Bame K J, Hassall A, Sanderson C, Venkatesan I, Sun C
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA.
Biochem J. 1998 Nov 15;336 ( Pt 1)(Pt 1):191-200. doi: 10.1042/bj3360191.
Heparanases are mammalian endoglycosidases that cleave heparan sulphate glycosaminoglycans from proteoglycan core proteins and degrade them into shorter chains. The enzymes have been proposed to act in a variety of cellular processes, including proteoglycan catabolism, remodelling of basement membranes and release of heparan sulphate-binding ligands from their extracellular storage sites. Additional functions for heparanases may be to generate short heparan sulphate chains that stabilize or activate other proteins. While heparanase activities have been described in a number of tissues and cell lines, it is not known how many different enzymes are responsible for these activities. Our recent studies characterizing the short glycosaminoglycans produced in Chinese hamster ovary (CHO) cells suggested that multiple heparanases are necessary for the formation of the short heparan sulphate chains [Bame and Robson (1997) J. Biol. Chem. 272, 2245-2251]. We examined whether this is the case by purifying heparanase activity from CHO cell homogenates. Based on their ability to bind ion-exchange resins and their elution from gel-filtration columns, four separate heparanase activities were partially purified. All four activities cleave free glycosaminoglycans over a broad pH range of 3.5-6.0 or 6. 5, suggesting that they act in the endosomal/lysosomal pathway. The sizes of the short heparan sulphate chains generated by the partially purified heparanases ranged from 6 to 9 kDa, and for two of the activities the product size is pH-dependent. Three of the four activities degrade proteoglycans as well as the free glycosaminoglycan chain. Interestingly, all four enzymes generate short glycosaminoglycans with a sulphate-rich, modified domain at the non-reducing end of the newly formed chain. Since our previous studies showed that in CHO cells there is also a population of short heparan sulphates with a modified domain at the reducing end of the chain, this suggests that there may be another heparanase in CHO cells that was not purified. Alternatively, our findings suggest that the formation of short heparan sulphate glycosaminoglycans inside CHO cells may be a result of the concerted action of multiple heparanases, and may depend on the proportions of the different enzymes and the environment in which the chains are degraded.
乙酰肝素酶是一种哺乳动物内切糖苷酶，可从蛋白聚糖核心蛋白上切割硫酸乙酰肝素糖胺聚糖，并将其降解为较短的链。有人提出，这些酶参与多种细胞过程，包括蛋白聚糖分解代谢、基底膜重塑以及从细胞外储存位点释放硫酸乙酰肝素结合配体。乙酰肝素酶的其他功能可能是生成稳定或激活其他蛋白质的短硫酸乙酰肝素链。虽然已在许多组织和细胞系中描述了乙酰肝素酶的活性，但尚不清楚有多少种不同的酶负责这些活性。我们最近对中国仓鼠卵巢（CHO）细胞中产生的短糖胺聚糖进行表征的研究表明，形成短硫酸乙酰肝素链需要多种乙酰肝素酶[巴姆和罗布森（1997年）《生物化学杂志》272卷，2245 - 2251页]。我们通过从CHO细胞匀浆中纯化乙酰肝素酶活性来研究是否如此。基于它们与离子交换树脂结合的能力以及从凝胶过滤柱上的洗脱情况，部分纯化了四种不同的乙酰肝素酶活性。所有这四种活性在3.5 - 6.0或6.5的较宽pH范围内切割游离糖胺聚糖，这表明它们在内体/溶酶体途径中起作用。部分纯化的乙酰肝素酶产生的短硫酸乙酰肝素链的大小范围为6至9 kDa，其中两种活性的产物大小与pH有关。四种活性中的三种既能降解蛋白聚糖，也能降解游离糖胺聚糖链。有趣的是，所有四种酶在新形成链的非还原端产生具有富含硫酸盐修饰结构域的短糖胺聚糖。由于我们之前的研究表明，在CHO细胞中也存在一群在链的还原端具有修饰结构域的短硫酸乙酰肝素，这表明CHO细胞中可能存在另一种未被纯化的乙酰肝素酶。或者，我们的研究结果表明，CHO细胞内短硫酸乙酰肝素糖胺聚糖的形成可能是多种乙酰肝素酶协同作用的结果，并且可能取决于不同酶的比例以及链被降解的环境。
