MAPPING EPITOPES TO DISTINCT REGIONS OF THE EXTRACELLULAR DOMAIN OF ENDOGLIN USING BACTERIALLY EXPRESSED RECOMBINANT FRAGMENTS

Endoglin (CD105) is a homodimeric cell surface component of the TGF-be ta 1 receptor complex, which is expressed at high levels on vascular e ndo-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 distinc t endoglin mutation, most of which generate premature stop codons. The purpose of the current study was to identify monoclonal antibodies ca pable of binding to normal and mutated forms of the protein. We genera ted stable transfectants of full-length human endoglin in murine fibro blasts and engineered and expressed in bacteria several fragments of t he extracellular domain. Relatively pure polypeptides were recovered w ith good yield from inclusion bodies and were tested by ELISA and West ern blot; 11 monoclonal antibodies were shown to react specifically wi th the endoglin transfectants. Ten of these mono-clonal antibodies rea cted 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 e xons 1 to 5. Monoclonal antibodies P4A4, 44G4, E-9, MAEND3 and PN-E2 a ll bound to a region of 54 amino acids encoded mostly by exon 7. Monoc lonal antibodies CLE4 and RMAC8 reacted with the C-terminal region of the extracellular domain, coded for by exons 8 to 12. Knowing the loca lization of these epitopes will facilitate the structural and function al analysis of normal and mutated forms of endoglin.