Identification of Different Binding Sites in the Dendritic Cell-specific Receptor DC-SIGN for Intercellular Adhesion Molecule 3 and HIV-1
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m111532200
ISSN1083-351X
AutoresTeunis B. H. Geijtenbeek, Gerard C.F. van Duijnhoven, Sandra J. van Vliet, Elmar Krieger, Gert Vriend, Carl G. Figdor, Yvette van Kooyk,
Tópico(s)Immune Cell Function and Interaction
ResumoThe novel dendritic cell (DC)-specific human immunodeficiency virus type 1 (HIV-1) receptor DC-SIGN plays a key role in the dissemination of HIV-1 by DC. DC-SIGN is thought to capture HIV-1 at mucosal sites of entry, facilitating transport to lymphoid tissues, where DC-SIGN efficiently transmits HIV-1 to T cells. DC-SIGN is also important in the initiation of immune responses by regulating DC-T cell interactions through intercellular adhesion molecule 3 (ICAM-3). We have characterized the mechanism of ligand binding by DC-SIGN and identified the crucial amino acids involved in this process. Strikingly, the HIV-1 gp120 binding site in DC-SIGN is different from that of ICAM-3, consistent with the observation that glycosylation of gp120, in contrast to ICAM-3, is not crucial to the interaction with DC-SIGN. A specific mutation in DC-SIGN abrogated ICAM-3 binding, whereas the HIV-1 gp120 interaction was unaffected. This DC-SIGN mutant captured HIV-1 and infected T cells in trans as efficiently as wild-type DC-SIGN, demonstrating that ICAM-3 binding is not necessary for HIV-1 transmission. This study provides a basis for the design of drugs that inhibit or alter interactions of DC-SIGN with gp120 but not with ICAM-3 or vice versa and that have a therapeutic value in immunological diseases and/or HIV-1 infections. The novel dendritic cell (DC)-specific human immunodeficiency virus type 1 (HIV-1) receptor DC-SIGN plays a key role in the dissemination of HIV-1 by DC. DC-SIGN is thought to capture HIV-1 at mucosal sites of entry, facilitating transport to lymphoid tissues, where DC-SIGN efficiently transmits HIV-1 to T cells. DC-SIGN is also important in the initiation of immune responses by regulating DC-T cell interactions through intercellular adhesion molecule 3 (ICAM-3). We have characterized the mechanism of ligand binding by DC-SIGN and identified the crucial amino acids involved in this process. Strikingly, the HIV-1 gp120 binding site in DC-SIGN is different from that of ICAM-3, consistent with the observation that glycosylation of gp120, in contrast to ICAM-3, is not crucial to the interaction with DC-SIGN. A specific mutation in DC-SIGN abrogated ICAM-3 binding, whereas the HIV-1 gp120 interaction was unaffected. This DC-SIGN mutant captured HIV-1 and infected T cells in trans as efficiently as wild-type DC-SIGN, demonstrating that ICAM-3 binding is not necessary for HIV-1 transmission. This study provides a basis for the design of drugs that inhibit or alter interactions of DC-SIGN with gp120 but not with ICAM-3 or vice versa and that have a therapeutic value in immunological diseases and/or HIV-1 infections. Transmission of human immunodeficiency virus type 1 (HIV-1) 1The abbreviations used are: HIV-1human immunodeficiency virus type 1DCdendritic cell(s)ICAMintercellular adhesion moleculeELISAenzyme-linked immunosorbent assayCRDcarbohydrate recognition domainRHL-1rat hepatic lectin-1MBPmannose-binding protein 1The abbreviations used are: HIV-1human immunodeficiency virus type 1DCdendritic cell(s)ICAMintercellular adhesion moleculeELISAenzyme-linked immunosorbent assayCRDcarbohydrate recognition domainRHL-1rat hepatic lectin-1MBPmannose-binding protein infection in humans requires the dissemination of virus from sites of infection at mucosal surfaces to T cell zones in secondary lymphoid organs, where extensive viral replication occurs in CD4+ T-helper cells. HIV-1 enters these cells through the interaction of the viral envelope glycoprotein gp120 with its primary receptor CD4 and members of the chemokine receptor family, primarily CCR5 and CXCR4 (1.Chan D.C. Kim P.S. Cell. 1998; 93: 681-684Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar, 2.Littman D.R. Cell. 1998; 93: 677-680Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Mechanisms of early viral dissemination remain uncertain, but anatomical distribution and in vitro infectivity studies infer that immature dendritic cells (DC) residing in the skin and at mucosal surfaces are the first cells to be targeted by HIV-1 (3.Cameron P.U. Freudenthal P.S. Barker J.M. Gezelter S. Inaba K. Steinman R.M. Science. 