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

2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês

10.1074/jbc.m111999200

ISSN

1083-351X

Autores

Ines Nasdala, Karen Wolburg‐Buchholz, Hartwig Wolburg, Annegret Kuhn, Klaus Ebnet, Gertrud Brachtendorf, Ulrike Samulowitz, Bernhard Küster, Britta Engelhardt, Dietmar Vestweber, Stefan Butz,

Tópico(s)

Cancer-related molecular mechanisms research

Resumo

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 β-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. 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 β-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. monoclonal antibody Madin-Darby canine kidney endothelial cell-selective adhesion molecule glutathioneS-transferase matrix-assisted laser desorption/ionization expressed sequence tag mass spectrometry Chinese hamster ovary phosphate-buffered saline Tris-buffered saline high endothelial venule(s) The integrity of interendothelial cell contacts is vital for the physiological role of the endothelium as the interface between the blood and tissue structures. The control of vascular permeability, leukocyte extravasation, and the formation and outgrowth of blood vessels are dependent on the opening and closure or the dissociation and formation of interendothelial cell junctions (1Dejana E. Lampugnani M.G. Martinez-Estrada O. Bazzoni G. Int. J. Dev. Biol. 2000; 44: 743-748PubMed Google Scholar, 2Vestweber D. J. Pathol. 2000; 190: 281-291Crossref PubMed Scopus (114) Google Scholar). Adherens junctions are essential for the integrity of endothelial cell contacts, and VE-cadherin, the most prominent transmembrane protein of adherens junctions, is directly involved in the maintenance of these contactsin vitro as well as in vivo (3Lampugnani M.G. Resnati M. Raiteri M. Pigott R. Pisacane A. Houen G. Ruco L.P. Dejana E. J. Cell Biol. 1992; 118: 1511-1522Crossref PubMed Scopus (550) Google Scholar, 4Gotsch U. Borges E. Bosse R. Böggemeyer E. Simon M. Mossmann H. Vestweber D. J. Cell Sci. 1997; 110: 583-588Crossref PubMed Google Scholar, 5Corada M. Mariotti M. Thurston G. Smith K. Kunkel R. Brockhaus M. Lampugnani M.G. Martin-Padura I. Stoppacciaro A. Ruco L. McDonald D.M. Ward P.A. Dejana E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9815-9820Crossref PubMed Scopus (559) Google Scholar). Paracellular permeability across endothelial or epithelial cell layers is determined by tight junctions that seal the intercellular space (6Anderson J.M. Van Itallie C.M. Am. J. Physiol. 1995; 269: G467-G475Crossref PubMed Google Scholar). Tight junctions show ion and size selectivity, and their barrier function varies in tightness between epithelial and endothelial cells and also between endothelia in different types of blood vessels and different tissues (7Kniesel U. Wolburg H. Cell. Mol. Neurobiol. 2000; 20: 57-76Crossref PubMed Scopus (431) Google Scholar). To understand how the paracellular permeability of tight junctions is regulated and how they are formed, we need to know their molecular composition. Several proteins have been identified that are associated with the cytoplasmic side of tight junctions, but only a few tight junction proteins are known that span the membrane (8Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-292Crossref PubMed Scopus (2041) Google Scholar). Occludin was the first tight junction membrane protein that was identified. It contains four transmembrane domains (9Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2129) Google Scholar) and is found in epithelia as well as endothelia. It is likely to be a functional component of tight junctions, although junctional strands and functional tight junctions also form in the absence of the occludin gene (10Saitou M. Fujimoto K. Doi Y. Itoh M. Fujimoto T. Furuse M. Takano H. Noda T. Tsukita S. J. Cell Biol. 1998; 141: 397-408Crossref PubMed Scopus (475) Google Scholar). By contrast, members of the newly discovered gene family of the claudins are indeed essential for tight junction formation and can lead to the formation of tight junction strands upon transfection into fibroblasts (11Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1715) Google Scholar, 12Furuse M. Sasaki H. