Tumor Biomarker Glycoproteins in the Seminal Plasma of Healthy Human Males Are Endogenous Ligands for DC-SIGN
2011; Elsevier BV; Volume: 11; Issue: 1 Linguagem: Inglês
10.1074/mcp.m111.008730
ISSN1535-9484
AutoresGary F. Clark, Paola Grassi, Poh‐Choo Pang, Maria Panico, David Lafrenz, Erma Z. Drobnis, Michael R. Baldwin, Howard R. Morris, Stuart M. Haslam, Sophia Schedin‐Weiss, Wei Sun, Anne Dell,
Tópico(s)Genetic factors in colorectal cancer
ResumoDC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewisx and Lewisy carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewisx and Lewisy sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus. DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewisx and Lewisy carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewisx and Lewisy sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus. Human C-type lectins are associated with a wide variety of different immune cell types and act as pattern-recognition receptors that play an essential role in the detection of many different pathogens that induce innate immune responses (1Garcia-Vallejo J.J. van Kooyk Y. Endogenous ligands for C-type lectin receptors: the true regulators of immune homeostasis.Immunol. Rev. 2009; 230: 22-37Crossref PubMed Scopus (98) Google Scholar). Prominent among this repertoire of receptors is DC-SIGN 1The abbreviations used are:DC-SIGNdendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrinPAPprostatic acid phosphatasePCIprotein C inhibitorPSAprostate specific antigen. (2Curtis B.M. Scharnowske S. Watson A.J. Sequence and expression of a membrane-associated C-type lectin that exhibits CD4-independent binding of human immunodeficiency virus envelope glycoprotein gp120.Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 8356-8360Crossref PubMed Scopus (342) Google Scholar, 3Geijtenbeek T.B. Torensma R. van Vliet S.J. van Duijnhoven G.C. Adema G.J. van Kooyk Y. Figdor C.G. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses.Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1467) Google Scholar). DC-SIGN binds to high mannose type N-glycans and several Lewis type carbohydrate sequences (Lewisa, Lewisb, Lewisx, Lewisy, sulfo-Lewisa, and pseudo-Lewisy antigens) (Table I) (4Appelmelk B.J. Van Die I. Van Vliet S.J. Vandenbroucke-Grauls C.M. Geijtenbeek T.B. Van Kooyk Y. Cutting edge: carbohydrate profiling identifies new pathogens that tnteract with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells.J. Immunol. 2003; 170: 1635-1639Crossref PubMed Scopus (383) Google Scholar, 5Mitchell D.A. Fadden A.J. Drickamer K. A novel mechanism of carbohydrate recognition by the C-type lectins DC- SIGN and DC-SIGNR: Subunit organization and binding to multivalent ligands.J. Biol. Chem. 2001; 276: 28939-28945Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar).Table ICarbohydrate Ligands for DC-SIGN Open table in a new tab dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin prostatic acid phosphatase protein C inhibitor prostate specific antigen. Though these Lewis type sequences are expressed on many human glycoproteins, only a select subset has been reported to bind to DC-SIGN. They include bile-salt stimulated lipase (6Naarding M.A. Dirac A.M. Ludwig I.S. Speijer D. Lindquist S. Vestman E.L. Stax M.J. Geijtenbeek T.B. Pollakis G. Hernell O. Paxton W.A. Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells.Antimicrob. Agents Chemother. 2006; 50: 3367-3374Crossref PubMed Scopus (65) Google Scholar), butyrophilin (7Malcherek G. Mayr L. Roda-Navarro P. Rhodes D. Miller N. Trowsdale J. The B7 homolog butyrophilin BTN2A1 is a novel ligand for DC-SIGN.J. Immunol. 2007; 179: 3804-3811Crossref PubMed Scopus (42) Google Scholar), carcinoembryonic antigen (8van Gisbergen K.P. Aarnoudse C.A. Meijer G.A. Geijtenbeek T.B. van Kooyk Y. Dendritic cells recognize tumor-specific glycosylation of carcinoembryonic antigen on colorectal cancer cells through dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin.Cancer Res. 2005; 65: 5935-5944Crossref PubMed Scopus (141) Google Scholar), carcinoma embryonic antigen related cell adhesion molecule, (9Bogoevska V. Horst A. Klampe B. Lucka L. Wagener C. Nollau P. CEACAM1, an adhesion molecule of human granulocytes, is fucosylated by fucosyltransferase IX and interacts with DC-SIGN of dendritic cells via Lewis x residues.Glycobiology. 2006; 16: 197-209Crossref PubMed Scopus (53) Google Scholar), ICAM-2 (10Garcia-Vallejo J.J. van Liempt E. da Costa Martins P. Beckers C. van het Hof B. Gringhuis S.I. Zwaginga J.J. van Dijk W. Geijtenbeek T.B. van Kooyk Y. van Die I. DC-SIGN mediates adhesion and rolling of dendritic cells on primary human umbilical vein endothelial cells through LewisY antigen expressed on ICAM-2.Mol. Immunol. 2008; 45: 2359-2369Crossref PubMed Scopus (58) Google Scholar, 11Geijtenbeek T.B. Krooshoop D.J. Bleijs D.A. van Vliet S.J. van Duijnhoven G.C. Grabovsky V. Alon R. Figdor C.G. van Kooyk Y. DC-SIGN-ICAM-2 interaction mediates dendritic cell trafficking.Nat. Immunol. 2000; 1: 353-357Crossref PubMed Scopus (434) Google Scholar), ICAM-3 (12Bogoevska V. Nollau P. Lucka L. Grunow D. Klampe B. Uotila L.M. Samsen A. Gahmberg C.G. Wagener C. DC-SIGN binds ICAM-3 isolated from peripheral human leukocytes through Lewis x residues.Glycobiology. 