Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparanase from human neutrophils. Possible role in invasion through basement membranes.
Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparanase from human neutrophils. Possible role in invasion through basement membranes.
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DOI：
10.1172/jci112104
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发表时间：
1985-10
期刊：
影响因子：
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通讯作者：
Y. Matzner;M. Bar‐Ner;J. Yahalom;R. Ishai‐Michaeli;Z. Fuks;I. Vlodavsky
中科院分区：
文献类型：
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作者：
Y. Matzner;M. Bar‐Ner;J. Yahalom;R. Ishai‐Michaeli;Z. Fuks;I. Vlodavsky
研究了新鲜分离的人中性粒细胞降解培养的角膜和血管内皮细胞产生的内皮下细胞外基质（ECM）中硫酸乙酰肝素蛋白多糖的能力。用Na 2（35 S）O 4代谢标记ECM，并通过Sepharose 6 B上的凝胶过滤分析标记的降解产物。超过90%的释放的放射性由硫酸乙酰肝素片段组成，所述硫酸乙酰肝素片段比通过木瓜蛋白酶或碱性硼氢化物从ECM释放的完整硫酸乙酰肝素侧链小5-6倍。这些片段对亚硝酸脱氨基敏感，并且在肝素或丝氨酸蛋白酶抑制剂存在下不产生。与此相反，降解的可溶性高分子量硫酸乙酰肝素蛋白聚糖，这是第一次从ECM释放，抑制肝素，但没有影响的蛋白酶抑制剂。这些结果表明，人嗜中性粒细胞与内皮下ECM的相互作用与其硫酸乙酰肝素的降解有关，通过一种新鉴定的特异性乙酰肝素酶活性，这种降解在很大程度上由丝氨酸蛋白酶促进。通过简单地将细胞在4 ℃下在没有添加刺激的情况下温育，嗜中性粒细胞乙酰肝素酶容易且优先地释放（60分钟内细胞内容物的15-25%）。在这些条件下，乳酸脱氢酶、溶菌酶和球蛋白降解蛋白酶的细胞含量的小于5%被释放。中性粒细胞乙酰肝素酶的进一步纯化通过其与肝素-琼脂糖凝胶结合并在1 M NaCl中洗脱来实现。有人建议，乙酰肝素酶活性参与早期事件的外渗和渗出的中性粒细胞在响应阈值信号从血管外发炎器官。
Freshly isolated human neutrophils were investigated for their ability to degrade heparan sulfate proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells. The ECM was metabolically labeled with Na2(35S)O4 and labeled degradation products were analyzed by gel filtration over Sepharose 6B. More than 90% of the released radioactivity consisted of heparan sulfate fragments 5-6 times smaller than intact heparan sulfate side chains released from the ECM by either papain or alkaline borohydride. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of either heparin or serine protease inhibitors. In contrast, degradation of soluble high molecular weight heparan sulfate proteoglycan, which was first released from the ECM, was inhibited by heparin but there was no effect of protease inhibitors. These results indicate that interaction of human neutrophils with the subendothelial ECM is associated with degradation of its heparan sulfate by means of a specific, newly identified, heparanase activity and that this degradation is facilitated to a large extent by serine proteases. The neutrophil heparanase was readily and preferentially released (15-25% of the cellular content in 60 min) by simply incubating the cells at 4 degrees C in the absence of added stimuli. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase, lysozyme, and globin degrading proteases was released. Further purification of the neutrophil heparanase was achieved by its binding to heparin-Sepharose and elution at 1 M NaCl. It is suggested that heparanase activity is involved in the early events of extravasation and diapedesis of neutrophils in response to a threshold signal from an extravascular inflamed organ.