Proteomic Analysis of DC-SIGN on Dendritic Cells Detects Tetramers Required for Ligand Binding but No Association with CD4
2004; Elsevier BV; Volume: 279; Issue: 50 Linguagem: Inglês
10.1074/jbc.m402741200
ISSN1083-351X
AutoresOliver K. Bernhard, Joey Lai, John Wilkinson, Margaret M. Sheil, Anthony L. Cunningham,
Tópico(s)vaccines and immunoinformatics approaches
ResumoDC-SIGN (dendritic cell specific intracellular adhesion molecule 3 grabbing non-integrin) or CD209 is a type II transmembrane protein and one of several C-type lectin receptors expressed by dendritic cell subsets, which bind to high mannose glycoproteins promoting their endocytosis and potential degradation. DC-SIGN also mediates attachment of HIV to dendritic cells and binding to this receptor can subsequently lead to endocytosis or enhancement of CD4/CCR5-dependent infection. The latter was proposed to be facilitated by an interaction between DC-SIGN and CD4. Endocytosis of HIV virions does not necessarily lead to their complete degradation. A proportion of the virions remain infective and can be later presented to T cells mediating their infection in trans. Previously, the extracellular domain of recombinant DC-SIGN has been shown to assemble as tetramers and in the current study we use a short range covalent cross-linker and show that DC-SIGN exists as tetramers on the surface of immature monocyte-derived dendritic cells. There was no evidence of direct binding between DC-SIGN and CD4 either by cross-linking or by fluorescence resonance energy transfer measurements suggesting that there is no constitutive association of the majority of these proteins in the membrane. Importantly we also show that the tetrameric complexes, in contrast to DC-SIGN monomers, bind with high affinity to high mannose glycoproteins such as mannan or HIV gp120 suggesting that such an assembly is required for high affinity binding of glycoproteins to DC-SIGN, providing the first direct evidence that DC-SIGN tetramers are essential for high affinity interactions with pathogens like HIV. DC-SIGN (dendritic cell specific intracellular adhesion molecule 3 grabbing non-integrin) or CD209 is a type II transmembrane protein and one of several C-type lectin receptors expressed by dendritic cell subsets, which bind to high mannose glycoproteins promoting their endocytosis and potential degradation. DC-SIGN also mediates attachment of HIV to dendritic cells and binding to this receptor can subsequently lead to endocytosis or enhancement of CD4/CCR5-dependent infection. The latter was proposed to be facilitated by an interaction between DC-SIGN and CD4. Endocytosis of HIV virions does not necessarily lead to their complete degradation. A proportion of the virions remain infective and can be later presented to T cells mediating their infection in trans. Previously, the extracellular domain of recombinant DC-SIGN has been shown to assemble as tetramers and in the current study we use a short range covalent cross-linker and show that DC-SIGN exists as tetramers on the surface of immature monocyte-derived dendritic cells. There was no evidence of direct binding between DC-SIGN and CD4 either by cross-linking or by fluorescence resonance energy transfer measurements suggesting that there is no constitutive association of the majority of these proteins in the membrane. Importantly we also show that the tetrameric complexes, in contrast to DC-SIGN monomers, bind with high affinity to high mannose glycoproteins such as mannan or HIV gp120 suggesting that such an assembly is required for high affinity binding of glycoproteins to DC-SIGN, providing the first direct evidence that DC-SIGN tetramers are essential for high affinity interactions with pathogens like HIV. Dendritic cells (DCs) 1The abbreviations used are: DC, dendritic cell; CLR, C-type (calcium-dependent) lectin receptor; DC-SIGN, dendritic cell specific intracellular adhesion molecule 3 grabbing non-integrin; MDDC, monocyte-derived dendritic cell; MS, mass spectrometry; DSS, disuccinimidyl suberate; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; FRET, fluorescence resonance energy transfer; MBP, mannose-binding protein; PMA, phorbol 12-myristate 13-acetate; HIV, human immunodeficiency virus; DTT, dithiothreitol.1The abbreviations