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 143: 391-401Crossref PubMed Scopus (791) Google Scholar, 13Tsukita S. Furuse M. Trends Cell Biol. 1999; 9: 268-273Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). There are presently some 20 members of this family known. They are small tetraspanning membrane proteins with short cytoplasmic N and C termini. Some of the claudins have highly restricted expression patterns. Claudin-5 was found to be expressed by endothelia in a large range of blood vessels in different tissues including muscle and brain (14Morita K. Sasaki H. Furuse M. Tsukita S. J. Cell Biol. 1999; 147: 185-194Crossref PubMed Scopus (696) Google Scholar). The third type of tight junction-associated membrane proteins is represented by the immunoglobulin supergene family (IgSF) member JAM (junctional adhesion molecule), a protein with two V-type Ig domains (15Martin-Padura I. Lostaglio S. Schneemann M. Williams L. Romano M. Fruscella P. Panzeri C. Stoppacciaro A. Ruco L. Villa A. Simmons D. Dejana E. J. Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1147) Google Scholar). JAM is expressed by endothelial as well as epithelial cells and was found to be enriched at tight junctions. JAM was also identified on mouse platelets and dendritic cells (16Malergue F. Galland F. Martin F. Mansuelle P. Aurrand-Lions M. Naquet P. Mol. Immunol. 1998; 35: 1111-1119Crossref PubMed Scopus (87) Google Scholar) and on human neutrophils, monocytes, subsets of lymphocytes, platelets, and red blood cells (17Williams L.A. Martin-Padura I. Dejana E. Hogg N. Simmons D.L. Mol. Immunol. 1999; 36: 1175-1188Crossref PubMed Scopus (155) Google Scholar), (18Liu Y. Nusrat A. Schnell F.J. Reaves T.A. Walsh S. Pochet M. Parkos C.A. J. Cell Sci. 2000; 113: 2363-2374Crossref PubMed Google Scholar). A mAb1 against JAM can block the extravasation of myeloid cells in two mouse inflammation models (15Martin-Padura I. Lostaglio S. Schneemann M. Williams L. Romano M. Fruscella P. Panzeri C. Stoppacciaro A. Ruco L. Villa A. Simmons D. Dejana E. J. Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1147) Google Scholar, 19De Del Maschio A. Luigi A. Martin-Padura I. Brockhaus M. Bartfai T. Fruscella P. Adorini L. Martino G. De Furlan R. Simoni M.G. Dejana E. J. Exp. Med. 1999; 190: 1351-1356Crossref PubMed Scopus (255) Google Scholar). It is not yet known whether the inhibitory effect of the antibody is due to interference with control mechanisms that regulate the opening of interendothelial cell contacts or whether it is due to the inhibition of leukocyte-endothelial interactions possibly mediated by JAM. The antibody does not change paracellular permeability of endothelial cell monolayers; however, another mAb against human JAM was reported to inhibit transepithelial resistance recovery in epithelial cell monolayers (18Liu Y. Nusrat A. Schnell F.J. Reaves T.A. Walsh S. Pochet M. Parkos C.A. J. Cell Sci. 2000; 113: 2363-2374Crossref PubMed Google Scholar). In combination with the recently found association of JAM with the PDZ domain protein ASIP/PAR-3 (20Ebnet K. Suzuki A. Horikoshi Y. Hirose T. Meyer-zu-Brickwedde M.