Characterization of CD1e, a Third Type of CD1 Molecule Expressed in Dendritic Cells
2000; Elsevier BV; Volume: 275; Issue: 48 Linguagem: Inglês
10.1074/jbc.m007082200
ISSN1083-351X
AutoresCatherine Angénieux, Jean Salamero, Dominique Fricker, Jean‐Pierre Cazenave, Bruno Goud, Daniel Hanau, Henri de la Salle,
Tópico(s)Immune Cell Function and Interaction
ResumoDendritic cells express several alternatively spliced CD1e mRNAs. These molecules encode proteins characterized by the presence of either one, two, or three α domains and either a 51- or 63-amino acid cytoplasmic domain. Moreover, mRNAs encoding isoforms lacking the transmembrane domain are observed. Several of these CD1e isoforms were expressed in transfected cells, and two of them, with three α domains, displayed a particular processing pathway. These latter isoforms slowly leave the endoplasmic reticulum due to the presence of atypical dilysine motifs in the cytoplasmic tail. These molecules are associated with the β2-microglobulin and accumulate in late Golgi and late endosomal compartments. In the latter compartments, they are cleaved into soluble forms that appear to be stable. In dendritic cells, these isoforms are mainly located in the Golgi apparatus, and upon maturation they are redistributed to late endosomal compartments. This work demonstrates the existence of CD1e molecules. As compared with other CD1 molecules, CD1e displays fundamentally different properties and therefore may represent a third type of CD1 molecules. Dendritic cells express several alternatively spliced CD1e mRNAs. These molecules encode proteins characterized by the presence of either one, two, or three α domains and either a 51- or 63-amino acid cytoplasmic domain. Moreover, mRNAs encoding isoforms lacking the transmembrane domain are observed. Several of these CD1e isoforms were expressed in transfected cells, and two of them, with three α domains, displayed a particular processing pathway. These latter isoforms slowly leave the endoplasmic reticulum due to the presence of atypical dilysine motifs in the cytoplasmic tail. These molecules are associated with the β2-microglobulin and accumulate in late Golgi and late endosomal compartments. In the latter compartments, they are cleaved into soluble forms that appear to be stable. In dendritic cells, these isoforms are mainly located in the Golgi apparatus, and upon maturation they are redistributed to late endosomal compartments. This work demonstrates the existence of CD1e molecules. As compared with other CD1 molecules, CD1e displays fundamentally different properties and therefore may represent a third type of CD1 molecules. CD1 molecules (reviewed in Ref. 1Porcelli S.A. Modlin R.L. Annu. Rev. Immunol. 1999; 17: 297-329Crossref PubMed Scopus (603) Google Scholar) are nonclassical major histocompatibility complex class I molecules, composed of a membrane-associated heavy chain comprising three immunoglobulin-like extracellular α domains, which associate with β2-microglobulin (β2m).1 The number ofCD1 genes varies among species, only two closely relatedCD1 genes having been described in mice but five different genes in humans. The human CD1A, -B, -C, -D, and -E genes are constitutively expressed in a limited number of cell types, including cortical thymocytes and dendritic cells (DCs), and they can be induced by granulocyte-macrophage colony-stimulating factor in monocytes. The CD1A, -B, and -C genes encode structurally and functionally related proteins and are classified as type I CD1 genes. CD1a, -b, and -c molecules are found in the plasma membrane and in the endosomal compartments of DCs. However, depending on the DC type and stage of maturation, these molecules display differences in terms of their intracellular localization and traffic (2Sugita M. Grant E.P. van Donselaar E. Hsu V.W. Rogers R.A. Peters P.J. Brenner M.B. Immunity. 1999; 11: 743-752Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). 2D. Hanau and J. Salamero, unpublished observations. These proteins can present glycolipids of microbial origin and, in the case of CD1b, also self-glycolipids (3Shamshiev A. Donda A. Carena I. Mori L. Kappos L. De Libero G. Eur. J. Immunol. 