Metastatic melanoma cell heparanase. Characterization of heparan sulfate degradation fragments produced by B16 melanoma endoglucuronidase.
Metastatic melanoma cell heparanase. Characterization of heparan sulfate degradation fragments produced by B16 melanoma endoglucuronidase.
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DOI：
10.1016/s0021-9258(17)43350-3
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发表时间：
1984-02
期刊：
影响因子：
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通讯作者：
M. Nakajima;T. Irimura;N. Ferrante;G. Nicolson
中科院分区：
文献类型：
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作者：
M. Nakajima;T. Irimura;N. Ferrante;G. Nicolson
硫酸乙酰肝素（HS）是血管内皮基层的重要成分，被转移性小鼠B16黑色素瘤细胞从内皮基层样基质中裂解成大Mr片段并溶解。我们研究了B16细胞产生的HS和其他纯化的糖胺聚糖的降解产物。在还原末端进行3h标记或用[35S]硫酸盐进行代谢标记的糖胺聚糖与B16细胞提取物一起培养，在不含或不存在d -糖酸1,4-内酯（一种有效的外β -葡萄糖醛酸酶抑制剂）的情况下，用高速凝胶渗透色谱法分析糖胺聚糖片段。从牛肺、Engelbreth-Holm-Swarm肉瘤和内皮下基质中分离的HS被降解为特征Mr片段，而透明质酸、6-硫酸软骨素、4-硫酸软骨素、硫酸皮肤蛋白、硫酸角蛋白和肝素基本上未被降解。肝素抑制HS降解，而其他糖胺聚糖抑制HS降解。HS降解为特定Mr片段的时间依赖性表明HS在特定的链内位点被裂解。为了确定HS的具体切割点，用NaBH4预还原的HS与B16细胞提取物孵育，分离HS片段。将HS片段新形成的还原端用NaB[3H]4还原，经三氟乙酸处理和亚硝酸脱氨水解为单糖。由于HS片段中3h还原的末端单糖绝大多数（大于90%）是l -谷氨酸，因此HS降解酶是一种内切葡萄糖醛酸酶（肝素酶）。
Heparan sulfate (HS), a prominent component of vascular endothelial basal lamina, is cleaved into large Mr fragments and solubilized from subendothelial basal lamina-like matrix by metastatic murine B16 melanoma cells. We have examined the degradation products of HS and other purified glycosaminoglycans produced by B16 cells. Glycosaminoglycans 3H-labeled at their reducing termini or metabolically labeled with [35S]sulfate were incubated with B16 cell extracts in the absence or presence of D-saccharic acid 1,4-lactone, a potent exo-beta-glucuronidase inhibitor, and glycosaminoglycan fragments were analyzed by high speed gel permeation chromatography. HS isolated from bovine lung, Engelbreth-Holm-Swarm sarcoma, and subendothelial matrix were degraded into fragments of characteristic Mr, in contrast to hyaluronic acid, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, keratan sulfate, and heparin which were essentially undegraded. Heparin, but not other glycosaminoglycans, inhibited HS degradation. The time dependence of HS degradation into particular Mr fragments indicated that HS was cleaved at specific intrachain sites. In order to determine specific HS cleavage points, HS prereduced with NaBH4 was incubated with a B16 cell extract and HS fragments were separated. The newly formed reducing termini of HS fragments were then reduced with NaB[3H]4, and the fragments hydrolyzed to monosaccharides by trifluoroacetic acid treatment and nitrous acid deamination. Since 3H-reduced terminal monosaccharides from HS fragments were overwhelmingly (greater than 90%) L-gulonic acid, the HS-degrading enzyme responsible is an endoglucuronidase (heparanase).