used are: DC, dendritic cell; CLR, C-type (calcium-dependent) lectin receptor; DC-SIGN, dendritic cell specific intracellular adhesion molecule 3 grabbing non-integrin; MDDC, monocyte-derived dendritic cell; MS, mass spectrometry; DSS, disuccinimidyl suberate; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; FRET, fluorescence resonance energy transfer; MBP, mannose-binding protein; PMA, phorbol 12-myristate 13-acetate; HIV, human immunodeficiency virus; DTT, dithiothreitol. and their subsets are potent antigen-presenting cells functioning at the interface between the adaptive and innate immune system (1Banchereau J. Steinman R.M. Nature. 1998; 392: 245-252Crossref PubMed Scopus (12186) Google Scholar, 2Turville S.G. Cameron P.U. Handley A. Lin G. Pöhlmann S. Doms R.W. Cunningham A.L. Nat. Immunology. 2002; 3: 975-983Crossref PubMed Scopus (444) Google Scholar), which recognize and internalize pathogens and subsequently activate T cells with pathogen-derived peptides. To internalize pathogens, DCs express a series of pathogen recognition receptors such as Toll-like receptors that recognize lipoproteins, lipopolysaccharide, or bacterial DNA commonly found on various bacteria. C-type (calcium-dependent) lectin receptors (CLRs) are also expressed and these bind to conserved oligosaccharides that are commonly found on the surface glycoproteins of viruses. CLRs expressed by DCs include the mannose receptor (CD206), DEC-205 (CD205), Langerin (CD207), and DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN; CD209) (3Figdor C.G. van Kooyk Y. Adema G.J. Nat. Rev. Immunology. 2002; 2: 77-84Crossref PubMed Scopus (693) Google Scholar). These receptors differ not only in their expression on various subsets of DCs and other tissues, but they also recognize different oligosaccharides thus discriminating between different ligands (3Figdor C.G. van Kooyk Y. Adema G.J. Nat. Rev. Immunology. 2002; 2: 77-84Crossref PubMed Scopus (693) Google Scholar, 4Frison N. Taylor M.E. Soilleux E. Bousser M.-T. Mayer R. Monsigny M. Drickamer K. Roche A.-C. J. Biol. Chem. 2003; 278: 23922-23929Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). DC-SIGN is a 44 kDa type II transmembrane protein that consists of a carbohydrate recognition domain, a neck domain involved in oligomerization, a transmembrane domain, and a cytoplasmic tail mediating interactions with the endocytosis machinery important for ligand internalization (4Frison N. Taylor M.E. Soilleux E. Bousser M.-T. Mayer R. Monsigny M. Drickamer K. Roche A.-C. J. Biol. Chem. 2003; 278: 23922-23929Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 5Mitchell D.A. Fadden A.J. Drickamer K. J. Biol. Chem. 2001; 276: 28939-28945Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 6Geijtenbeek T.B.H. Kwon D.S. Torensma R. van Vliet S.J. van Kuijnhoven G.C.F. Middel J. Cornelissen I.L.M.H.A. Nottet H.L.S.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 and internalizes several viruses such as HIV, Ebola virus, cytomegalovirus, Dengue virus, and hepatitis C virus (7Alvarez C.P. Lasala F. Carrillo J. Muniz O. Corbi A.L. Delgado R. J. Virol. 2002; 76: 6841-6844Crossref PubMed Scopus (526) Google Scholar, 8Halary F. Amara A. Lortat-Jacob H. Messerle M. Delaunay T. Houles C. Fieschi F. Arenzana-Seisdedos F. Moreau J.F. Dechanet-Merville J. Immunity. 2002; 17: 653-664Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 9Navarro-Sanchez E. Altmeyer R. Amara A. Schwartz O. Fieschi F. Virelizier J.L. Arenzana-Seisdedos F. Embo Rep. 2003; 4: 1-6Crossref Scopus (391) Google Scholar, 10Pohlmann S. Zhang J. Baribaud F. Chen Z. Leslie G.J. Lin G. Granelli-Piperno A. Doms R.W. Rice C.M. McKeating J.A. J. Virol. 2003; 77: 4070-4080Crossref PubMed Scopus (341) Google Scholar) though other receptors are also involved (2Turville S.G. Cameron P.U. Handley A. Lin G. Pöhlmann S. Doms R.W. Cunningham A.L. Nat. Immunology. 2002; 3: 975-983Crossref PubMed Scopus (444) Google Scholar). Other non-viral pathogens can also interact with DC-SIGN (11van Kooyk Y. Geijtenbeek T.B. Nat. Rev. Immunol. 