1999; 29: 1667-1675Crossref PubMed Scopus (251) Google Scholar). The presentation of glycolipids by CD1b is dependent on its internalization into acidic late endosomal compartments and recycling to the cell surface (2Sugita M. Grant E.P. van Donselaar E. Hsu V.W. Rogers R.A. Peters P.J. Brenner M.B. Immunity. 1999; 11: 743-752Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). On the basis of homology studies, CD1d has been proposed to represent a second type of CD1 molecule, and structural and functional studies have confirmed this classification. Notably, whereas association with β2m is required for the cell surface expression of type I CD1 molecules (4Bauer A. Huttinger R. Staffler G. Hansmann C. Schmidt W. Majdic O. Knapp W. Stockinger H. Eur. J. Immunol. 1997; 27: 1366-1373Crossref PubMed Scopus (37) Google Scholar), human β2m-free CD1d can be expressed at the plasma membrane as a nonglycosylated protein (5Balk S.P. Burke S. Polischuk J.E. Frantz M.E. Yang L. Porcelli S. Colgan S.P. Blumberg R.S. Science. 1994; 265: 259-262Crossref PubMed Scopus (147) Google Scholar). In polarized human epithelial cells, nonglycosylated β2m-free CD1d molecules are observed on the apical cell surface, while glycosylated CD1d molecules are present on both sides of the cells (6Somnay-Wadgaonkar K. Nusrat A. Kim H.S Canchis W.P. Balk S.P. Colgan S.P. Blumberg R.S. Int. Immunol. 1999; 11: 383-392Crossref PubMed Scopus (68) Google Scholar). In transfected melanoma cells, β2m-free CD1d molecules expressed at the cell surface are endoglycosidase H (Endo H)-sensitive, while β2m-associated CD1d molecules are Endo H-resistant (7Kim H.S. Garcia J. Exley M. Johnson K.W. Balk S.P. Blumberg R.S. J. Biol. Chem. 1999; 274: 9289-9295Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Thus, three biochemically different forms of CD1d molecules have been described in humans. In human intestinal epithelial cells, CD1d is expressed on the cell surface and internalized into endosomal compartments. This traffic is controlled by aYXXZ motif in the cytoplasmic tail, which is likewise found in CD1b and CD1c (1Porcelli S.A. Modlin R.L. Annu. Rev. Immunol. 1999; 17: 297-329Crossref PubMed Scopus (603) Google Scholar). The localization of CD1d in basolateral membranes is also regulated by the cytoplasmic domain (8Rodionov D.G. Nordeng T.W. Pedersen K. Balk S.P. Bakke O. J. Immunol. 1999; 162: 1488-1495PubMed Google Scholar). The murine molecules orthologous to CD1d are the CD1.1 and CD1.2 proteins. In mice, β2m-associated CD1 molecules are expressed at the surface of cells of different types including DCs, while in transfected cells murine CD1 is found on the plasma membrane and in endosomal compartments (9Mandal M. Chen X.R. Alegre M.L. Chiu N.M. Chen Y.H. Castano A.R. Wang C.R. Mol. Immunol. 1998; 35: 525-536Crossref PubMed Scopus (82) Google Scholar). There is indirect evidence that β2m-free CD1d molecules exist in mice in that CD1-restricted T cells can develop in aged β2m-deficient mice (10Murakami M. Paul W.E. J. Immunol. 1998; 160: 2649-2654PubMed Google Scholar). Murine CD1 molecules bind different kinds of hydrophobic antigens, including peptides with hydrophobic anchor residues, glycosylphosphatidylinositol, and ceramide-containing glycolipids, which are also presented by human CD1d molecules (11Castano A.R. Tangri S. Miller J.E. Holcombe H.R. Jackson M.R. Huse W.D. Kronenberg M. Peterson P.A. Science. 1995; 269: 223-226Crossref PubMed Scopus (226) Google Scholar, 12Joyce S. Woods A.S. Yewdell J.W. Bennink J.R. De Silva A.D. Boesteanu A. Balk S.P. Cotter R.J. Brutkiewicz R.R. Science. 1998; 279: 1541-1544Crossref PubMed Scopus (364) Google Scholar, 13Naidenko O.V. Maher J.K. Ernst W.A. Sakai T. Modlin R.L. Kronenberg M. J. Exp. Med. 1999; 190: 1069-1080Crossref PubMed Scopus (141) Google Scholar). The presentation of ceramide glycolipids by mouse CD1 or human CD1d antigens stimulates NKT cells, a T cell subpopulation expressing an invariant T cell receptor α chain and producing interleukin-4 and interferon-γ upon stimulation. In mice, the activation of NKT cells by CD1 proteins appears to play key role in the induction of systemic immune tolerance following immunization through an immune-privileged site (14Sonoda K.H. Exley M. Snapper S. Balk S.P. Stein-Streilein J. J. Exp. Med. 1999; 190: 1215-1226Crossref PubMed Scopus (318) Google Scholar), although the ligands of CD1 involved in this process are not known. At least in mice, CD1 molecules also stimulate other T cell subsets (15Chiu Y.H. Jayawardena J Weiss A Lee D Park S.H. Dautry-Varsat A Bendelac A. J. Exp. Med. 1999; 189: 103-110Crossref PubMed Scopus (251) Google Scholar). CD1e molecules have not been studied to date. The human CD1E gene was described more than 14 years ago and shown to be transcribed in Jurkat and Molt4 tumor T cell lines (16Martin L.H. Calabi F. Milstein C. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9154-9158Crossref PubMed Scopus (160) Google Scholar,17Woolfson A. Milstein C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6683-6687Crossref PubMed Scopus (37) Google Scholar), and several partially characterized transcripts were registered in GenBankTM. However, the existence of protein(s) encoded by the CD1E gene has not yet been demonstrated. The aims of the present work were thus to analyze the pattern of CD1e transcripts in DCs, to determine whether CD1E gene product(s) could direct the synthesis of proteins, and to define the different cellular and biochemical properties of CD1e molecules. Total RNA from DC were prepared using RNeasy extraction kit (Qiagen, Les Ullis, France). RNA was reverse transcribed with avian myeloblastosis virus reverse transcriptase (Eurogentec, Seraing, Belgium) using random hexanucleotides as primers. CD1e cDNAs were amplified using 100 ng of reverse transcribed RNA, Taq DNA polymerase (Goldstar, Eurogentec), and GGGGGATATCCTCCTTTAACAGAGCTTCA and ATTTGGGGAGTACAGAAGAG oligonucleotides (94 °C for 30 s, 56 °C for 30 s, and 72 °C for 2 min, 35 cycles). Amplified products were digested withEcoRV and EcoRI and cloned into the homologous restriction sites of pEGFP-N3 (CLONTECH, Palo Alto, CA). To express CD1e/CD1c or CD1c/CD1e fusion molecules, first a plasmid encoding CD1c with Eco47III and ScaI restriction sites located in the N-terminal and the C-terminal parts of the transmembrane domain, respectively, was constructed. This construction, obtained by polymerase chain reaction mutagenesis, encodes for the exact sequence of CD1c protein. Plasmids encoding CD1c (extracellular and transmembrane domains)/CD1e (part of the CD1e cytoplasmic domain) fusion molecules were then obtained by inserting fragments encoding part of the CD1e cytoplasmic domain and amplified by polymerase chain reaction in the ScaI site. Before cloning into theScaI site, the fragments encoding the N-terminal half of the long or short cytoplasmic domains (DSRLKKQSSNKNILSPHTPSPVFLMGANTQDTKN and DSRLKKQSPVFLMGANTQDTKN, respectively) were cut withEcoRI and treated with T4 DNA polymerase, producing a stop codon at the end of the fragments. The amino acid sequence of theEco-end fragment (C-terminal half of the CD1e cytoplasmic domain) fused to CD1c was NSRHQFCLAQVSWIKNRVLKKWKTRLNQLW. The plasmid pL213 encodes the first 305 amino acids of CD1e, four extra unrelated amino acids (DLEAK), and then the amino acids of the transmembrane and cytoplasmic domains of CD1c fused to eGFP. Natural CD1e isoforms were expressed by inserting the different reconstituted full-length CD1e cDNA clones downstream from the cytomegalovirus promotor of pEGFP-N3 expression vector. Plasmid cDNAs were transfected in cell lines using Fugene (Roche Diagnostics, Meylan, France) or Exgen (Euromedex, Schiltigheim, France) reagents. Stable M10 transfectants were isolated using 500 μg/ml G418 (Life Technologies, Paisley, UK). The transfected clones expressing the membrane-associated isoforms were selected by immunofluorescence staining on fixed and permeabilized cells using VIIC7 monoclonal antibody (mAb). Clones expressing pL213 were first selected using the fluorescence of eGFP. Fluorescence microscopy showed the hybrid molecule to be expressed on the cell surface. HeLa cells were obtained from ATCC (number CCL-2). The melanoma M10 cell line was kindly provided by Dr. T. Hercend (Villejuif, France). HeLa cells were grown in Dulbecco's culture medium, and M10 cells were grown in RPMI 1640, all supplemented with 10% fetal calf serum (Life Technologies). Monocyte-derived DCs and epidermal Langerhans cells (LCs) were prepared as previously (18Saudrais C. Spehner D. de la Salle H. Bohbot A. Cazenave J.-P. Goud B. Hanau D. Salamero J. J. Immunol. 