-K. Ohno S. Vestweber D. EMBO J. 2001; 20: 3738-3748Crossref PubMed Scopus (327) Google Scholar), an essential cytoplasmic factor for the establishment of cell polarity, it is conceivable that JAM is involved in the regulation or formation of tight junctions. Recently two JAM-related proteins were identified, each containing one V and one C2 type Ig domain (21Palmeri D. van Zante A. Huang C.C. Hemmerich S. Rosen S.D. J. Biol. Chem. 2000; 275: 19139-19145Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 22Cunningham S.A. Arrate M.P. Rodriguez J.M. Bjercke R.J. Vanderslice P. Morris A.P. Brock T.A. J. Biol. Chem. 2000; 275: 34750-34756Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 23Aurrand-Lions M. Duncan L. Ballestrem C. Imhof B.A. J. Biol. Chem. 2001; 276: 2733-2741Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), for which the names JAM-2 and JAM-3 (24Aurrand-Lions M.A. Du Duncan L. Pasquier L. Imhof B.A. Curr. Top. Microbiol. Immunol. 2000; 251: 91-98PubMed Google Scholar, 25Arrate M.P. Rodriguez J.M. Tran T.M. Brock T.A. Cunningham S.A. J. Biol. Chem. 2001; 276: 45826-45832Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) or VE-JAM (21Palmeri D. van Zante A. Huang C.C. Hemmerich S. Rosen S.D. J. Biol. Chem. 2000; 275: 19139-19145Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) have been suggested. The subcellular localization at junctions was analyzed for JAM-1 by immunogold electron microscopy (15Martin-Padura I. Lostaglio S. Schneemann M. Williams L. Romano M. Fruscella P. Panzeri C. Stoppacciaro A. Ruco L. Villa A. Simmons D. Dejana E. J. Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1147) Google Scholar), revealing its localization at tight junctions and even its close spatial relationship with tight junction strands (26Itoh M. Sasaki H. Furuse M. Ozaki H. Kita T. Tsukita S. J. Cell Biol. 2001; 154: 491-497Crossref PubMed Scopus (321) Google Scholar). Based on confocal laser scanning microscopy, JAM-2 was reported to be enriched at the apical site of intercellular contacts of transfected MDCK cells, suggesting its potential to be targeted to the area of tight junctions (23Aurrand-Lions M. Duncan L. Ballestrem C. Imhof B.A. J. Biol. Chem. 2001; 276: 2733-2741Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). As judged by immunohistochemistry, this JAM was restricted to endothelial cells in sections of mouse kidney and lymph nodes (23Aurrand-Lions M. Duncan L. Ballestrem C. Imhof B.A. J. Biol. Chem. 2001; 276: 2733-2741Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). VE-JAM was reported to be absent from human leukocytes and from epithelia in several human tissues and was found on endothelial cells of various blood vessels (21Palmeri D. van Zante A. Huang C.C. Hemmerich S. Rosen S.D. J. Biol. Chem. 2000; 275: 19139-19145Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Based on a classical mAb approach, we have found two endothelially specific mAbs with which we identified a 55-kDa protein at interendothelial cell contacts. Identification of the purified protein by mass spectrometry revealed that it is an Ig-SF member containing one V-type and one C2-type Ig domain that is related to the JAMs although different in several aspects from the three known members of this family. Our antigen is identical to the recently described endothelial cell-selective adhesion molecule (ESAM) whose endothelial specificity has been analyzed so far on the RNA level by in situhybridization (27Hirata K. Ishida T. Penta K. Rezaee M. Yang E. Wohlgemuth J. Quertermous T. J. Biol. Chem. 2001; 276: 16223-16231Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Analyzing a panel of mouse tissues by immunohistochemistry, we show here that the ESAM protein is indeed specifically expressed on endothelium and not on epithelium. Although a tagged recombinant form of ESAM had been reported not to colocalize with ZO-1 in transfected MDCK-II cells (27Hirata K. Ishida T. Penta K. Rezaee M. Yang E. Wohlgemuth J. Quertermous T. J. Biol. Chem. 2001; 276: 16223-16231Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), we