1992; 257: 383-387Crossref PubMed Scopus (554) Google Scholar, 4.Weissman D. Li Y. Ananworanich J. Zhou L.J. Adelsberger J. Tedder T.F. Baseler M. Fauci A.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 826-830Crossref PubMed Scopus (168) Google Scholar, 5.Granelli-Piperno A. Finkel V. Delgado E. Steinman R.M. Curr. Biol. 1999; 9: 21-29Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). human immunodeficiency virus type 1 dendritic cell(s) intercellular adhesion molecule enzyme-linked immunosorbent assay carbohydrate recognition domain rat hepatic lectin-1 mannose-binding protein human immunodeficiency virus type 1 dendritic cell(s) intercellular adhesion molecule enzyme-linked immunosorbent assay carbohydrate recognition domain rat hepatic lectin-1 mannose-binding protein Upon pathogen infiltration, immature DC migrate specifically to sites of inflammation to capture pathogens. Captured pathogens are processed into antigenic peptides and presented on major histocompatibility complex class II molecules at the surface of DC. DC mature and migrate to the secondary lymphoid organs, where they interact with T cells to initiate an immune response. HIV-1 is thought to subvert the trafficking capacity of DC to gain access to the CD4+ T cell compartment of lymphoid tissues where DC enhance the infection of CD4+ T cells by HIV-1 (3.Cameron P.U. Freudenthal P.S. Barker J.M. Gezelter S. Inaba K. Steinman R.M. Science. 1992; 257: 383-387Crossref PubMed Scopus (554) Google Scholar, 4.Weissman D. Li Y. Ananworanich J. Zhou L.J. Adelsberger J. Tedder T.F. Baseler M. Fauci A.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 826-830Crossref PubMed Scopus (168) Google Scholar, 6.Cameron P. Pope M. Granelli-Piperno A. Steinman R.M. J. Leukocyte Biol. 1996; 59: 158-171Crossref PubMed Scopus (111) Google Scholar). The molecular basis behind this process remained unclear until we recently identified DC-SIGN (DC-specificICAM-3-grabbing nonintegrin) through its high affinity interaction with the intercellular adhesion molecule 3 (ICAM-3) (7.Geijtenbeek T.B.H. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar, 8.Geijtenbeek T.B.H. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Middel J. Cornelissen I.L.M.H.A. Nottet H.S.L.M. KewalRamani V.N. Littman D.R. Figdor C.G. van Kooyk Y. Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2028) Google Scholar). DC-SIGN binds with high affinity to the HIV-1 envelope glycoprotein gp120 (Kd = 1.7 nm (9.Curtis B.M. Scharnowske S. Watson A.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8356-8360Crossref PubMed Scopus (336) Google Scholar)), and we demonstrated that DC-SIGN binds both M- and T-tropic HIV-1 and enhances infection of T cells in trans (8.Geijtenbeek T.B.H. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Middel J. Cornelissen I.L.M.H.A. Nottet H.S.L.M. KewalRamani V.N. Littman D.R. Figdor C.G. van Kooyk Y. Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2028) Google Scholar). Furthermore, DC-SIGN is expressed by immature DC localized in those mucosal tissues involved in sexual transmission of HIV-1. Thus, DC-SIGN endows DC with the ability to efficiently capture HIV-1, even when the virus is present in minute amounts. HIV-1 subsequently exploits the migratory capacity of DC to gain access to the T cell areas of lymphoid tissues, where DC-SIGN enhances the infection of residing CD4+ T cells by HIV-1. The cellular ligand of DC-SIGN, ICAM-3, is highly expressed on naive T cells, and we demonstrated that DC-SIGN binding to ICAM-3 initiates the DC-T cell interaction necessary for T cell activation (7.Geijtenbeek T.B.H. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar). Blocking antibodies against DC-SIGN inhibit the DC-T cell clustering and the subsequent T cell activation, demonstrating the importance of this first step in DC-T cell contact (7.Geijtenbeek T.B.H. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar). Here, we have investigated the mechanism of both ligand and Ca2+ binding by DC-SIGN. Strikingly, ICAM-3 recognition by DC-SIGN is dependent on N-glycosylation of the ligand, whereas HIV-1 gp120 binding is independent of both N- and O-linked glycosylations. Differences in ligand binding were further confirmed by site-directed mutagenesis and three-dimensional modeling. We generated a DC-SIGN mutant that specifically bound HIV-1 gp120 but not ICAM-3 and that efficiently mediated HIV-1 infection of T cells in trans. Thus, DC-SIGN has different binding sites for HIV-1 gp120 and ICAM-3. This information will be essential in the successful development of preventative and therapeutic strategies in the treatment of HIV-1 and opens up the possibility of designing drugs specifically inhibiting interactions of DC-SIGN with one ligand without affecting that of the other ligand. The monoclonal antibodies against DC-SIGN are AZN-D1 and AZN-D2 (7.Geijtenbeek T.B.H. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar). Glycosylated and nonglycosylated HIV-1SF2 gp120 was provided by M. Quiroga through the NIH AIDS Research and Reference Reagent Program. Nonglycosylated HIV-1 gp120 was produced in yeast using an intracellular expression vector lacking the signal sequence, thereby preventing passage through the secretory pathway and thus the addition of carbohydrates (10.Haigwood N.L. Shuster J.R. Moore G.K. Lee H. Skiles P.V. Higgins K.W. Barr P.J. George-Nascimento C. Steimer K.S. AIDS Res. Hum. Retroviruses. 1990; 6: 855-869Crossref PubMed Scopus (81) Google Scholar). Peptide-N-glycosidase F and endo-N-acetylgalactosaminidase were obtained from Oxford Glycosciences (Wakefield, MA). Mutations in the cDNA encoding DC-SIGN were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). K562 transfectants either expressing wild-type or mutant DC-SIGN were generated by transfection of K562 cells with 10 μg of plasmid by electroporation as described previously (8.Geijtenbeek T.B.H. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Middel J. Cornelissen I.L.M.H.A. Nottet H.S.L.M. KewalRamani V.N. Littman D.R. Figdor C.G. van Kooyk Y. Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2028) Google Scholar). Positive cells were sorted several times to obtain stable transfectants with similar expression levels of DC-SIGN. DC-SIGN-Fc consists of the extracellular portion of DC-SIGN (amino acid residues 64–404) fused at the C terminus to a human IgG1-Fc fragment into the Sig-pIgG1-Fc vector (11.Fawcett J. Holness C.L. Needham L.A. Turley H. Gatter K.C. Mason D.Y. Simmons D.L. Nature. 1992; 360: 481-484Crossref PubMed Scopus (302) Google Scholar). DC-SIGN-Fc was produced in Chinese hamster ovary K1 cells by co-transfection of DC-SIGN-Sig-pIgG1 Fc (20 μg) and pEE14 (5 μg) vector. DC-SIGN-Fc concentrations in the supernatant were determined by an anti-IgG1 ELISA. The fluorescent bead adhesion assay was performed as described earlier (7.Geijtenbeek T.B.H. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar). Adhesion was determined in the presence of either 5 mm mannan, 5 mm EGTA, or 20 μg/ml antibodies against DC-SIGN. The soluble DC-SIGN adhesion assay was performed as follows. Soluble ligands were coated in ELISA plates (1 μg/well) for 1 h at 4 °C, followed by blocking with 1% bovine serum albumin for 30 min at 4 °C. Soluble DC-SIGN-Fc supernatant was added, and the adhesion was performed for 30 min at 37 °C. Unbound DC-SIGN-Fc was washed away, and binding was determined by an anti-IgG1 ELISA. Specificity was determined in the presence of either 20 μg/ml blocking antibodies or 5 mmEGTA. The infection assays were performed as described previously (8.Geijtenbeek T.B.H. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Middel J. Cornelissen I.L.M.H.A. Nottet H.S.L.M. KewalRamani V.N. Littman D.R. Figdor C.G. van Kooyk Y. Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2028) Google Scholar). Pseudotyped viral stocks were generated by calcium phosphate transfections of 293T cells with the proviral plasmid pNL-Luc-E−R−, containing a luciferase reporter gene and an expression plasmid for the ADA gp160 envelope. Briefly, DC-SIGN transfectants were incubated for 2 h with identical pseudotyped HIV-1ADA virus concentrations, unbound virus was washed away, and phytohemagglutinin-activated T cells were added. Cell lysates were obtained after 2 days and analyzed for luciferase activity. We have built the model of the C-type lectin domain of DC-SIGN by exploiting the sequence similarity to the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor, which was solved using x-ray crystallography (12.Meier M. Bider M.D. Malashkevich V.N. Spiess M. Burkhard P. J. Mol. Biol. 2000; 300: 857-865Crossref PubMed Scopus (150) Google Scholar) (Protein Data Bank entry 1DV8). The modeling was done with WHAT IF (13.Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3357) Google Scholar), following the protocol as previously described (14.Vriend G. Eijsink V. J. Comput. Aided Mol. Des. 1993; 7: 367-396Crossref PubMed Scopus (82) Google Scholar). Three insertions (Arg312-Phe313, Leu321-Asn322, and Tyr342) were added to the model of the asialoglycoprotein receptor by copying the respective loop conformations from two other homologous templates (Protein Data Bank entries 1ESL and 1HLJ) to provide a structure with high homology to the CRD of DC-SIGN. The model was minimized with the YASARA NOVA force field, which was parameterized for the refinement of homology models and was shown to routinely reduce the C root mean square deviation. 2E. Krieger, unpublished results. With 50% sequence identity between DC-SIGN and the template 1DV8, homology modeling is commonly considered straightforward (14.Vriend G. Eijsink V. J. Comput. Aided Mol. Des. 1993; 7: 367-396Crossref PubMed Scopus (82) Google Scholar). Validation of the model with WHAT_CHECK (15.Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1798) Google Scholar) revealed no large problems in the model structure. The model coordinates are available on the World Wide Web at www.cmbi.kun.nl/gv/service/dcsign/. DC-SIGN, a type II transmembrane protein with a C-terminal C-type lectin domain, binds with high affinity to both the immunoglobulin superfamily member ICAM-3 (7.Geijtenbeek T.B.H. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar) and the HIV-1 envelope glycoprotein gp120 (8.Geijtenbeek T.B.H. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Middel J. Cornelissen I.L.M.H.A. Nottet H.S.L.M. KewalRamani V.N. Littman D.R. Figdor C.G. van Kooyk Y. Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2028) Google Scholar). We used K562 transfectants stably expressing DC-SIGN to specifically investigate the binding characteristics of DC-SIGN in the absence of any other ICAM-3- or HIV-1-receptors, since this erythroleukemic cell line does not express leukocyte function-associated molecule 1 and CD4. DC-SIGN expressed by these cells is fully functional (Fig. 1A), with a binding activity similar to that observed for DC-SIGN expressed by DC (7.Geijtenbeek T.B.H. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar, 8.Geijtenbeek T.B.H. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Middel J. Cornelissen I.L.M.H.A. Nottet H.S.L.M. KewalRamani V.N. Littman D.R. Figdor C.G. van Kooyk Y. Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2028) Google Scholar). DC-SIGN-mediated adhesion to both ICAM-3 and gp120 is completely inhibited by the blocking antibodies against DC-SIGN, the calcium chelator EGTA, and the polysaccharide mannan (Fig. 1A). The Ca2+ dependence confirms that ligand binding is mediated by the C-type lectin domain of DC-SIGN, which interacts also with mannose-type carbohydrates, since adhesion to its natural ligands was inhibited by the polycarbohydrate mannan (Fig. 1A). Both ICAM-3 and gp120 are heavily glycosylated (16.Funatsu O. Sato T. Kotovuori P. Gahmberg C.G. Ikekita M. Furukawa K. Eur. J. Biochem. 