Fosang A J, Last K, Gardiner P, Jackson D C, Brown L
University of Melbourne, Department of Paediatrics, Royal Children's Hospital, Parkville, Australia.
Biochem J. 1995 Aug 15;310 ( Pt 1)(Pt 1):337-43. doi: 10.1042/bj3100337.
We have developed a monoclonal antibody AF-28 that specifically recognizes a neo-epitope on polypeptides with N-terminal FFGVG ... sequences. This sequence is found at the N-terminus of aggrecan fragments that have been digested with matrix metalloproteinases (MMPs). By immunoblotting, monoclonal antibody AF-28 specifically detected G2 fragments derived from an aggrecan G1-G2 substrate digested with stromelysin, collagenase, gelatinase and matrilysin, but failed to detect G2 fragments obtained from elastase, trypsin or cathepsin B digests. Undigested G1-G2 was not detected. In addition, AF-28 antibody detected fragments derived from whole aggrecan and this detection did not require prior treatment with chondroitinase or keratanase. Competition experiments confirmed that peptides containing internal ... FFGVG ... sequences were not detected by the antibody, while native MMP-digested aggrecan fragments and a synthetic 32-mer peptide with FFGVG ... N-termini were equally competitive on a molar basis. An FFGVG 5-mer, and an FGVGGEEDI9-mer which lacked the N-terminal phenylalanine residue, were 50 times and 230 times respectively less competitive than the FFGVG ... 32-mer. Two fragments from the interglobular domain, F342-F373 and F342-D441, that are predicted products of G1-G2 digestion by neutrophil collagenase but have not previously been detected, could be detected with AF-28. The epitope recognized by AF-28 was also detected in human synovial fluids by Western blot analysis. A broad band of 100-200 kDa was detected in some patients and a dominant band of 40-60 kDa was found in two patients. The size of this small fragment corresponds with that seen for the porcine F342-E373 product and may represent the natural physiological product of aggrecan cleaved in vivo at both the MMP site (... DIPEN341 decreases F342FGVG ...) and the aggrecanase site (... ITEGE373 decreases A374RGSVI ...).
我们研发了一种单克隆抗体AF-28，它能特异性识别N端具有FFGVG……序列的多肽上的新表位。该序列存在于经基质金属蛋白酶（MMPs）消化的聚集蛋白聚糖片段的N端。通过免疫印迹法，单克隆抗体AF-28能特异性检测到由基质溶解素、胶原酶、明胶酶和基质溶素消化聚集蛋白聚糖G1-G2底物产生的G2片段，但未能检测到由弹性蛋白酶、胰蛋白酶或组织蛋白酶B消化产生的G2片段。未消化的G1-G2未被检测到。此外，AF-28抗体能检测到来自完整聚集蛋白聚糖的片段，且这种检测不需要事先用软骨素酶或角蛋白酶处理。竞争实验证实，抗体未检测到含有内部……FFGVG……序列的肽段，而天然MMP消化的聚集蛋白聚糖片段和具有FFGVG……N端的合成32聚体肽在摩尔基础上具有同等竞争力。一个FFGVG 5聚体和一个缺少N端苯丙氨酸残基的FGVGGEEDI 9聚体的竞争力分别比FFGVG……32聚体低50倍和230倍。来自球间结构域的两个片段F342-F373和F342-D441，它们是中性粒细胞胶原酶消化G1-G2的预测产物，但此前未被检测到，可用AF-28检测到。通过蛋白质印迹分析在人滑液中也检测到了AF-28识别的表位。在一些患者中检测到一条100-200 kDa的宽带，在两名患者中发现一条40-60 kDa的主带。这个小片段的大小与猪F342-E373产物的大小一致，可能代表聚集蛋白聚糖在体内MMP位点（……DIPEN341使F342FGVG……减少）和聚集蛋白聚糖酶位点（……ITEGE373使A374RGSVI……减少）处裂解产生的天然生理产物。