found that native ESAM does colocalize with ZO-1 but not with β-catenin in transfected MDCK-C7 cells. More importantly, ESAM was clearly colocalized with the three tight junction markers occludin, claudin-5, and ZO-1 in brain and muscle blood capillaries, as documented by immunogold electron microscopy. ESAM was not found on peripheral blood mouse leukocytes but was strongly expressed on megakaryocytes and activated platelets. The association of ESAM with endothelial tight junctions and its ability to support homophilic adhesion between transfected CHO cells suggest that ESAM might be involved in the control of interendothelial cell contacts. Monoclonal antibodies against mouse endothelial cell surface antigens were generated by immunizing rats with intact bEnd.3 mouse endothelioma cells and screening hybridoma supernatants for antibody binding in cell surface enzyme-linked immunosorbent assays as described (28Morgan S.M. Samulowitz U. Darley L. Simmons D.L. Vestweber D. Blood. 1999; 93: 165-175Crossref PubMed Google Scholar). Positive antibodies were further screened for cell contact staining by immunofluorescence and for endothelial specificity by immunohistochemistry (see below). In this way two antibodies were selected: 1G8 (IgG2a) and 4C10 (IgG2a). Rabbit antibodies against the extracellular domain of ESAM (VE-19) were raised against an ESAM-IgG fusion protein, and rabbit antibodies against the cytoplasmic domain of ESAM (VE-2) were raised against a GST fusion protein containing the cytoplasmic domain of ESAM except for the last 21 C-terminal amino acids. To purify the polyclonal antibodies, non-IgG proteins were removed from the sera by caprylic acid precipitation. Antigen-specific antibodies of serum VE-19 were affinity-purified on ESAM-IgG immobilized on CNBr-Sepharose (Amersham Biosciences), and antibodies against the IgG1 Fc part were removed by incubation with immobilized human IgG1. Antibodies from antiserum VE-2 were affinity-purified on CNBr-Sepharose-immobilized MBP-ESAM fusion protein. The following antibodies were commercially obtained: mouse mAb clone 14 (IgG1) against β-catenin (Transduction Laboratories, Lexington, KY); rat mAb RAM 34 (IgG2a) against mouse CD34 (Pharmingen, Heidelberg, Germany); rat mAb MECA-79 (29Streeter P.R. Rouse B.T.N. Butcher E.C. J. Cell Biol. 1988; 107: 1853-1862Crossref PubMed Scopus (538) Google Scholar) against peripheral node addressin (ATCC, Manassas, VA); rabbit polyclonal antibodies against ZO-1 and occludin (Zymed Laboratories Inc., San Francisco, CA); rabbit serum against von Willebrand factor (DAKO, Hamburg, Germany); labeled secondary antibodies (Cy3-conjugated goat anti-rat from Jackson Immunoresearch Lab., Inc. West Grove, PA; Alexa goat anti-rabbit from MoBiTec, Göttingen, Germany); gold-conjugated antibodies: goat anti-rat 5 nm, goat anti-rabbit 10 nm, and 15 nm (British Biocell Int., Cardiff, UK). Generation and affinity purification of rabbit polyclonal antibodies against mouse claudin-5 have been described (30Liebner S. Fischmann A. Rascher G. Duffner F. Grote E.H. Kalbacher H. Wolburg H. Acta Neuropathol. 2000; 100: 323-331Crossref PubMed Scopus (372) Google Scholar). Confluent monolayers of bEnd.3 cells were rinsed two times with phosphate-buffered saline, collected by scraping in the same buffer supplemented with 2 mm dithiothreitol and protease inhibitors (2 mmphenylmethylsulfonyl fluoride, 2 μg/ml pepstatin A, 20 μg/ml aprotinin, 20 μg/ml leupeptin), harvested by centrifugation, and extracted in lysis buffer (20 mm imidazole, pH 6.8, 100 mm NaCl, 5 mm EDTA, 1 mmdithiothreitol, 0.5% Triton X-100, 1 mmphenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 10 μg/ml aprotinin, 10 μg/ml leupeptin). The lysates were clarified by two consecutive centrifugation steps (30,000 × g for 20 min; 90,000 × g