Positional and functional epitope mapping of endoglin, a proliferation-associated antigen of human endothelial cells.

摘要

Endoglin (EDG, CD105), a homodimeric glycoprotein, is a proliferation-associated cell membrane antigen of endothelial cells and strongly expressed on the angiogenic vascular endothelial cells of solid tumors. EDG is essential for angiogenesis and is an auxiliary TGF-β co-receptor. We have previously raised 12 anti-EDG monoclonal antibodies (mAbs), termed SN6 series mAbs, some of which were shown to inhibit angiogenesis and tumor growth. However, the molecular nature of the epitopes to which the mAbs bind remains to be clarified. Eight recombinant gene fragments representing different parts of EDG gene were prepared and expressed in E. coli. Reactivities of twelve SN6 series anti-EDG mAbs with the recombinant polypeptide fragments were analyzed by Western blot. The results allowed us to assign epitopes defined by individual mAbs to one of the seven regions of the extracellular domain of EDG. Region 2 (Ala94-Gly205) contained an epitope defined by mAb SN6h (SN6h epitope) and SN6k epitope while region 3 (Pro206-Glu251) contained SN6j epitope. Region 4 (Tyr252-Cys305) contained epitopes defined by SN6, SN6c, SN6d, SN6f and SN6i while region 7, the juxtamembrane region, (Gln426-Gly561) contained epitopes defined by SN6c, SN6h and SN6e. SN6g defines a carbohydrate epitope which is not synthesized in E. coli. SN6c and SN6k defined conformational epitopes, whereas the remaining nine mAbs defined linear sequence epitopes.; Anti-EDG mAbs were able to inhibit growth of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Differences in the suppression between mAbs defining different epitopes of EDG were observed. Antigen-binding avidities of the mAbs were not the dominant factor in the HUVEC suppression. TGF-β1 also inhibited HUVEC growth in a dose-dependent manner. The isobologram analysis showed that combination of TGF-β1 and each of the four anti-EDG mAbs (i.e., SN6, SN6a, SN6h and SN6j) exerted synergistic suppression of HUVECs. ADCC and apoptosis were induced in HUVEC by SN6j. Anti-EDG mAbs induced signal transduction in HUVECs by up-regulating the protein level of Smad4 and by phosphorylation of Smad2/3, which play key roles in the TGF-β-mediated signal transduction in the cells. Among the anti-EDG mAbs, SN6a and SN6h, which define epitopes in the juxtamembrane region, induced the strongest signaling. The present novel functions of EDG and anti-EDG mAbs will be useful for understanding the functions of EDG and the mechanisms by which anti-EDG mAbs modulate angiogenesis and tumor growth.; In summary, novel findings of the present study include (1) mapping of epitopes defined by twelve anti-EDG mAbs, (2) correlation of epitopes with function, (3) direct suppression of endothelial cells by anti-EDG mAbs, (4) synergy between anti-EDG mAbs and TGF-β in suppression of endothelial cells and (5) EDG/anti-EDG mAb mediated activation of TGF-β signal pathway. In addition, present results help selecting appropriate anti-EDG mAbs for different objectives such as clinical therapy and signal transduction studies.