2003; 3: 697-709Crossref PubMed Scopus (765) Google Scholar). Many of these viruses, however, have evolved a mechanism leading to their escape from lysosomal degradation and allowing them either to infect DCs or to hide inside the cells followed by transfer to and infection of T cells when these cells are being activated by the mature DC. In mature monocyte-derived DCs (mMDDCs) it has been shown that HIV binding to DC-SIGN and subsequent internalization into the DC does not lead to the complete degradation and part of it is protected and subsequently infects T cells (12Moris A. Nobile C. Buseyene F. Porrot F. Abastado J.-P. Schwartz O. Blood. 2004; 103: 2648-2654Crossref PubMed Scopus (157) Google Scholar, 13Turville S.G. Santos J.J. Frank I. Cameron P.U. Wilkinson J. Miranda-Saksena M. Dable J. Stossel H. Romani N. Piatak M. Lifson J.D. Pope M. Cunningham A.L. Blood. 2003; 103: 2170-2179Crossref PubMed Scopus (329) Google Scholar). The mechanism of this protection is poorly understood (11van Kooyk Y. Geijtenbeek T.B. Nat. Rev. Immunol. 2003; 3: 697-709Crossref PubMed Scopus (765) Google Scholar, 14Kwon D.S. Gregorio G. Bitton N. Hendrickson W.A. Littman D.R. Immunity. 2002; 16: 135-144Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar) but it has been suggested that it is an HIV-induced change in endocytic routing (11van Kooyk Y. Geijtenbeek T.B. Nat. Rev. Immunol. 2003; 3: 697-709Crossref PubMed Scopus (765) Google Scholar, 13Turville S.G. Santos J.J. Frank I. Cameron P.U. Wilkinson J. Miranda-Saksena M. Dable J. Stossel H. Romani N. Piatak M. Lifson J.D. Pope M. Cunningham A.L. Blood. 2003; 103: 2170-2179Crossref PubMed Scopus (329) Google Scholar). Alternatively, a minor proportion of HIV is transferred to the CD4/CCR5-mediated infection pathway. A detailed knowledge of the DC-SIGN interaction with glycoproteins is thus crucial for the understanding of DC-SIGN-induced routing of internalized ligands and involves both the selectivity for individual sugar structures as well as the stoichiometry of the DC-SIGN-ligand complex including any DC-SIGN-associated proteins in the cell membrane. Crystal structural data have revealed that DC-SIGN binds to internal tri-mannose glycosylation structures present in N-linked high mannose oligosaccharides. The receptor makes contact with the three adjacent mannose residues at an internal branched structure but fails to bind to the core tri-mannose motif in complex oligosaccharides because of steric interference resulting from different anomeric linkages (15Feinberg H. Mitchell D.A. Drickamer K. Weis W.I. Science. 2001; 294: 2163-2166Crossref PubMed Scopus (573) Google Scholar). This observation explained the preference of DC-SIGN for high mannose carbohydrate structures and is in contrast to binding characteristics of other CLRs such as the mannose receptor, which has been suggested to bind to single terminal mannose residues (11van Kooyk Y. Geijtenbeek T.B. Nat. Rev. Immunol. 2003; 3: 697-709Crossref PubMed Scopus (765) Google Scholar). Based on the oligomerization of its extracellular domain it was suggested that DC-SIGN forms tetramers and that this oligomerization enhances the affinity for neoglycoproteins (5Mitchell D.A. Fadden A.J. Drickamer K. J. Biol. Chem. 2001; 276: 28939-28945Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Support for this is provided by electron microscopy detection of clusters of DC-SIGN residing in rafts on the surface of immature monocyte-derived DCs (iMDDCs) (16Cambi A. de Lange F. van Maarseveen N.M. Nijhuis M. Joosten B. van Dijk E.M.H.P. de Bakker B.I. Fransen J.A.M. Bovee-Geurts P.H.M. van Leeuwen F.N. Van Hulst N.F. Figdor C.G. J. Cell Biol. 2004; 164: 145-155Crossref PubMed Scopus (197) Google Scholar). It is therefore proposed that the interaction between DC-SIGN and glycoproteins involves several molecules of DC-SIGN binding to differential sugar moieties on the glycoprotein spaced at appropriate distances determined by the DC-SIGN oligomerization (5Mitchell D.A. Fadden A.J. Drickamer K. J. Biol. Chem. 2001; 276: 28939-28945Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). However, tetramer formation on the cell surface has not yet been proven. Furthermore, it is still unclear whether DC-SIGN associates with any other proteins and how this could influence ligand binding. This is especially important as the mechanism of cis infection of DCs by HIV suggests a possible association between CD4/CCR5 and DC-SIGN in the plasma membrane and such an association has been shown by colocalization on alveolar macrophages but the authors were unable to co-immunoprecipitate those molecules in transiently transfected cells (17Lee B. Leslie G. Soilleux E. O'Doherty U. Baik S. Levroney E. Flummerfelt K. Swiggard W. Coleman N. Malim M. Doms R.W. J. Virol. 