for 2 h). The cell lysates were incubated with mAb 1G8 or 4C10 immobilized to CNBr-Sepharose. The immunocomplexes were washed four times with lysis buffer (protease inhibitors omitted), subjected to SDS-PAGE, and analyzed by silver staining. Gel-separated proteins were reduced, alkylated, and digested in gel according to Ref. 31Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7822) Google Scholar and subsequently identified by a two-tier mass spectrometric approach. In the first round, small aliquots (1–2%) of the generated peptide mixtures were analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on a Bruker REFLEX III (Bruker Daltonik, Bremen, Germany) to yield a peptide mass map (32Jensen O.N. Podtelejnikov A.V. Mann M. Anal. Chem. 1997; 69: 4741-4750Crossref PubMed Scopus (244) Google Scholar). A list of monoisotopic peptide masses was obtained from the spectrum, and this list was used to query a nonredundant protein sequence data base (NRDB, >650,000 entries) at the European Bioinformatics Institute (Hinxton, UK). Correlating the measured peptide masses with theoretical digests of all proteins present in the data base did not lead to the identification of a protein. Therefore, the isolated protein was subjected to nanoelectrospray tandem mass spectrometry (33Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Nature. 1996; 379: 466-469Crossref PubMed Scopus (1507) Google Scholar). Protein digests were desalted and concentrated using microcolumns packed with approximately 100 nl of POROS R2 perfusion chromatography material (Perseptive Biosystems, Framingham, MA). Peptides were eluted with 60% MeOH/5% HCOOH directly into nanospray capillaries (MDS Proteomics, Odense, Denmark). Peptide sequencing by tandem mass spectrometry was performed using a nanoelectrospray ion source (MDS Proteomics) coupled to a quadrupole time-of-flight mass spectrometer (QSTAR, Sciex, Toronto, Canada). The sequence and mass information contained in the tandem mass spectra were assembled into peptide sequence tags (34Mann M. Wilm M. Anal. Chem. 1994; 66: 4390-4399Crossref PubMed Scopus (1317) Google Scholar) and used for queries in the NRDB and dbEST (expressed sequence tags data base, NCBI). Proteins/ESTs were identified by comparing the retrieved sequences with the mass spectrometric data. 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. This second approach was done by TopLab (Martinsried, Germany). Mass spectrometric data of immunoisolated proteins were applied in EST data base searches, ESTs of interest were sequenced, and the retrieved sequences were aligned with each other, with mouse genomic sequences, or with sequences of homologous proteins. This led to the identification of an EST clone containing an open reading frame of 1185 bp coding for a protein sequence that was covered to 49% by all MS generated sequence data. The sequence and its expression in bEnd.3 cells were confirmed by amplifying the corresponding cDNA by reverse transcription-PCR from total RNA of bEnd.3 cells; using the sense primer (5′-GCG GGTACC CTC CCT GAG TAC TCC GGG CC-3′) and the antisense primer (5′-GCG AAGCTT ACA CAA GAG ACC CA CCT GAC T-3′) yielded a single 1.2-kilobase pair product that was subcloned into the pCR®2.1-TOPO vector (Invitrogen) after the addition of 3′A overhangs with Platinum Taq (Invitrogen). DNA sequence analysis on an ABI-377 automated DNA sequencer (Applied Biosystems, Foster City, CA) confirmed that the EST contained start and stop codons. Full-length ESAM eukaryotic expression vectors were constructed by cloning theEcoRI/XbaI insert of EST (AA472099) into corresponding sites of pCMV5 (pCMV5-ESAM) and pcDNA3 (pcDNA3-ESAM). C-terminally truncated ESAM (amino acid residues 1–389, referred to as pcDNA3-ESAM antigen/Δ5) was cloned by PCR using pcDNA3-ESAM as template and the