Fosang A J, Neame P J, Last K, Hardingham T E, Murphy G, Hamilton J A
Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Parkville, Australia.
J Biol Chem. 1992 Sep 25;267(27):19470-4.
The action of three matrix metalloproteinases (MMPs), 72- and 95-kDa gelatinases (MMP-2 and MMP-9) and PUMP (MMP-7), and a cysteine proteinase, cathepsin B, were investigated on aggrecan the major proteoglycan of cartilage. All the enzymes cleaved aggrecan although the activity of the 95-kDa gelatinase was very low. Specific cleavage sites were investigated following incubation with a purified aggrecan G1-G2 domain fragment (150 kDa). Both gelatinases produced 110-kDa G2 and 56-kDa G1 products by a single cleavage at an Asn-Phe bond within the interglobular domain close to the G1 domain. This was similar to the action of stromelysin (MMP-3) (Fosang, A. J., Neame, P. J., Hardingham, T. E., Murphy, G., and Hamilton, J. A. (1991) J. Biol. Chem. 266, 15579-15582). Cathepsin B also produced two fragments from a single cleavage at a Gly-Val bond only three amino acids C-terminal to the metalloproteinase cleavage site. PUMP cleaved at the metalloproteinase Asn-Phe site, but in addition produced a low yield of a smaller G2 fragment (56 kDa) corresponding to cleavage between Asp441 and Leu442 (human sequence), within the interglobular domain, close to the G2 domain. The apparent difference in size between the two G2 fragments released by PUMP (110 and 56 kDa) was much greater than predicted from the peptide length between the cleavage sites (100 amino acids). However, keratanase digestion greatly reduced the size of the 110-kDa G2 fragment, while producing only a small reduction in size of the 56-kDa product, showing that there was approximately 30-40 kDa of keratan sulfate attached to the interglobular domain between the PUMP cleavage sites. This new structural information on aggrecan may account for the previously observed stiffness of the interglobular domains when viewed by rotary shadowing electron microscopy (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D. G., Hardingham, T. E., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). These results show that in spite of a high keratan sulfate content the interglobular domain provides important sites for cleavage by different proteinases, including several members of the matrix metalloproteinase family.
研究了三种基质金属蛋白酶（MMP），即72 kDa和95 kDa明胶酶（MMP - 2和MMP - 9）、PUMP（MMP - 7）以及一种半胱氨酸蛋白酶组织蛋白酶B对软骨主要蛋白聚糖聚集蛋白聚糖的作用。所有这些酶都能切割聚集蛋白聚糖，不过95 kDa明胶酶的活性非常低。在用纯化的聚集蛋白聚糖G1 - G2结构域片段（150 kDa）孵育后，研究了特异性切割位点。两种明胶酶均通过在靠近G1结构域的球间结构域内的一个天冬酰胺 - 苯丙氨酸键处单次切割，产生110 kDa的G2和56 kDa的G1产物。这与基质溶素（MMP - 3）的作用类似（Fosang, A. J., Neame, P. J., Hardingham, T. E., Murphy, G., and Hamilton, J. A. (1991) J. Biol. Chem. 266, 15579 - 15582）。组织蛋白酶B也通过在仅位于金属蛋白酶切割位点C末端三个氨基酸处的一个甘氨酸 - 缬氨酸键处单次切割产生两个片段。PUMP在金属蛋白酶的天冬酰胺 - 苯丙氨酸位点切割，但此外还产生了低产量的较小的G2片段（56 kDa），对应于在靠近G2结构域的球间结构域内天冬氨酸441和亮氨酸442（人类序列）之间的切割。PUMP释放的两个G2片段（110 kDa和56 kDa）之间明显的大小差异远大于根据切割位点之间的肽长度（100个氨基酸）所预测的差异。然而，角质酶消化极大地减小了110 kDa G2片段的大小，而对56 kDa产物的大小仅产生了小幅度减小，表明在PUMP切割位点之间的球间结构域上附着有约30 - 40 kDa的硫酸角质素。当通过旋转阴影电子显微镜观察时，关于聚集蛋白聚糖的这一新结构信息可能解释了先前观察到的球间结构域的刚性（Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore - Gray, M., Dunham, D. G., Hardingham, T. E., Heinegard, D., Timpl, R., and Engel, J. (