2001; 75: 12028-12038Crossref PubMed Scopus (160) Google Scholar). Here we report the use of novel "proteomics" techniques based on mass spectrometry (MS) along with fluorescence resonance energy transfer (FRET) measurements to investigate lateral protein associations of native DC-SIGN on iMDDCs. We have reported recently the versatility of those tools in determining lateral membrane protein associations on lymphoid cells (18Bernhard O.K. Sheil M.M. Cunningham A.L. Biochemistry. 2004; 43: 256-264Crossref PubMed Scopus (15) Google Scholar). In this article, we use cross-linking, immunopurification, and Western blotting to show that DC-SIGN assembles into oligomers of very high apparent molecular weight on the cell-surface of iMDDCs. Mass spectrometry analysis of the purified complexes identified them as homo-oligomers of DC-SIGN and cross-linking at different concentrations of the cross-linker suggests that they are tetramers. However, CD4 could not be detected in the complexes. Furthermore FRET measurements and cross-linking followed by co-immunoprecipitation also did not detect any CD4/DC-SIGN association. We also show for the first time that the DC-SIGN complexes bind immobilized mannan (a yeast-derived polysaccharide) as well as gp120 with high affinity whereas DC-SIGN monomers are not bound. This confirms the formation of DC-SIGN tetramers on immature iMDDCs, shows that they do not associate with other proteins and that the oligomerization mediated by the lateral organization in the membrane is required for high affinity ligand binding. Cells and Reagents—Unless otherwise specified, all reagents were obtained from Sigma. Immature MDDCs were prepared from blood monocytes as described previously (2Turville S.G. Cameron P.U. Handley A. Lin G. Pöhlmann S. Doms R.W. Cunningham A.L. Nat. Immunology. 2002; 3: 975-983Crossref PubMed Scopus (444) Google Scholar). The T lymphoblastoid cell line CemT4 and the CD4-negative lymphoblastoid derivative cell line A2.01 were obtained from the National Institutes of Health AIDS reagents and reference program (Rockville, MD). OKT4 hybridoma cells were obtained from the American Type Tissue Collection. Q4120 hybridoma cells were a generous gift from Dr. Quentin Sattentau (Centre d'Immunologie de Marseille-Luminy, Marseille, France), and WM82 hybridoma cells were donated by Dr. Tony Hennicker (Westmead Hospital, Westmead, NSW, Australia). The DC-SIGN specific monoclonal antibody 120507 was obtained from R&D Systems (Minneapolis, MN), and the rabbit polyclonal antibody against DC-SIGN was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HIV-1 gp120 (Clade E, 3234, used for co-immunoprecipitations) was obtained from the National Institutes of Health AIDS Research and Reference Program. Sheep polyclonal antibody raised against gp120 (Lot DV-012) had been obtained from the Center for Biologicals Evaluation and Research (FDA, Bethesda, MD). Mouse IgG1 was from Sigma. The horseradish peroxidase-labeled secondary antibody (donkey anti-rabbit) was purchased from Amersham Biosciences (Castle Hill, NSW, Australia). Disuccinimidyl suberate (DSS), dithiobis(succinimidylpropionate) (DSP), were from Pierce, protein G-Sepharose and Cy3 monoreactive NHS ester from Amersham Biosciences. 6-(Fluorescein-5-carboxamido)hexanoic acid was from Molecular Probes (Eugene, OR) and PMA was purchased from Sigma-Aldrich. Cross-linking and Cell Lysate Preparation—Cells were washed twice with cross-link buffer (CLB) (10 mm HEPES, pH 8.0, 140 mm NaCl, 1 mm MgCl2, 0.1 mm EGTA, 0.02% (w/v) NaN3) and resuspended in CLB at 5 × 106 cells/ml. DSS or DSP were dissolved in Me2SO at 25 mm or at lower concentrations when indicated and added to the suspension (20 μl per 1 ml suspension). Cross-linking was carried out for 30 min at room temperature followed by quenching of the cross-linker, which was done by adding TBS, pH 7.5, pelleting the cells, and resuspending them in TBS, pH 7.5 for 15 min. Cells were pelleted