1998; 160: 2597-2607PubMed Google Scholar, 19Hanau D. Fabre M. Schmitt D.A. Garaud J.C. Pauly G. Tongio M.-M. Mayer S. Cazenave J.-P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2901-2905Crossref PubMed Scopus (73) Google Scholar). Maturation of DCs was induced with 1 μg/mlEscherichia coli LPS (Sigma) or 30 ng/ml TNFα (R&D Systems, Abingdon, UK). Mature LCs were obtained by 48-h culture in RPMI 1640 supplemented with 10% fetal calf serum, 50 ng/ml granulocyte-macrophage colony-stimulating factor (generously provided by Novartis, Rueil Malmaison, France), and 30 ng/ml TNFα. The following mAbs were used: B1G6 (anti-β2m, IgG2a) (Immunotech, Marseille, France); mouse IgG1κ (anti-GFP, clones 7.1 and 13.1) (Roche Molecular Biochemicals); L161 (anti-CD1c, IgG1) (Immunotech); W6/32 (pan-anti-HLA class I, IgG2a) (Dako, Trappes, France); biotinylated goat anti-mouse IgG (Pharmingen, San Diego, CA); fluorescein isothiocyanate-conjugated F(ab′)2 goat anti-mouse IgG (Silenus, Melbourne, Australia); phycoerythrin-conjugated F(ab′)2 goat anti-mouse IgG (Dako), F(ab′)2 fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG and Cy3-conjugated F(ab′)2 donkey anti-mouse IgG (Jackson Immunoresearch, West Baltimore, PA); Alexa-594-conjugated goat anti-mouse IgGs and Alexa-488-conjugated goat anti-mouse IgG (Molecular Probes, Inc., Eugene, OR); and control IgG1 and IgG2a (Immunotech). H5C6 (IgG1, anti-CD63, was kindly provided by Dr F. Lanza, EFS-Alsace, Strasbourg) and the rabbit anti-EEA1 antiserum (20Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 39: 494-498Crossref Scopus (918) Google Scholar) by Dr. H. Stenmark (EMBL, Heidelberg, Germany). H5C6 was directly coupled to cyanin 3 using a Cy3 labeling kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Polyclonal rabbit IgGs against Rab-6 were produced and purified as described (21Martinez O. Schmidt A. Salamero J. Hoflack B. Roa M. Goud B. J. Cell Biol. 1994; 127: 1575-1588Crossref PubMed Scopus (221) Google Scholar). Polyclonal anti TGN-46 (22Prescott A.R. Lucocq J.M. James J. Lister J.M. Ponnambalam S. Eur. J. Cell Biol. 1997; 72: 238-246PubMed Google Scholar) and Lamp-1 Abs (23Karlsson K. Carlsson S.R. J. Biol. Chem. 1998; 273: 18966-18973Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) were obtained from Dr. J. Lucocq (University of Dundee, United Kingdom), and Dr S. Carlsson, (University of Umea, Sweden). The mAb VIIC7 (IgG1, anti-CD1e cytoplasmic domain) was obtained by immunizing mice with a synthetic peptide (YIKNRVLKKWKTRL, corresponding to amino acids −17/−5 of the cytoplasmic domain) coupled to keyhole limpet hemocyanin. Hybridomas were screened with a dot blot assay using a glutathione S-transferase-CD1e cytoplasmic domain fusion protein expressed in E. coli. The mAbs 1.2 (IgG1), 2.9 (IgG2a), and 20.6 (IgG1) (anti-CD1e α domains) were obtained by immunizing mice with an M10 cell line transfected with pL213. Hybridomas were screened by incubating untransfected or pL213-transfected M10 cells first with hybridoma supernatants and then with biotinylated anti-mouse Abs and finally with Cy-Chrome-conjugated streptavidin (Pharmingen). The specificities of these mAbs were determined using HeLa cells transfected with plasmids obtained in the cloning step. All membrane-associated CD1e isoforms were expressed as protein lacking the C-terminal end of the cytoplasmic domain (downstream from theEcoRI restriction site) fused to enhanced green fluorescent protein (eGFP) (CD1e ΔCyt-eGFP molecules). The cells were fixed, permeabilized, and incubated with antibody 1.22, 2.9, or 20.6 followed by Cy3-conjugated anti-mouse Abs. Simultaneous eGFP and Cy3 labeling revealed the specificities of the mAbs for the different membrane-associated isoforms of CD1e. Cells were washed in cold phosphate-buffered saline (PBS) and incubated with the relevant mAb in PBS for 30 min at 4 °C. In indirect staining experiments, the cells were incubated with fluorescein isothiocyanate- or phycoerythrin-conjugated goat anti-mouse IgG for 30 min at 4 °C. Controls included staining with an isotype-matched irrelevant Ab. In intracellular staining, the cells were first fixed with 1% paraformaldehyde in PBS for 15 min and washed in cold PBS, and the primary Ab was added in staining buffer (RPMI 1640, 5% normal goat serum, 0.2% sodium azide, 0.1% saponin) for 30 min at 4 °C. The cells were then washed twice in saponin buffer (0.1% saponin in PBS), and the secondary Ab was added in the same buffer. The cells were washed twice in saponin buffer and twice in PBS before analysis on a FACScan cytometer (Becton Dickinson). Immunofluorescence (IF) microscopy of fixed permeabilized DCs was carried out as described previously (18Saudrais C. Spehner D. de la Salle H. Bohbot A. Cazenave J.