Freeman C, Parish C R
Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT2601, Australia.
Biochem J. 1997 Jul 1;325 ( Pt 1)(Pt 1):229-37. doi: 10.1042/bj3250229.
Heparan sulphate (HS) is an important component of the extracellular matrix and the vasculature basal laminar which functions as a barrier to the extravasation of metastatic and inflammatory cells. Cleavage of HS by endoglycosidase or heparanase activity produced by invading cells may assist in the disassembly of the extracellular matrix and basal laminar, and thereby facilitate cell migration. Heparanase activity has previously been shown to be related to the metastatic potential of murine and human melanoma cell lines [Nakajima, Irimura and Nicolson (1988) J. Cell. Biochem. 36, 157-167]. To determine heparanase activity, porcine mucosal HS was partially de-N-acetylated and re-N-acetylated with [3H]acetic anhydride to yield a radiolabelled substrate. This procedure prevented the masking of, or possible formation of, new heparanase-sensitive cleavage sites as has been observed with previous methods of radiolabelling. Heparanase activity in a variety of tissues and cell homogenates including human platelets, colonic carcinoma cells, umbilical vein endothelial cells and rat mammary adenocarcinoma cells (both metastatic and non-metastatic variants) and liver homogenates all degraded the substrate in a stepwise fashion from 18.5 to approximately 13, 8 and finally to 4.5 kDa fragments, as assessed by gel-filtration analysis, confirming the substrate as suitable for the detection of heparanase activity present in a variety of cells and tissues. A rapid quantitative assay was developed with the HS substrate using a novel method for separating degradation products from the substrate by taking advantage of the decreased affinity of the heparanase-cleaved products for the HS-binding plasma protein chicken histidine-rich glycoprotein (cHRG). Incubation mixtures were applied to cHRG-Sepharose columns, with unbound material corresponding to heparanase-degradation products. Heparanase activity was determined for a variety of human, rat and murine cell and tissue homogenates. The highly metastatic rat mammary adenocarcinoma and murine lung carcinoma cell lines had four to ten times the heparanase activity of non-metastatic variants, confirming the correlation of heparanase activity with metastatic potential. Human cancer patients had twice the serum heparanase levels of normal healthy adults. The assay will be valuable for the determination of heparanase activity from a variety of tissue and cell sources, as a diagnostic tool for the determination of heparanase potential, and for the development of specific inhibitors of heparanase activity and metastasis.
硫酸乙酰肝素（HS）是细胞外基质和血管基底膜的重要组成部分，其作用是作为转移细胞和炎性细胞外渗的屏障。侵入细胞产生的内切糖苷酶或乙酰肝素酶活性对HS的切割可能有助于细胞外基质和基底膜的解体，从而促进细胞迁移。此前已表明，乙酰肝素酶活性与鼠类和人类黑色素瘤细胞系的转移潜能有关[中岛、入村和尼科尔森（1988年）《细胞生物化学杂志》36卷，第157 - 167页]。为了测定乙酰肝素酶活性，将猪黏膜HS进行部分去N - 乙酰化，并用[3H]乙酸酐重新N - 乙酰化，以产生放射性标记的底物。该方法避免了如之前放射性标记方法所观察到的对新的乙酰肝素酶敏感切割位点的掩盖或可能形成。包括人血小板、结肠癌细胞、脐静脉内皮细胞和大鼠乳腺腺癌细胞（转移和非转移变体）以及肝匀浆在内的多种组织和细胞匀浆中的乙酰肝素酶活性，通过凝胶过滤分析评估，均以逐步方式将底物降解为从18.5 kDa至约13 kDa、8 kDa，最终至4.5 kDa的片段，证实该底物适用于检测多种细胞和组织中存在的乙酰肝素酶活性。利用乙酰肝素酶切割产物对HS结合血浆蛋白鸡富含组氨酸糖蛋白（cHRG）亲和力降低的特点，采用一种从底物中分离降解产物的新方法，开发了一种基于HS底物的快速定量测定法。将孵育混合物应用于cHRG - 琼脂糖柱，未结合的物质对应于乙酰肝素酶降解产物。测定了多种人、大鼠和小鼠细胞及组织匀浆中的乙酰肝素酶活性。高转移性大鼠乳腺腺癌细胞系和小鼠肺癌细胞系的乙酰肝素酶活性是非转移变体的4至10倍，证实了乙酰肝素酶活性与转移潜能的相关性。癌症患者血清中的乙酰肝素酶水平是正常健康成年人的两倍。该测定法对于从多种组织和细胞来源测定乙酰肝素酶活性、作为测定乙酰肝素酶潜能的诊断工具以及开发乙酰肝素酶活性和转移的特异性抑制剂将具有重要价值。