again and lysed in CLB containing 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS for 1 h at 4°C. Insoluble debris was pelleted for 10 min at 4 °C and 10 000 × g and the cleared lysate was supplemented with 10 mm CaCl2. For the preparation of a mock lysate, Me2SO was added without dissolved DSS. For direct analysis, lysate was then supplemented with 5× SDS sample buffer, and proteins were separated on an 8–16% gradient gel or a 5% gel, Western-blotted, and DC-SIGN was detected with a polyclonal antibody. To generate CemT4 and A2.01 lysates, cells were harvested, washed twice with CLB, and lysed in CLB containing 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS for 1 h at 4 °C. Insoluble debris was pelleted by centrifugation at 10 000 × g for 10 min at 4 °C, and the supernatant was used for immunoprecipitations. Immunoprecipitations and Co-immunoprecipitations—Affinity beads were generated by coupling the monoclonal antibodies Q4120 (anti-CD4) and the anti-DC-SIGN antibody as well as mouse IgG1 to cyanogen bromide-activated Sepharose according to the manufacturer's instructions. For precipitation of DC-SIGN complexes, 0.5 ml lysates were precipitated with 2.5 μg of anti-DC-SIGN antibody bound to 20 μl of protein G beads. For detecting cross-linking of the CD4 and DC-SIGN receptors, 0.5 ml lysates were precipitated with 20 μl of Q4120 beads (specific for CD4) or 20 μl of anti-DC-SIGN beads. For co-immunoprecipitating gp120 and DC-SIGN, 10 μl of anti-gp120 serum-bound to 20 μl of protein G beads were incubated with 1 ml of lysate. All incubations were performed for 1 h at room temperature. For co-immunoprecipitations, 2.5 μg of gp120 was incubated with the lysate for 1 h at room temperature prior to addition to the antibody bound to the beads. For precipitations with mannan beads the beads were washed with HBS, pH 7.5, 10 mm CaCl2, and 0.1% (v/v) Triton X-100 and then incubated with the lysate. After binding the beads were washed 5× with HBS, pH 7.5, 10 mm CaCl2, and 0.1% (v/v) Triton X-100 and resuspended in 1× SDS sample buffer. If indicated, DTT was added to a concentration of 50 mm for reduction of the disulfide bonds. Proteins were separated on an 8–16% gradient gel and again Western-blotted. DC-SIGN and CD4 were detected using polyclonal antibodies. Affinity Purification of DC-SIGN Complexes—Lysate from about 9 × 107 iMDDCs that had been treated with DSS was first passed over ∼100 μl of IgG1-Sepharose to absorb nonspecific binding proteins and subsequently passed over ∼100 μl of anti-DC-SIGN beads (containing ∼100–150 μg of immobilized antibody). The beads were then washed with TBS, pH 8.0 containing 0.1% (v/v) Triton X-100. Complexes were eluted by boiling the beads for 5 min in 1× SDS sample buffer. SDS-PAGE and Protein Identification Using Mass Spectrometry— The eluate (50 μl) was loaded onto a 8–16% gradient gel (Gradipore, Frenchs Forest, NSW), and proteins were separated with a constant current of 25 mA per gel. Proteins were stained with Coomassie Brilliant Blue as described previously (18Bernhard O.K. Sheil M.M. Cunningham A.L. Biochemistry. 2004; 43: 256-264Crossref PubMed Scopus (15) Google Scholar). Excised gel slices were washed, subjected to in-gel digest, and the tryptic peptides were extracted and purified as described elsewhere (18Bernhard O.K. Sheil M.M. Cunningham A.L. Biochemistry. 2004; 43: 256-264Crossref PubMed Scopus (15) Google Scholar). Electrospray ionization mass spectrometry (ESI-MS) analyses were carried out using a Quadrupole-Time-of-flight (Q-Tof 2) instrument (Micromass/Waters, Manchester, UK) in nanoelectrospray mode. Typically 5–8 μl of sample were loaded into a coated glass capillary (Protana, Odense, Denmark). The mass spectrometry data acquisition was performed as described in our previous studies (18Bernhard O.K. Sheil M.M. Cunningham A.L. Biochemistry. 2004; 43: 256-264Crossref PubMed Scopus (15) Google Scholar, 19Bernhard O.K. Burgess J.A. Hochgrebe T. Sheil M.M. Cunningham A.L. Proteomics. 