sense primer (5′-GCG CCA TGG GAA GCA AGA CCT TGG AAG AGC TG-3′) and antisense primer (5′-GCG TCT AGA CTA CTG ACT CTG TGC AGG CAC C-3′). The PCR product was subcloned into the pCR®2.1-TOPO vector (Invitrogen) after the addition of 3′A overhangs with platinum Taq (Invitrogen). After digestion of the plasmid with EcoRV/XbaI, the insert was ligated into corresponding sites of pcDNA-ESAM. Expression vectors encoding fragments of murine ZO-1 (ZO-1/1–3, ZO-1/6–1256), AF-6 (AF-6/full-length), and ASIP (ASIP/full-length) were described previously (20Ebnet K. Suzuki A. Horikoshi Y. Hirose T. Meyer-zu-Brickwedde M.-K. Ohno S. Vestweber D. EMBO J. 2001; 20: 3738-3748Crossref PubMed Scopus (327) Google Scholar, 35Ebnet K. Schulz C.U. Meyer-zu-Brickwedde M.-K. Pendl G.G. Vestweber D. J. Biol. Chem. 2000; 275: 27979-27988Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Expression vectors encoding the PDZ domains of ZO-2 (ZO-2/1–3) and ZO-3 (ZO-3/1–3) were generated by subcloning amino acid residues 1–636 of ZO-2 or amino acid residues 1–495 of ZO-3 into pSecTag (Invitrogen). The cloned cDNAs encoding canine ZO-2 and ZO-3 were kindly provided by Dr. B. Stevenson. A cDNA fragment coding for the extracellular part of ESAM covering amino acid residues 1–249 (bp 1–747) was amplified from pCMV5-ESAM using the BamHI site containing sense oligonucleotide 5′-CG GGATCC ATG ATT CTT CAG GCT GGA AC-3′ and the EcoRI site containing antisense oligonucleotide 5′-G GAATTC ACTTACCT TTG GAC CCT GTC ATC ACG-3′. The product was inserted in a pcDNA3 vector in frame and upstream of a fragment of human IgG1 covering bases 553–1803 (hinge, CH2, CH3). Prokaryotic expression vectors encoding the C termini of JAM, claudin-1, and claudin-5 fused to GST were described previously (20Ebnet K. Suzuki A. Horikoshi Y. Hirose T. Meyer-zu-Brickwedde M.-K. Ohno S. Vestweber D. EMBO J. 2001; 20: 3738-3748Crossref PubMed Scopus (327) Google Scholar). GST-ESAM antigen was generated by cloning a cDNA fragment coding for the cytoplasmic part of ESAM (amino acid residues 278–394) into pGEX-KG (36Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1640) Google Scholar). The respective fragment was generated from total RNA of bEND.3 cells by reverse transcription-PCR using the XhoI site-containing sense primer (5′-GCG CTCGAG AGC AAG ACC TTG GAA GAG CTG-3′) and theHindIII site-containing antisense primer (5′-GCG AAGCTT ACA CAA GAG ACC CA CCT GAC T-3′). For generation of rabbit antibodies against the cytoplasmic part of ESAM, prokaryotic GST and MBP (mannose-binding protein) fusion proteins were generated that contained the truncated cytoplasmic part of ESAM lacking the 21 C-terminal amino acids, which show high homology to mCAR. For construction of GST-ESAM-21 and MBP-ESAM-21 (amino acid residues 278–373), the respective fragment of ESAM was amplified by polymerase chain reaction using the XhoI site-containing sense primer (5′-GCG CTCGAG AGC AAG ACC TTG GAA GAG CTG-3′) and theHindIII site-containing antisense primer (5′-GCG AAGCTT AAG CAG AAG AAG AAA CCC CAC C-3′) and subcloned into the pGEX-KG vector or into the pMal-c2 vector, respectively (New England Biolabs, Frankfurt, Germany). The following cells were used: murine endothelioma bEnd.3 derived from brain capillaries (37Williams R.L. Risau W. Zerwes H.G. Drexler H. Aguzzi A. Wagner E.F. Cell. 1989; 57: 1053-1063Abstract Full Text PDF PubMed Scopus (223) Google Scholar) provided by Dr. Werner Risau (Max-Planck-Institute for Physiological and Clinical Research, Bad Nauheim, Germany); murine endothelioma myEnd from mouse myocard, a gift from Drs. N. Golenhofen and D. Drenckhahn (Julius-Maximilians-University, Würzburg, Germany); and murine rectal carcinoma CMT, provided by Dr. Rolf Kemler (Max-Planck-Institute for Immunobiology, Freiburg, Germany). All of these cells as well as the myeloma cell line SP2/0 and the hybridomas were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mml-glutamine (for myEnd, additionally 1 mm sodium pyruvate), and penicillin/streptomycin (Invitrogen) at 37 °C in a 10% CO2 atmosphere. MDCK C7 cells representing a high resistance subclone of MDCK cells (38Gekle M. Wunsch S. Oberleithner H. Silbernagl S. Pfluegers Arch. Eur. J. Physiol. 