-P. Goud B. Hanau D. Salamero J. J. Immunol. 1998; 160: 2597-2607PubMed Google Scholar). Confocal laser scanning microscopy and IF analyses (24Salamero J. Le Borgne R. Saudrais C. Goud B. Hoflack B. J. Biol. Chem. 1996; 271: 30318-30321Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) were performed as described on a Leica TCS4D confocal microscope (Leica Lazer Tecknik, Heidelberg, Germany). Confluent 75-cm2 flasks of transfected or untransfected cells were washed twice in PBS and incubated for 1 h in 20 ml of methionine and cysteine-free medium supplemented with 10% dialyzed fetal calf serum and 1 mm glutamine. The cells were washed twice and labeled with 250 μCi of [35S] methionine and cysteine (Promix; Amersham Pharmacia Biotech) in 3 ml of medium for 30 min to 1 h. The reaction was stopped by the addition of ice-cold PBS followed by two washing steps. After chase in 20 ml of RPMI containing 10% fetal calf serum, the cells were lysed in 1 ml of lysis buffer (20 mm Tris, pH 8, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 1 mmphenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 2 μg/ml leupeptin) for 20 min on ice. Lysates were centrifuged at 20,000 × g for 15 min and were incubated twice with 50 μl of protein A-Sepharose (Amersham Pharmacia Biotech) for 2 h. Supernatants were incubated with protein A-Sepharose and 5 μg of mAb or irrelevant isotype-matched mAb for 2 h. After extensive washing, the immunoprecipitates were treated or not with Endo Hf or Endo F (Biolabs, Beverly, CA). Samples were separated on 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) gels under reducing conditions. Gels were treated with Amplify (Amersham Pharmacia Biotech) and exposed for autofluorography. Since humanCD1A, -B, and -C genes are expressed in DCs, we first tested whether this was the case for theCD1E gene. RNA from monocyte-derived DCs was reverse transcribed, and CD1e cDNA was amplified by polymerase chain reaction using oligonucleotides hybridizing with the 5′- and 3′-untranslated regions of the gene. Several fragments were co-amplified, digested with EcoRV (present in the sequence of the 5′ oligonucleotide) and EcoRI (present in the middle of the coding sequence in the 3′ exon), and cloned into the homologous restriction sites of the pEGFP-N3 expression vector. Sequence analysis of the clones revealed a multiplicity of alternatively spliced transcripts of CD1e. Among 45 clones analyzed, more than 15 alternatively spliced mRNA species could be characterized (Fig.1 A). Two mRNAs encode a membrane protein with three extracellular α domains, a transmembrane domain and either a long or a short cytoplasmic domain. The two cytoplasmic domains (CytL and CytS) are encoded by two alternatively spliced exon VI region, and both are quite long (63 and 51 amino acids, respectively) as compared with those of other CD1 molecules (6–10 amino acids). Two potential glycosylation sites are located in the α1 domain. Other forms include only one (α3) or two α domains (α2α3, or α1α3), the transmembrane domain and either the short or the long cytoplasmic domain. An increase in diversity results from the use of two alternative donor splicing sites from intron 4, which gives rise to mRNA sequences encoding molecules with truncated α3 domains. One of these forms retains only 37 amino acids of the 93 amino acids of the complete α3 domain (α3"). Since the reading frame is conserved, these molecules remain membrane-associated. The second alternative splicing (α3′) induces a frameshift in the fifth exon, which produces mRNAs encoding a molecule with 57 amino acids of the α3 domain and 24 additional amino acids and thus leads to a possible soluble secreted form. The N-terminal sequence of all isoforms is 10 amino acids longer than that deduced from the genomic sequence