2003; 2: 139-146Crossref Scopus (14) Google Scholar), and all multi-charged ions that were clearly distinguishable from the background were selected for subsequent fragmentation and analyses (in so-called tandem mass spectrometry or MS/MS experiments). Spectra were interpreted and matched using the MASCOT search engine (www.matrixscience.com/cgi/index.pl?page=/search_form_select.html) and checked manually if the score obtained was below 50. FRET Measurements on CD4-DC-SIGN Association—The monoclonal antibodies Q4120, OKT4, WM82, and anti-DC-SIGN were labeled with either fluorescein or Cy3 according to the manufacturer's instructions. Immature MDDCs were labeled similarly as described for flow cytometry (18Bernhard O.K. Sheil M.M. Cunningham A.L. Biochemistry. 2004; 43: 256-264Crossref PubMed Scopus (15) Google Scholar) without fixing the cells. Cells were stained with unconjugated CD4 and DC-SIGN antibody (blank sample), with unconjugated DC-SIGN antibody and fluorescein conjugated CD4 antibody (donor sample), with Cy3-conjugated DC-SIGN antibody and unconjugated CD4 antibody (acceptor sample) and Cy3-conjugated DC-SIGN antibody and fluorescein-conjugated CD4 antibody (FRET sample). Flow cytometry analysis was carried out using a FACScalibur (BD Biosciences) with a 488 nm laser. Cells were gated to exclude debris and dead cells and mean fluorescence intensities were used for analysis. Two fluorescence intensities were collected, FL1 at 530 nm and FL2 at 585 nm. All samples were stored on ice until analyzed. Cells were subsequently incubated at 37 °C for a given time and then analyzed again. For cell stimulation, 100 ng/ml PMA was added at the same time to each sample before the incubation period. Nothing was added to mock-treated samples. Energy transfer efficiencies were determined as donor fluorescence quenching and calculated using the formula in Equation 1.E=[{FL1(Donor)−FL1(FRET)}/{FL1(Donor)−FL1(Acceptor)}]×100% (Eq. 1) Complexes of High Molecular Weight-containing DC-SIGN Can Be Detected after Cross-linking—Because DC-SIGN was reported to form tetramers based on the properties of the recombinantly expressed extracellular domain, we investigated here, whether DC-SIGN is engaged in lateral protein interactions on the surface of iMDDCs. Cells were treated with DSS or subjected to a mock treatment (see above), and DC-SIGN was visualized after PAGE separation and Western blotting using a DC-SIGN-specific polyclonal antibody. Fig. 1 shows the detection of DC-SIGN in lysates and immunoprecipitates from mock or DSS-treated cells. In a whole cell lysate from mock-treated iMDDCs, DC-SIGN shows an apparent molecular mass of around 50 kDa, which is a good approximation to the theoretical molecular mass of 44 kDa from PAGE results alone. DC-SIGN present in a clarified lysate from mock-treated cells shows a similar molecular mass to that in untreated cells, whereas DC-SIGN present in the lysate from cells treated with DSS has an apparent molecular mass of more than 300 kDa. This indicates that DC-SIGN was cross-linked into large complexes. Immunoprecipitation of DC-SIGN with a murine monoclonal antibody confirms that the detected species are monomers and large complexes of DC-SIGN. The band at ∼250 kDa in the DSS sample from precipitation with anti-DC-SIGN antibody (and similar bands in later figures) is the murine anti-DC-SIGN antibody that is weakly recognized by the anti-rabbit secondary antibody. The Complexes are Homo-oligomers of DC-SIGN—To investigate whether the identified large complexes of DC-SIGN are homo-oligomers or associations with other molecules, complexes were affinity-purified from ∼9 × 107 iMDDCs after DSS treatment, separated on a polyacrylamide gel and visualized with Coomassie Brilliant Blue. Purification with IgG1 Sepharose prior to the DC-SIGN purification was included as a control for nonspecific binding. Fig. 2 shows the visualized proteins from the DC-SIGN and the control purification. A band of high molecular mass can be seen in the lane containing the purified DC-SIGN complexes whereas a band of around 250 kDa can be seen in the control lane. Mass spectrometry analysis was carried out on the excised bands to determine their protein composition. Table I shows the peptides detected in the sample derived from the in-gel digest from the visible DC-SIGN complex. Using the sensitive nano-ESI-MS and nano-ESI-MS/MS-techniques, ten tryptic peptides from DC-SIGN were detected whereas no other protein was identified showing that the complexes are most likely homo-oligomers