1994; 428: 157-162Crossref PubMed Scopus (173) Google Scholar, 39Wunsch S. Gekle M. Kersting U. Schuricht B. Oberleithner H. J. Cell. Physiol. 1995; 164: 164-171Crossref PubMed Scopus (54) Google Scholar) provided by Dr. Hans Oberleithner (University of Münster, Germany) were cultured in minimum essential medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum and penicillin/streptomycin. Stable transfectants of MDCK C7, expressing full-length ESAM or C-terminally truncated ESAM (ESAM/Δ5) were 5selected and cultured in medium as described above containing 800 μg/ml G418 (PAN Biotech, Aidenbach, Germany). CHO dhfr− cells were maintained in α-minimal essential medium supplemented with 10% fetal calf serum, 2 mml-glutamine, and penicillin/streptomycin. Stable transfected CHO dhfr− cells expressing full-length ESAM or ESAM-IgG were grown in α-minimal essential medium as described above supplemented with 800 μg/ml active G418. For the production of ESAM-IgG fusion protein, the same medium with ultralow IgG-fetal calf serum was used (Biochrom, Berlin, Germany). CHO dhfr− cells were transfected with 20 μg of ESAM-IgG plasmid or pcDNA3 full-length ESAM by electroporation. Stable transfectants were selected with 800 μg/ml G418 (PAN Biotech, Aidenbach, Germany) and maintained in the continuous presence of the selecting drug. Stable transfectants of MDCK C7 expressing full-length ESAM or C-terminally truncated ESAM (ESAM/Δ5) were generated using the same protocol. Mouse peripheral blood leukocytes were isolated and separated by density gradient centrifugation (Histopaque; Sigma) according to the manufacturer's instructions. Platelet-rich plasma was obtained from 1 ml of acid citrate dextrose-anticoagulated mouse blood, diluted 1:1 with PBS, pH 7.7, and centrifuged for 10 min at 40 × gat room temperature. The platelets were either left unstimulated or activated with thrombin (1 unit/ml) for 5 min at room temperature in the presence of glycine-proline-arginine-proline (1.25 mm) (Bachem) to prevent fibrin polymerization as described (40Frenette P. Denis C.V. Weiss L. Jurk K. Subbarao S. Kehrel B. Hartwig J.H. Vestweber D. Wagner D.D. J. Exp. Med. 2000; 191: 1413-1422Crossref PubMed Scopus (358) Google Scholar). The platelets were fixed with Cell Fix (Becton Dickinson, San Jose, CA) for 30 min at room temperature and washed with PBS. The staining was done with the following antibodies: RB40 (anti-mouse P-selectin), VE-2, VE-19 (rabbit polyclonal anti-mouse ESAM antibodies), 1G8, 4C10 (rat monoclonal anti-ESAM antibodies), fluorescein isothiocyanate-conjugated clone MWReg30 (rat IgG1 mAb against mouse CD41), fluorescein isothiocyanate-conjugated clone R3–34 (isotype standard rat IgG1), V7C7 (rat IgG2b mAb against mouse endomucin), and V1G5 (rat anti-mouse PECAM-1). All primary antibodies were used at a concentration of 10 μg/ml. As secondary antibodies dichlorotriazinyl amino fluorescein- and phycoerythrin-conjugated donkey anti-rat (H + L) and phycoerythrin-conjugated donkey anti-rabbit IgG (H + L) were used at a dilution of 1:100. All antibody incubations were performed for 30 min at room temperature. Cells metabolically labeled with [35S] methionine/cysteine were washed with phosphate-buffered saline and lysed directly in the culture dish in lysis buffer (see above). Insoluble material was removed by two consecutive centrifugation

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