Immunoselection of GRP94/endoplasmin from a KNRK cell‐specific λGTLL library using antibodies directed against a putative heparanase amino‐terminal peptide
Immunoselection of GRP94/endoplasmin from a KNRK cell‐specific λGTLL library using antibodies directed against a putative heparanase amino‐terminal peptide
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使用针对假定的乙酰肝素酶氨基末端肽的抗体对 KNRK 细胞特异性 λGTLL 文库中的 GRP94/内质蛋白进行免疫选择
DOI：
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发表时间：
1994
影响因子：
6.4
通讯作者：
H. Birnboim
中科院分区：
文献类型：
--
作者：
Michael W. de Vouoe;A. Yamazaki;S. Bennett;Jiahua Chen;P. Shwed;Chantal Couture;H. Birnboim
转移性肿瘤细胞诱导侵袭性表型的部分原因是细胞外基质降解酶的不适当表达，这些酶通常参与胚胎形态发生、组织重塑、血管生成和伤口愈合。此类酶包括降解内皮基底膜中硫酸乙酰肝素（HS）的内切糖苷酶，以及更好表征的蛋白酶。乙酰肝素酶是一种最初在B16黑色素瘤细胞中检测到的内切-β-D-葡萄糖醛酸酶，被描述为Mr 96 000糖蛋白，pI为5.2，并已免疫定位于细胞表面和细胞质。我们利用基于聚丙烯酰胺凝胶的HS降解试验来证明KNRK（一种由v‐K‐ras转化的大鼠肾成纤维细胞系）表现出与B16 F10小鼠黑色素瘤细胞相似的HS降解活性。为了从KNRK细胞特异性λgtII cDNA文库中免疫选择乙酰肝素酶表达克隆，我们还制备了针对B16 F10细胞乙酰肝素酶的推定氨基末端肽的兔抗血清。来自一个克隆的溶原表达β-半乳糖苷酶融合蛋白，其与肽抗血清的染色通过与过量肽的竞争而被抑制。对该重组子的插入末端进行双脱氧介导的测序显示，它代表了Mr 94，000葡萄糖调节蛋白（GRP 94/胞浆内蛋白）的大鼠同源物，GRP 94/胞浆内蛋白是一种分子伴侣，含有先前归因于乙酰肝素酶的确切氨基末端序列。我们的结果质疑了该肽序列的特异性，以及先前使用此类抗血清进行的乙酰肝素酶免疫定位研究。
Induction of an invasive phenotype by metastatic tumour cells results in part from inappropriate expression of extracellular matrix‐degrading enzymes normally involved in embryonic morphogenesis, tissue remodelling, angiogenesis and wound healing. Such enzymes include endoglycosidases that degrade heparan sulfate (HS) in endothelial basement membrane, as well as better characterized proteases. Heparanase, an endo‐β‐D‐glucuronidase initially detected in B16 melanoma cells, has been described as a Mr 96 000 glycoprotein with pl of 5.2, and has been immunolocalized to the cell surface and cytoplasm. We have utilized a polyacrylamide‐gel‐based HS degradation assay to demonstrate that KNRK, a rat kidney fibroblast cell line transformed by v‐K‐ras, exhibits HS‐degrading activity similar to that of B16F10 mouse melanoma cells. To immuno‐select heparanase‐expressing clones from a KNRK‐cell‐specific λgtII cDNA library, we have also prepared a rabbit anti‐serum directed against a putative amino‐terminal peptide of B16F10 cellular heparanase. Lysogens from one clone expressed a β‐galactosidase fusion protein whose staining with peptide anti‐serum was inhibited by competition with excess peptide. Dideoxy‐mediated sequencing of the insert termini of this reco.mbinant revealed that it represents a rat homologue of Mr94,000 glucose‐regulated protein (GRP94/endoplasmin), a molecular chaperone that contains the exact amino‐terminal sequence previously attributed to heparanase. Our results call into question the specificity of this peptide sequence, as well as previous immunolocalization studies of heparanase carried out using such anti‐sera.
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DOI：
10.1016/s0021-9258(17)43350-3
发表时间：
1984-02
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
M. Nakajima;T. Irimura;N. Ferrante;G. Nicolson
通讯作者：
M. Nakajima;T. Irimura;N. Ferrante;G. Nicolson
DOI：
10.1172/jci112104
发表时间：
1985-10
期刊：
The Journal of clinical investigation
影响因子：
--
作者：
Y. Matzner;M. Bar‐Ner;J. Yahalom;R. Ishai‐Michaeli;Z. Fuks;I. Vlodavsky
通讯作者：
Y. Matzner;M. Bar‐Ner;J. Yahalom;R. Ishai‐Michaeli;Z. Fuks;I. Vlodavsky
乙酰肝素酶活性与转染各种癌基因的成纤维细胞转移潜能增加之间的关系。
影响因子：
9.7
作者：
Schwarz,LC;Inoue,T;Irimura,T;Damen,JE;Greenberg,AH;Wright,JA
通讯作者：
Wright,JA
影响因子：
13.8
作者：
VLODAVSKY, I;BARSHAVIT, R;FUKS, Z
通讯作者：
FUKS, Z
脱粒肥大细胞分泌一种糖苷内切酶，可降解内皮下细胞外基质中的硫酸乙酰肝素。
影响因子：
20.3
作者：
Bashkin,P;Razin,E;Eldor,A;Vlodavsky,I
通讯作者：
Vlodavsky,I