| Abstract
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The 44G4 antigen, initially defined by a monoclonal antibody produced against the pre-B leukemic cell line HOON, is predominantly expressed by vascular endothelial cells in normal tissues. Studies were undertaken to determine if the 44G4 antigen represents a specific, novel marker of endothelial cells and to characterize the protein identified by monoclonal antibody 44G4. The following specific objectives were achieved: (1) the cellular localization of the 44G4 antigen, (2) the biochemical characterization of the 44G4 protein from the HOON cell line, and (3) the elucidation of the primary structure of the 44G4 protein, also known as endoglin. MAb 44G4 was strongly reactive with vascular endothelium of capillaries and small vessels in lymph node, tonsil, spleen, thymus, kidney, lung, liver, brain, and placenta. All other cell types observed in tissue sections, with the exception of scattered mononuclear cells in lymph node and kidney and syncytiotrophoblasts in the placenta, were negative. The 44G4 antigen was expressed at very low density by a subset of bone marrow cells, but was absent from peripheral blood granulocytes, monocytes, lymphocytes, and from PHA-stimulated T cells. Monoclonal antibody 44G4 reacted weakly with leukemic cells from a majority of patients with non-T acute lymphoblastic leukemia and acute myeloblastic leukemia, but not T cell acute lymphoblastic leukemia. Cell lines of pre-B and myeloblastic phenotype were also reactive with the 44G4 monoclonal antibody. Thus, MAb 44G4 defines an antigen which is virtually restricted to endothelial cells in all normal tissues except bone marrow; it is shared by placental syncytiotrophoblasts, and leukemic cells and cell lines of pre-B and myeloblastic cell phenotype. The antigens isolated from HOON leukemic cells and umbilical vein endothelial cells are similarly composed of two covalently linked subunits of apparent molecular weight 95,000. The 44G4 protein from HOON cells binds Ricinus communis, wheat germ, and peanut agglutinins, indicating the presence of N- and O-linked oligosaccharides. Treatment with glycosidases results in a loss in apparent molecular weight of 33,000. Monoclonal antibody 44G4 recognizes a conformational epitope of the 44G4 molecule of HOON cells. Endoglin/44G4 cDNA was cloned from an umbilical vein endothelial cell usd\lambdausdgt11 cDNA library using a rabbit antibody prepared against placental endoglin. The N-terminal sequence of placental endoglin was found within the amino acid sequence deduced from the cDNA, thus confirming its identity. The deduced endothelial cell endoglin polypeptide is a Type I integral membrane protein of molecular weight 68,051 with an extracellular domain of 561 amino acids, a membrane-spanning domain, and a cytoplasmic tail of 47 amino acid residues. The extracellular domain contains four potential N-linked glycosylation sites, a region of potential O-linked glycosylation, and the putative cell adhesion recognition tripeptide, Arg-Gly-Asp. Gene and protein sequence database searches and a comparison with properties of known endothelial cell surface proteins suggest that endoglin/44G4 is a newly described protein. Future studies will address the potential role of endoglin in RGD-mediated adhesive interactions of endothelial cells and leukocytes.
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