of DC-SIGN. Further, no ions that could represent cross-linked peptides could be detected. The band in the control purification was identified as murine IgG1 (data not shown) suggesting that some antibody eluted from the beads during boiling of the sample. The intensity of the antibody-derived band was subject to run-to-run differences and was weaker in subsequent control purifications. Further, in some DC-SIGN purifications confirming the isolation of complexes containing only DC-SIGN, IgG-derived peptides could be detected suggesting that the intensity of the antibody-derived band varies from sample to sample (data not shown).Table IDC-SIGN-derived peptides identified in the purified complexesPrecursorPeptideSequencePositionMASCOT SCOREm/zaExperimentally determined m/z value.massbTheoretical uncharged monoisotopic peptide mass.818.91635.8SAEEQNFLQLQSSR296-30987784.91567.9LQQLGLLEEEQLR9-2158760.91519.8QQEIYQELTQLK235-24632753.41504.8LQEIYQELTQLK97-10862663.81325.6M*QEIYQELTRcM* indicates oxidized methionine.166-17570655.81309.6MQEIYQELTR166-17518654.31306.7QQEIYQELTR189-198dProtein produces multiple peptides of the same sequence, only the first position is shown.37646.31291.7LQEIYQELTR120-12952609.31216.6VPSSISQEQSR63-7353528.31054.6EVGAQLVVIK286-29547457.3912.5AAVGELPEK109-117dProtein produces multiple peptides of the same sequence, only the first position is shown.60a Experimentally determined m/z value.b Theoretical uncharged monoisotopic peptide mass.c M* indicates oxidized methionine.d Protein produces multiple peptides of the same sequence, only the first position is shown. Open table in a new tab The High Molecular Weight Complexes Are Tetramers—In order to deduce the oligomerization state of the complexes, iMDDCs were subjected to cross-linking with different concentrations of DSS. Cells were then lysed and the cleared lysate separated on 8–16% gradient gels as well as on 5% gels in order to increase the resolution in the high molecular weight region of the gel. Fig. 3 shows representative DC-SIGN oligomers detected in the cleared lysate after cells had been treated with increasing concentrations of DSS. It can be seen in Fig. 3A that the amount of monomeric DC-SIGN gradually decreases and that bands of high apparent molecular weight appear with increasing concentration of the cross-linker. Fig. 3B shows resolution of the high molecular mass bands into three individual bands indicating that they consist of DC-SIGN dimers, trimers and tetramers. Dimers are detected at an apparent molecular mass of around 100 kDa slightly below a nonspecific recognized band. Trimer bands migrate at ∼150 kDa whereas the tetramer bands are detected slightly below 250 kDa. In each case the migration is slightly above the predicted molecular mass, which is probably a consequence of some globularity being retained through the cross-linking that interferes with the normal migration of the proteins through the gel. A similar effect can be seen in Fig. 2, where the antibody of a molecular mass of 150 kDa migrates at an apparent molecular mass of 250 kDa. This is probably because of the globularity of the antibody that is retained through the intact interchain disulfide bridges. The Tetramers Bind to Mannan and HIV-1 gp120 —It has been proposed that DC-SIGN tetramerization is required for high affinity ligand binding. An increased affinity of the extracellular domain of recombinant DC-SIGN for mannosylated BSA was detected when the neck region required for oligomerization was present (5Mitchell D.A. Fadden A.J. Drickamer K. J. Biol. Chem. 2001; 276: 28939-28945Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). To directly investigate whether HIV gp120 as well as yeast-derived mannan binds with higher affinity to DC-SIGN tetramers than to monomers on dendritic cells, we conducted co-immunoprecipitation experiments between the molecules. Fig. 4 shows the co-immunoprecipitation of DC-SIGN tetramers (arrow) with HIV gp120 whereas DC-SIGN monomers, detected by precipitation with a murine anti-DC-SIGN antibody, were not detected in Fig. 4B. In Fig. 4A nonspecific bands from contaminating antibody obscured in Fig. 4A at ∼50 kDa where the gel had not been reduced. This shows tha
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