人白细胞黏附糖蛋白GP90（CD18）的大规模纯化及特性分析

Purification in large scale and characterization of the human leukocyte adhesion glycoprotein GP90 (CD18).

作者信息

Kantor C, Suomalainen-Nevanlinna H, Patarroyo M, Osterlund K, Bergman T, Jörnvall H, Schröder J, Gahmberg C G

机构信息

Department of Biochemistry, University of Helsinki, Finland.

出版信息

Eur J Biochem. 1988 Jan 4;170(3):653-9. doi: 10.1111/j.1432-1033.1988.tb13747.x.

DOI:10.1111/j.1432-1033.1988.tb13747.x

PMID:2892679

Abstract

The leukocyte adhesion 90-kDa glycoprotein GP90 (antigen CD18) is non-covalently associated separately with cell-surface glycoproteins GP160 (antigens CD11a, TA-1, LFA-1), GP155 (antigens CD11b, OKM1, MO1) or GP130 (antigens CD 11c, Leu-M5). Large amounts of these protein complexes were purified to homogeneity from blood mononuclear leukocytes by immunoaffinity chromatography using a monoclonal antibody. GP90 was further isolated by preparative gel electrophoresis in the presence of sodium dodecyl sulfate. Rabbit antiserum towards the complex inhibited phorbol-ester-induced adhesion of leukocytes. The antiserum towards purified GP90 reacted more strongly with the denatured GP90 protein, but showed reactivity also with GP160 protein, indicating structural homologies between GP90 and GP160. The amino acid composition of GP90 was determined. Its N-terminus was found to be blocked. Treatment of GP90 with endo-beta-N-acetylglucosaminidase F, but not with endo-beta-N-acetylglucosaminidase H, reduced the apparent molecular mass to 75 kDa, indicating the presence of five or six N-linked complex-type oligosaccharides/molecule.

摘要

白细胞黏附90-kDa糖蛋白GP90（抗原CD18）分别与细胞表面糖蛋白GP160（抗原CD11a、TA-1、淋巴细胞功能相关抗原-1）、GP155（抗原CD11b、OKM1、MO1）或GP130（抗原CD11c、Leu-M5）非共价结合。使用单克隆抗体通过免疫亲和层析从血液单核白细胞中大量纯化这些蛋白质复合物至均一状态。在十二烷基硫酸钠存在下，通过制备性凝胶电泳进一步分离GP90。针对该复合物的兔抗血清抑制佛波酯诱导的白细胞黏附。针对纯化的GP90的抗血清与变性的GP90蛋白反应更强，但也与GP160蛋白反应，表明GP90和GP160之间存在结构同源性。测定了GP90的氨基酸组成。发现其N端被封闭。用内切β-N-乙酰葡糖胺酶F处理GP90，但不用内切β-N-乙酰葡糖胺酶H处理，可使表观分子量降至75 kDa，表明每个分子存在五个或六个N-连接的复合型寡糖。