2001; 268: 1020-1029Crossref PubMed Scopus (22) Google Scholar, 17.Leonard C.K. Spellman M.W. Riddle L. Harris R.J. Thomas J.N. Gregory T.J. J. Biol. Chem. 1990; 265: 10373-10382Abstract Full Text PDF PubMed Google Scholar), and these post-translational modifications could be important for the interaction with DC-SIGN. Enzymatic removal of the O-linked glycosylations from both ligands had no effect on their binding to DC-SIGN (Fig. 1B). HIV-1 gp120 contains O-linked glycosylations (18.Bernstein H.B. Tucker S.P. Hunter E. Schutzbach J.S. Compans R.W. J. Virol. 1994; 68: 463-468Crossref PubMed Google Scholar), as was confirmed by SDS-PAGE analysis after deglycosylation (data not shown), whereas no shift in molecular weight was observed in ICAM-3 after endo-N-acetylgalactosaminidase treatment (data not shown), suggesting that ICAM-3 does not contain O-linked glycosylations. In contrast, removal of the N-linked glycosylations from ICAM-3 by peptide-N-glycosidase F completely abrogated the adhesion (Fig. 1B), demonstrating that N-linked carbohydrates are involved in this interaction. In contrast, treatment of HIV-1 gp120 with peptide-N-glycosidase F did not result in a decreased interaction. It is possible that the carbohydrates were not completely removed, although it appeared that the N-linked glycosylations had been removed from gp120 as determined by SDS-PAGE analysis (data not shown). Therefore, we used nonglycosylated recombinant gp120 and observed that DC-SIGN interacts with this nonglycosylated gp120 in a similar way as with native gp120, derived from mammalian cells (Fig. 1C). The interaction with nonglycosylated gp120 was blocked by the anti-DC-SIGN antibodies as well as by EGTA. These results confirm that glycosylations are not necessary for the interaction of HIV-1 gp120 with DC-SIGN, whereas N-linked but not O-linked oligosaccharides are essential for the binding of ICAM-3. DC-SIGN belongs to the C-type lectin family (Fig. 2A), and we decided to obtain structural information about the C-type lectin domain of DC-SIGN, through comparison with other C-type lectins, to identify crucial amino acid residues involved in ligand binding by site-directed mutagenesis. The CRD of DC-SIGN has 50% identity with that of the H1 subunit of the asialoglycoprotein receptor (rat hepatic lectin-1 (RHL-1)) and 39% with that of the rat serum mannose-binding protein (MBP-A). We performed molecular modeling with the crystal structure of the RHL-1 (12.Meier M. Bider M.D. Malashkevich V.N. Spiess M. Burkhard P. J. Mol. Biol. 2000; 300: 857-865Crossref PubMed Scopus (150) Google Scholar) as a template because of the high identity with RHL-1, and we generated a three-dimensional structure of the C-type lectin domain of DC-SIGN (amino acid residues 267–381; Fig. 2B). The CRD of DC-SIGN is a globular protein consisting of 12 β-strands, two α-helices, and three disulfide bridges. The β-strands are arranged in three β-sheets and form the core of the protein with an α-helix on each side of this core. One prominent loop sticks out from the protein surface and forms part of the two Ca2+-binding sites, designated site 1 and site 2. Ca2+ is essential to the activity of DC-SIGN (Fig. 1A); thus, these sites are probably part of the ligand binding site. Amino acid sequence alignment of DC-SIGN with other C-type lectins indicates that four amino acids, Glu347, Asn349, Glu354, and Asn365, interact with the Ca2+ at site 2 through their carbonyl groups (Figs.2A and 3A). We hypothesized that these four amino acid residues might also participate in the interaction of DC-SIGN with its ligands via hydrogen bonds as was shown for other C-type lectins such as MBP-A (19.Weis W.I. Drickamer K. Hendrickson W.A. Nature. 