Abstract
Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.
Highlights
Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells
With the help of two mAbs we have found a 55-kDa endothelial transmembrane protein, located at interendothelial cell contacts
endothelial cellselective adhesion molecule (ESAM) is located at tight junctions in close spatial relationship to ZO-1, occludin, and claudin-5, which is in contrast to what had been expected from published results on ESAM-transfected MDCK-II cells [27]
Summary
Antibodies—Monoclonal antibodies against mouse endothelial cell surface antigens were generated by immunizing rats with intact bEnd. mouse endothelioma cells and screening hybridoma supernatants for antibody binding in cell surface enzyme-linked immunosorbent assays as described [28]. All data base searches were performed using the program Pepsea (MDS Proteomics) This procedure led to the identification of ESTs that represent parts of the 1G8 sequence that were subsequently used for cloning. Based on these data, it was sufficient to perform MALDI mass spectrometry to identify the 4C10-immunoprecipitated protein as the 1G8 antigen. It was sufficient to perform MALDI mass spectrometry to identify the 4C10-immunoprecipitated protein as the 1G8 antigen This second approach was done by TopLab (Martinsried, Germany). The sequence and its expression in bEnd. cells were confirmed by amplifying the corresponding cDNA by reverse transcription-PCR from total RNA of bEnd. cells; using the sense primer (5Ј-GCG GGTACC CTC CCT GAG TAC TCC GGG CC-3Ј) and the antisense primer (5Ј-GCG AAGCTT ACA CAA GAG ACC CA CCT GAC T-3Ј) yielded a single 1.2-kilobase pair product that was subcloned into the pCR®2.1-TOPO vector (Invitrogen) after the addition of 3ЈA overhangs with Platinum Taq (Invitrogen)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
