新型肽抗体证实了该发现，并表明在第9天已经存在一个片段。对两个最丰富的片段的质谱分析显示，最小的一个片段几乎包含在精氨酸258和异亮氨酸259之间切割的整个NC 4结构域，其序列为-ETCNELPAR（258）-COOHNH 2-ITP-。较大的片段含有NC 4结构域和COL 3结构域的主要部分，在COL 3中的甘氨酸400和苏氨酸401之间具有切割位点（-RGPPGPPGPSG（400）-COOH NH 2-TIG-）。多个胶原α 1（IX）N-末端序列的存在表明，释放的分子在IL-1处理之前或由于IL-1处理而在非常接近原始N末端的位点被切割。基质金属蛋白酶13（MMP-13）是活跃的，并在研究的时间间隔内切割纤调蛋白。用MMP-13处理的软骨外植体显示出释放与IL-1处理后获得的那些具有相同大小和相同切割位点的胶原α 1（IX）片段。这些数据描述了MMP-13活性对非胶原和三螺旋结构域的切割。这些潜在的重要降解事件先于II型胶原的主要损失。
Degradation of bovine nasal cartilage induced by interleukin-1 (IL-1) was used to study catabolic events in the tissue over 16 days. Culture medium was fractionated by two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE). Identification of components by peptide mass fingerprinting revealed released fragments representing the NC4 domain of the type IX collagen alpha 1 chain at days 12 and 16. A novel peptide antibody against a near N-terminal epitope of the NC4 domain confirmed the finding and indicated the presence of one of the fragments already at day 9. Mass spectrometric analysis of the two most abundant fragments revealed that the smallest one contained almost the entire NC4 domain cleaved between arginine 258 and isoleucine 259 in the sequence-ETCNELPAR(258)-COOHNH2-ITP-. A larger fragment contained the NC4 domain and the major part of the COL3 domain with a cleavage site between glycine 400 and threonine 401 in COL3(-RGPPGPPGPPGPSG(400)-COOH NH2-TIG-). The presence of multiple collagen alpha 1 (IX) N-terminal sequences demonstrates that the released molecules were cleaved at sites very close to the original N terminus either prior to or due to IL-1 treatment. Matrix metalloproteinase 13(MMP-13) is active and cleaves fibromodulin in the time interval studied. Cartilage explants treated with MMP-13 were shown to release collagen alpha 1 (IX) fragments with the same sizes and with the same cleavage sites as those obtained upon IL-1 treatment. These data describe cleavage by an MMP-13 activity toward non-collagenous and triple helix domains. These potentially important degradation events precede the major loss of type II collagen.