reported in GenBankTM (X14975). Only one clone retaining the 3′-end of the mRNA could be isolated, suggesting that no alternative splicing occurs downstream from the EcoRI restriction site. In order to study the localization of the different isoforms, several mAbs were selected and then tested on transfected cells expressing individual CD1e isoforms, fused or not with eGFP. The eGFP-fused molecules were examined first for practical reasons, as they could be expressed using the recombinant plasmids obtained in the cloning step. Membrane-associated CD1e isoforms expressed using these vectors were fused to eGFP at the EcoRI restriction site in the middle of the sequence encoding the cytoplasmic domain. Consequently, these constructions lacked the 28 C-terminal amino acids of the cytoplasmic domain encoded by the sequences downstream from theEcoRI restriction site and were designated using the "Δ Cyt" suffix. All isoforms were further designated by listing their different constitutive domains (Fig. 1 B; only a few representative isoforms are shown). A mouse mAb (VIIC7) was raised against a peptide of the CD1e cytoplasmic domain (at positions −5/−17). Additional experiments confirmed that this mAb could be used to follow the expression of membrane-associated CD1e isoforms in transfected cells by IF, immunoprecipitation, and Western blotting. 3C. Angénieux, unpublished observations. Abs specific for the α domains were obtained by constructing a plasmid encoding a hybrid protein comprising the three α domains of CD1e, fused to the transmembrane and intracellular domains of CD1c and to eGFP. M10 cells transfected with this expression vector were shown by IF microscopy and biochemical experiments to express the hybrid molecule on the cell surface. Mice were immunized with the transfected cells and hybridomas were prepared from one mouse. Three mAbs, named (1.22, 2.9, and 20.6) were selected, which stained the transfected cells but not untransfected M10 cells. The specificity of these mAbs was tested on HeLa cells transiently transfected with plasmids encoding the different isoforms. In these experiments, the membrane-associated molecules were expressed as CD1e ΔCyt-eGFP fusion proteins. The cells were fixed, permeabilized, stained with the different mAbs, and analyzed by IF microscopy (Table I). None of the mAbs stained transfected HeLa cells expressing the α2α3 isoform, while in IF studies the mAb 1.22 recognized all other isoforms, thus appearing to be specific for the α1 domain. The mAb 20.6 stained cells expressing the α1α2α3, α1α2α3′, and α1α2α3" isoforms hence was more selective than 1.22. Although the mAb 2.9 specifically recognized the three α domain isoforms, the intensity of the signals were poor relative to those of the other mAbs, and 2.9 was therefore inappropriate for IF staining. On the basis of eGFP fluorescence or mAb staining, the CD1e α1α2-, α2α3-, and α1α2α3"-ΔCyt-eGFP molecules appeared to be retained in the ER. In contrast, CD1e α1α2α3ΔCytL-eGFP were found on the cell surface and intracellularly.Figure 1CD1e alternatively spliced forms. A, schematic representation of human CD1e transcripts generated by alternative splicing in DCs derived from monocytes. Theupper part reproduces the organization of theCD1E gene. Boxes correspond to exons encoding successively the signal peptide (S), the three α domains, the transmembrane domain (TM), seven amino acids of the cytoplasmic domain (in gray, exon V), and the end of the cytoplasmic domain (Cyt). The segment of CytL that is absent from CytS is represented by an open square. Thehorizontal arrows indicate the positions of the forward and backward primers used for the reverse transcription-polymerase chain reaction. The EcoRI restriction site used for the cloning of CD1e cDNAs into the pEGFP-N3 expression vector is denoted (▴). Below this scheme are shown the membrane-associated and the soluble isoforms.Diagonal lines linking exons indicate splicing patterns. Alternative splicing of exon VI to give mRNA encoding proteins lacking the transmembrane domain ("soluble forms") is not shown, and the asterisk represents the stop codon.B, schematic representation of some recombinant CD1e molecules.View Large Image Figure ViewerDownload (PPT)Table ISpecificities of anti-α domain mAbsmAbα1α2α3α1α2α3′α1α2α3"α1α3α2α31.22++++++++++++−2.9+−−−−20.6+++++++++−−The specificities of the mAbs against the CDle α domains were tested by IF. HeLa cells were transfected with CD1e cDNA cloned into theEcoRV and EcoRI restriction sites of pEGFP-N3. 