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-beta 1 receptor complex, which is expressed at high levels on vascular endothelium 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 monoclonal 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.
Pichuantes
S
S
Department of Immunodiagnostics Development, Chiron Corporation, Emeryville, California, USA.
Vera
S
S
Bourdeau
A
A
Pece
N
N
Kumar
S
S
Wayner
E A
EA
Letarte
M
M
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015703:Antigens, CD; D019281:Dimerization; D000071063:Endoglin; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D010446:Peptide Fragments / Q000096:biosynthesis; D011485:Protein Binding; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C064206:ENG protein, human; D000071063:Endoglin; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D019010:Vascular Cell Adhesion Molecule-1