A Transmembrane Tight Junction Protein Selectively Expressed on Endothelial Cells and Platelets

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 ␤-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.

VE-PTP controls blood vessel development by balancing Tie-2 activity

“…After stopping confocal analysis, allantoides were fixed and immunolabeled with anti-PECAM-1 and phospho-Histone 3 antibodies as described previously (Baumer et al, 2006). domains 1-8 of VE-PTP, pAb D17 against the extracellular membrane proximal fibronectin type III-like domain of VE-PTP, mAb 3G1 against Tie-2 ( Koblizek et al, 1997), mAb against human Tie-2 (Millipore), pAb against Tie-1 (Santa Cruz Biotechnology, Inc.), pAb against phospho-Tie-2-Tyr992, pAb against phospho-Erk1/2-Thr202/Tyr204 pAb and mAb against Erk1/2, pAb against phospho-Akt-Ser473, pAb against Akt (Cell Signaling Technology), mAb 11D4.1 and pAb C5 against murine VE-cadherin (Gotsch et al, 1997), pAb against human VE-cadherin (Santa Cruz Biotechnology, Inc.), mAb 1G8 against mouse ESAM (Nasdala et al, 2002), pAb against human ESAM , mAb against PECAM-1 1G5.1 and 5D2.6 (Wegmann et al, 2006), mAb against plakoglobin (BD Biosciences), mAb 4G10 against phosphotyrosine (Millipore), and mAb and pAb against phospho-Histone H3-Ser10 (Millipore). Secondary antibodies were purchased from Dianova; Alexa 488-, Alexa 633-, and Alexa 568-coupled antibodies from Invitrogen; and DyLight680 and DyLight800-coupled antibodies from Thermo Fisher Scientific.…”

Novel uses for anti‐platelet agents as anti‐inflammatory drugs

“…The physiological function of JAMs on platelets remains unclear; however, it is plausible that these receptors play a part in platelet adhesion to the sub-endothelium (Nasdala et al, 2002). JAM-1 has been described as a counter-receptor for CD11a/CD18 (Ostermann et al, 2002).…”

Migration of immature mouse DC across resting endothelium is mediated by ICAM‐2 but independent of β2‐integrins and murine DC‐SIGN homologues

“…Other mAb were 3D5 (mouse anti-His-tag; Invitrogen), R35-95 (rat IgG2a isotype-control mAb; BD Pharmingen), 2G9 (rat anti-mMHC class II I-A d /I-E d ; BD Pharmingen), ER-TR9 (rat anti-mSIGN-R1; BMA Biomedicals, Augst, Switzerland), M17/4 (rat anti-mCD11a; BD Pharmingen), M1/70 (rat antimCD11b; BD Pharmingen), HL3 (hamster anti-mCD11c; BD Pharmingen), 2.4G2 (rat anti-mCD16/32; BD Pharmingen); GAME-46 (rat anti-mCD18; BD Pharmingen), 4C10 (rat antimouse endothelial cell-selective adhesion molecule [47]), YN1/1.7 (rat anti-mICAM-1; Acris Antibodies, Hiddenhausen, Germany), 3C4 (rat anti-mICAM-2; BD Pharmingen), 4RB12 (rat anti-mPSGL-1 [23]), mCD99-19.2 (rat anti-mCD99, own production; G. Bixel). The affinity-purified anti-mCD99 serum was described previously [25].…”