Mapping epitopes to distinct regions of the extracellular domain of endoglin using bacterially expressed recombinant fragments

被引:63
作者
Pichuantes, S
Vera, S
Bourdeau, A
Pece, N
Kumar, S
Wayner, EA
Letarte, M
机构
[1] HOSP SICK CHILDREN,DIV IMMUNOL & CANC RES,TORONTO,ON M5G 1X8,CANADA
[2] CHIRON CORP,DEPT IMMUNODIAGNOST DEV,EMERYVILLE,CA 94608
[3] UNIV TORONTO,DEPT IMMUNOL,TORONTO,ON,CANADA
[4] UNIV MANCHESTER,DEPT PATHOL SCI,MANCHESTER M13 9PL,LANCS,ENGLAND
[5] FRED HUTCHINSON CANC RES CTR,DIV BIOL,SEATTLE,WA 98104
来源
TISSUE ANTIGENS | 1997年 / 50卷 / 03期
关键词
monoclonal antibody; endothelial cell; recombinant protein; transforming growth factor beta receptor;
D O I
10.1111/j.1399-0039.1997.tb02870.x
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Endoglin (CD105) is a homodimeric cell surface component of the TGF-beta 1 receptor complex, which is expressed at high levels on vascular endo-thelium and at lower levels on activated monocytes. It is also the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1. To date, each family has a distinct endoglin mutation, most of which generate premature stop codons. The purpose of the current study was to identify monoclonal antibodies capable of binding to normal and mutated forms of the protein. We generated stable transfectants of full-length human endoglin in murine fibroblasts and engineered and expressed in bacteria several fragments of the extracellular domain. Relatively pure polypeptides were recovered with good yield from inclusion bodies and were tested by ELISA and Western blot; 11 monoclonal antibodies were shown to react specifically with the endoglin transfectants. Ten of these mono-clonal antibodies reacted with the bacterial fragments, and their epitopes were assigned to 3 distinct regions of endoglin: Monoclonal antibodies P3D1, TEC4 and GRE reacted with the N-terminal region of 204 amino acids encoded by exons 1 to 5. Monoclonal antibodies P4A4, 44G4, E-9, MAEND3 and PN-E2 all bound to a region of 54 amino acids encoded mostly by exon 7. Monoclonal antibodies CLE4 and RMAC8 reacted with the C-terminal region of the extracellular domain, coded for by exons 8 to 12. Knowing the localization of these epitopes will facilitate the structural and functional analysis of normal and mutated forms of endoglin.
引用
收藏
页码:265 / 276
页数:12
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