1992; 360: 127-134Crossref PubMed Scopus (850) Google Scholar). Site-directed mutagenesis was used to assess the role of these residues in ligand binding by DC-SIGN. Changing either Glu347 into Gln or Asn349 and Asn365 into Asp resulted in complete loss of both ICAM-3 and gp120 binding (Table I). Structural integrity was retained within these mutants, since the interaction with antibodies against DC-SIGN was unaffected (Table I). Loss of ligand binding due to these mutations could be ascribed to elimination of a single hydrogen bond between DC-SIGN and its ligand through removal of either a hydrogen donor (amide group in Asn) or acceptor (acidic group in Glu) (Fig. 3A). Ca2+ binding in these mutants will not be affected, since the altered amino acid residues (Glu into Gln and Asn into Asp) are still able to donate a carbonyl group to the interaction with Ca2+, as was also shown for other C-type lectins (19.Weis W.I. Drickamer K. Hendrickson W.A. Nature. 1992; 360: 127-134Crossref PubMed Scopus (850) Google Scholar). The amino acid residue Asp366 coordinates the Ca2+ion without contributing to the ligand binding (Fig. 3A). Mutation of Asp366 into Ala resulted in complete loss of ligand binding, which is contributed to loss of Ca2+binding (Table I).Table IFunctional characterization of the different DC-SIGN mutantsDC-SIGN mutantaStable K562 transfectants.AdhesionExpressionFunctionICAM-3gp120AZN-D1AZN-D2%%%%Wild type49399592E347Q049392Ligand and Ca2+ site 2 bindingN349D109496N365D209697D366A139491Ca2+site 2 bindingD320A279898E324A009595N350A119492Ca2+ site 1 bindingD355A029897a Stable K562 transfectants. Open table in a new tab The CRD of DC-SIGN binds two calcium ions (7.Geijtenbeek T.B.H. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar), and, as we demonstrated, the ligand binding site of DC-SIGN is situated at Ca2+ site 2. The Ca2+ at site 1, the so-called auxiliary site, is located opposite the protein loop (Fig. 2B). Sequence alignment suggests that the amino acid residues Asp320, Glu324, Asn350, and Asp355 are involved in the binding of this Ca2+ (Fig. 2A). This Ca2+ is important in the interaction of DC-SIGN with its ligands, since mutating the respective amino acid residues into Ala residues results in complete loss of ligand binding (Table I). These mutants are not able to bind the Ca2+ at site 1 because the change of either Glu, Asp, or Asn into Ala removes the carbonyl group at their respective positions necessary for the interaction with Ca2+, as has previously been shown for MBP-A (19.Weis W.I. Drickamer K. Hendrickson W.A. Nature. 1992; 360: 127-134Crossref PubMed Scopus (850) Google Scholar). The structure of these mutants is not changed dramatically, since they are still recognized by antibodies against DC-SIGN (Table I). Presumably, the Ca2+ at site 1 is essential to the correct positioning of the loops forming the primary ligand binding site as was shown for MBP-A (19.Weis W.I. Drickamer K. Hendrickson W.A. Nature. 1992; 360: 127-134Crossref PubMed Scopus (850) Google Scholar). We have previously reported two antibodies, AZN-D1 and -D2 (7.Geijtenbeek T.B.H. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar), that interact specifically with DC-SIGN and completely inhibit the interaction of DC-SIGN with both HIV-1 gp120 and ICAM-3 (Fig. 1A). We determined the epitopes of these antibodies by site-directed mutagenesis and subsequent screening of the different DC-SIGN mutants for antibody recognition. Mutagenesis of Lys368 into Ala abrogated binding by the antibody AZN-D1, whereas AZN-D2 did not bind to the Gln323 mutant (Fig. 3B). The structure of DC-SIGN shows that the AZN-D1 epitope is located in the active ligand binding site close to Ca2+site 2, whereas AZN-D2 interacts with amino acid residues on the other site of the protein loop close to Ca2+ site 1 (Fig. 3C). The Ca2+ at site 2 constitutes part of the ligand binding, whereas Ca2+ site 1 is involved in stabilizing the binding site. Thus, interference with these sites blocks the interaction of DC-SIGN with its ligands, confirming the importance of both Ca2+ sites for DC-SIGN function. The ligand binding site of the CRD of DC-SIGN forms a flat hydrophobic surface resembling a shallow trough, with the amino acid residue Val351 forming the edge of the pocket (Fig. 3, A and C). We investigated the role of this Val351residue in the binding activity of DC-SIGN by changing it into Gly, thereby removing the β-carbon of Val, which has been shown in similar C-type lectins to be important (20.Iobst