48 h after transfection, the cells were fixed, permeabilized, and incubated with the different mAbs and then with Cy3-conjugated anti-mouse IgG. Cells were analyzed using IF microscopy. Open table in a new tab The specificities of the mAbs against the CDle α domains were tested by IF. HeLa cells were transfected with CD1e cDNA cloned into theEcoRV and EcoRI restriction sites of pEGFP-N3. 48 h after transfection, the cells were fixed, permeabilized, and incubated with the different mAbs and then with Cy3-conjugated anti-mouse IgG. Cells were analyzed using IF microscopy. Additional experiments using transfected M10 cells showed that all three mAbs could be used to detect CD1e isoforms by immunoprecipitation and displayed identical specificity as in IF, whereas none of them could be used for Western blotting. Moreover, cytofluorimetry on transfected cells expressing either CD1a, -b, or -c molecules demonstrated that these mAbs do not react with the other CD1 molecules expressed by DCs.3 Preliminary IF experiments using the VIIC7 mAb showed that whereas CD1e α1α2α3ΔCyt-eGFP fusion molecules transiently expressed in HeLa cells could reach the plasma membrane, complete CD1e molecules expressed in transfected cells appeared to be retained in the ER (data not shown). This suggested that the 28 C-terminal amino acids were involved in the retention of the full-length CD1e molecules. Since initially we had no mAb against the α domains of CD1e, it was not possible to directly determine which part of the protein mediated its retention in the ER. Therefore, we constructed plasmids expressing hybrid molecules consisting of the extracellular and transmembrane domains of CD1c fused to different parts of the CD1e cytoplasmic domains. The transmembrane domain of CD1c was fused to the N-terminal part of the cytoplasmic domain of the CytL and CytS isoforms, encoded by sequences upstream from theEcoRI restriction site (CD1c-ΔCytL* and CD1c-ΔCytS*), or to the C-terminal half of the cytoplasmic domain, encoded by sequences downstream from the EcoRI restriction site (CD1c-Eco-end) (Fig.2 A). These CD1c/CD1e hybrid molecules were expressed in M10 cells, an HLA-DR+ melanoma cell line that contains, like DCs, cellular compartments involved in the processing of exogenous antigens. Controls included cells transfected with CD1c alone. Stably transfected M10 cells were permeabilized or not, stained with an anti-CD1c mAb, and analyzed by flow cytometry or confocal microscopy. In M10 cells, CD1c molecules as well as CD1c-ΔCytL* and CD1c-ΔCytS* hybrid molecules were found on the cell surface (Fig. 2 B). In contrast, CD1c-Eco-end was only weakly present on the cell surface, although the intracellular staining showed it to be expressed equally as strongly as the other molecules. Confocal microscopy revealed intracellular CD1c molecules to be localized in HLA-DR+compartments, as expected, while CD1c-Eco-end molecules were principally in the ER.3 The C-terminal region of this part of the cytoplasmic domain of CD1e contains two overlapping dilysine motifs (KKXK), both of which are known to mediate the retention of molecules in the ER (25Teasdale R.D. Jackson M.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 27-54Crossref PubMed Scopus (447) Google Scholar). Dilysine motifs are generally located at amino acids −3/−4 (KK motif) or −3/−5 (KXK motif) of the cytoplasmic domain, whereas the KKXK sequence of CD1e lies at positions −8/−11. The function of this sequence was explored by expressing in M10 cells other hybrid molecules comprising the extracellular and transmembrane domains of CD1c fused to the cytoplasmic domain CytL, complete or truncated at different positions in the C-terminal end. Hybrid fusion proteins containing the complete cytoplasmic domain of CD1e (short or long) could not be detected on the surface of transfected M10 cells.3 Similarly, a CD1e long cytoplasmic tail deleted of the first five C-terminal amino acids induced a complete intracellul
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