Hughes C, Murphy G, Hardingham T E
Biochemistry Division, Kennedy Institute, London, U.K.
Biochem J. 1991 Nov 1;279 ( Pt 3)(Pt 3):733-9. doi: 10.1042/bj2790733.
The action of purified rabbit bone stromelysin was investigated on proteoglycan aggregates from pig laryngeal cartilage. The enzyme caused a rapid fall in viscosity of proteoglycan aggregate solution (6 mg/ml), and the products of a partial digest (60% loss of relative viscosity) and a complete digest (95% loss of relative viscosity) were characterized. Analysis by gel chromatography on Sepharose 2B under associative conditions showed that 95% of the glycosaminoglycans in the complete digest were in small-sized fragments, whereas most of the hyaluronan-binding G1 domain and link protein remained intact and bound to hyaluronan. In contrast, there was extensive digestion of the G2 domain which resulted in 76% loss in its detection by immunoassay. Analysis of the partial digest also showed considerable loss (40%) of detection of the G2 domain, but the glycosaminoglycan-rich fragments were much larger than in the complete digest. There was also much less cleavage to create small fragments containing the G1 domain. This was evident on SDS/PAGE analysis where a 58 kDa G1 domain fragment was abundant in the complete digest, but was only present in small amounts in the partial digest. There was also only very limited conversion of link protein from a 44 kDa form to a 40 kDa form. The digestion of proteoglycan aggregate (6 mg/ml) by stromelysin was unaffected by the addition of a high concentration of extra chondroitin sulphate chains (14 mg/ml), and the digestion of proteoglycan monomer showed that the G1 domain was resistant to stromelysin digestion even when not bound to hyaluronan and link protein. The results show that stromelysin degrades the proteoglycan protein core with major cleavages close to, but not within, the G1 domain, and extensive cleavage in other regions. Experiments with purified collagenase, a metalloproteinase structurally related to stromelysin, showed that it too cleaved proteoglycan at several sites within the glycosaminoglycan-rich region of the core protein. Metalloproteinase attack on proteoglycan thus not only occurs with stromelysin but also with collagenase.
研究了纯化的兔骨基质溶解素对猪喉软骨蛋白聚糖聚集体的作用。该酶导致蛋白聚糖聚集体溶液（6毫克/毫升）的粘度迅速下降，并对部分消化产物（相对粘度损失60%）和完全消化产物（相对粘度损失95%）进行了表征。在缔合条件下通过琼脂糖2B凝胶色谱分析表明，完全消化产物中95%的糖胺聚糖处于小尺寸片段中，而大多数与透明质酸结合的G1结构域和连接蛋白保持完整并与透明质酸结合。相比之下，G2结构域发生了广泛的消化，导致免疫测定检测其含量损失76%。部分消化产物分析也显示G2结构域检测量有相当大的损失（40%），但富含糖胺聚糖的片段比完全消化产物中的大得多。产生含G1结构域小片段的裂解也少得多。这在SDS/PAGE分析中很明显，其中58 kDa的G1结构域片段在完全消化产物中丰富，但在部分消化产物中仅少量存在。连接蛋白从44 kDa形式转化为40 kDa形式的情况也非常有限。添加高浓度的额外硫酸软骨素链（14毫克/毫升）不影响基质溶解素对蛋白聚糖聚集体（6毫克/毫升）的消化，对蛋白聚糖单体的消化表明，即使不与透明质酸和连接蛋白结合，G1结构域也对基质溶解素消化具有抗性。结果表明，基质溶解素降解蛋白聚糖蛋白核心，主要裂解发生在靠近G1结构域但不在其内部的位置，以及其他区域的广泛裂解。用纯化的胶原酶（一种在结构上与基质溶解素相关的金属蛋白酶）进行的实验表明，它也在核心蛋白富含糖胺聚糖的区域内的几个位点切割蛋白聚糖。因此，金属蛋白酶对蛋白聚糖的攻击不仅发生在基质溶解素上，也发生在胶原酶上。