S.T. Wormald M.R. Weis W.I. Dwek R.A. Drickamer K. J. Biol. Chem. 1994; 269: 15505-15511Abstract Full Text PDF PubMed Google Scholar). Indeed, this amino acid residue is essential for binding of DC-SIGN to ICAM-3, since the adhesion is completely abolished (Fig. 4A). Interestingly, the mutant is still able to bind the HIV-1 envelope glycoprotein gp120 to a similar extent as wild-type DC-SIGN (Fig. 4A). The Ca2+ dependence of the interaction of the V351G mutant with gp120 is lower than that of wild-type DC-SIGN, but even at high Ca2+ concentrations the V351G mutant is unable to interact with ICAM-3 (Fig. 4B), confirming a complete loss of ICAM-3 binding activity. These data demonstrate that the interaction of DC-SIGN with ICAM-3 is distinct from that with the HIV-1 envelope glycoprotein gp120 and that the V351G mutant plays an essential role in ICAM-3 but not in gp120 binding. Previously, we demonstrated that DC capture HIV-1 through DC-SIGN and that DC-SIGN-bound HIV-1 is efficiently transmitted to T cells, producing a vigorous infection of T cells (8.Geijtenbeek T.B.H. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Middel J. Cornelissen I.L.M.H.A. Nottet H.S.L.M. KewalRamani V.N. Littman D.R. Figdor C.G. van Kooyk Y. Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2028) Google Scholar). The V351G mutant is able to bind HIV-1 (Fig. 4A), and we investigated whether this mutant is able to transmit HIV-1 to T cells. K562 transfectants expressing the V351G mutant were incubated with M-tropic HIV-1 for 2 h, and unbound virus was washed away. Activated T cells were co-cultured with the HIV-1-pulsed K562 transfectants expressing the V351G mutant, and this resulted in a productive infection of T cells similar to that observed with wild-type DC-SIGN (Fig. 4C). Similar results were obtained with T-tropic HIV-1 strains (results not shown). Thus, the V351G mutant is able to both capture HIV-1 and transmit the virus to activated T cells. These results also demonstrate that the ICAM-3-binding activity is not essential for transmission of HIV-1 by DC-SIGN. Sexual transmission of HIV-1 involves dissemination of the virus from the mucosal tissues to the lymphoid organs where the target T cells reside. DC are thought to mediate this process (3.Cameron P.U. Freudenthal P.S. Barker J.M. Gezelter S. Inaba K. Steinman R.M. Science. 1992; 257: 383-387Crossref PubMed Scopus (554) Google Scholar, 6.Cameron P. Pope M. Granelli-Piperno A. Steinman R.M. J. Leukocyte Biol. 1996; 59: 158-171Crossref PubMed Scopus (111) Google Scholar), and the DC-specific HIV-1 receptor DC-SIGN facilitates capture of HIV-1, its subsequent transport, and the efficient transmission of HIV-1 to T cells (8.Geijtenbeek T.B.H. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Middel J. Cornelissen I.L.M.H.A. Nottet H.S.L.M. KewalRamani V.N. Littman D.R. Figdor C.G. van Kooyk Y. Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2028) Google Scholar). DC-SIGN is able to capture both M- and T-tropic HIV-1, HIV-2, and simian immunodeficiency virus and to transmit the viruses to recipient T cells (8.Geijtenbeek T.B.H. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Middel J. Cornelissen I.L.M.H.A. Nottet H.S.L.M. KewalRamani V.N. Littman D.R. Figdor C.G. van Kooyk Y. Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2028) Google Scholar, 21.Pohlmann S. Baribaud F. Lee B. Leslie G.J. Sanchez M.D. Hiebenthal-Millow K. Munch J. Kirchhoff F. Doms R.W. J. Virol. 2001; 75: 4664-4672Crossref PubMed Scopus (200) Google Scholar). The expression of DC-SIGN on DC in mucosal tissues is consistent with its key function in the early stages of viral infection after sexual transmission (8.Geijtenbeek T.B.H. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C.F. Middel J. Cornelissen I.L.M.H.A. Nottet H.S.L.M. KewalRamani V.N. Littman D.R. Figdor C.G. van Kooyk Y. Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2028) Google Scholar). Moreover, DC-SIGN might also play a role in chronic HIV-1 infections, since DC-SIGN+ DC are abundantly present in the
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