2007; 17: 324-333Crossref PubMed Scopus (29) Google Scholar), and Mac-1 (13van Gisbergen K.P. Ludwig I.S. Geijtenbeek T.B. van Kooyk Y. Interactions of DC-SIGN with Mac-1 and CEACAM1 regulate contact between dendritic cells and neutrophils.FEBS Lett. 2005; 579: 6159-6168Crossref PubMed Scopus (88) Google Scholar). These findings indicate that a very specific orientation and/or density of Lewis type sequences on a glycoconjugate could enable DC-SIGN binding, and not simply the presence or absence of specific carbohydrate epitopes (1Garcia-Vallejo J.J. van Kooyk Y. Endogenous ligands for C-type lectin receptors: the true regulators of immune homeostasis.Immunol. Rev. 2009; 230: 22-37Crossref PubMed Scopus (98) Google Scholar). Lewisx and Lewisy sequences are profusely expressed in the human male urogenital tract. The results of an early study confirmed that N-glycans bearing these sequences were linked to prostate specific antigen (PSA), also known as γ-seminoprotein, kallikrein-3 or semenogelase (14van Halbeek H. Gerwig G.J. Vliegenthart J.F. Tsuda R. Hara M. Akiyama K. Schmid K. Occurrence of the Y determinant on the N-glycosidic carbohydrate units of human γ-seminoprotein.Biochem. Biophys. Res. Commun. 1985; 131: 507-514Crossref PubMed Scopus (33) Google Scholar). Further analysis of seminal plasma glycoproteins and mucins revealed that O-glycans terminated with Lewisx and Lewisy sequences were linked only to high MW mucins (>500 kDa) (15Hanisch F.G. Egge H. Peter-Katalinić J. Uhlenbruck G. Structure of neutral oligosaccharides derived from mucus glycoproteins of human seminal plasma.Eur J Biochem. 1986; 155: 239-247Crossref PubMed Scopus (26) Google Scholar). Free oligosaccharides capped with these sequences were also present in human seminal plasma (16Chalabi S. Easton R.L. Patankar M.S. Lattanzio F.A. Morrison J.C. Panico M. Morris H.R. Dell A. Clark G.F. The expression of free oligosaccharides in human seminal plasma.J. Biol. Chem. 2002; 277: 32562-32570Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). More recent ultrasensitive mass spectrometric analyses have confirmed the prolific expression of N-glycans bearing antennae terminated with multiple Lewisx and Lewisy epitopes on glycoproteins associated with human sperm and seminal plasma (17Pang P.C. Tissot B. Drobnis E.Z. Morris H.R. Dell A. Clark G.F. Analysis of the human seminal plasma glycome reveals the presence of immunomodulatory carbohydrate functional groups.J. Proteome Res. 2009; 8: 4906-4915Crossref PubMed Scopus (44) Google Scholar, 18Pang P.C. Tissot B. Drobnis E.Z. Sutovsky P. Morris H.R. Clark G.F. Dell A. Expression of bisecting type and Lewisx/Lewisy terminated N-glycans on human sperm.J. Biol. Chem. 2007; 282: 36593-36602Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). These observations indicated that there could be many endogenous glycoprotein ligands for DC-SIGN in the male reproductive system. The current study was undertaken to identify the major seminal plasma glycoproteins that bind to this immune lectin. Lotus tetragonolobus seeds were purchased from B&T World Seeds (Aigues-Vives, France). The lectin associated with these seeds was isolated by employing the same procedure developed previously by Smith and coworkers (19Yan L. Wilkins P.P. Alvarez Manilla G. Do S.I. Smith D.F. Cummings R.D. Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Le(x) determinant.Glycoconj. J. 1997; 14: 45-55Crossref PubMed Scopus (71) Google Scholar). Lotus lectin-agarose was generated by coupling freshly prepared lectin to Affi-Gel 15 (BioRad, Richmond, CA) at a final concentration of 26 mg/ml of packed gel. Amicon Ultra cartridges were purchased from Millipore (Carrightwohill, Co. Cork, Ireland). Protease inhibitor mixture set III (EDTA free) and all glycosidases except bovine testes β-galactosidase were purchased from CalBiochem (Gibbstown, NJ). Dulbecco’s phosphate buffered saline (PBS) was obtained from Invitrogen (Carlsbad, CA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO) except as noted. This study was approved by the Health Sciences Institutional Review Board of the University of Missouri-Columbia. All research procedures involving materials originating from patients were conducted following the approved study protocol. Semen was obtained from fertile individuals who had fathered a child during the previous two years. All the specimens used in this study also had normal semen parameters based on World Health Organization (WHO) guidelines (20WHO (1999) World Health Organization laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge University Press, Cambridge, UK.Google Scholar). Each semen sample was allowed to liquefy for 30 min at room temperature, diluted with an equal volume of phosphate-buffered saline (PBS), and centrifuged at 600 × g for 5 min. The supernatant was collected and centrifuged at 2000 × g for 5 min. The acellular supernatants containing diluted seminal plasma were collected individually and stored frozen at −20°C. This separation was carried out essentially as described previously (19Yan L. Wilkins P.P. Alvarez Manilla G. Do S.I. Smith D.F. Cummings R.D. Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Le(x) determinant.Glycoconj. J. 1997; 14: 45-55Crossref PubMed Scopus (71) Google Scholar), with some modifications. Seminal plasma samples were thawed under running tap water and centrifuged at 18,000 × g at 4°C for 30 min to remove any insoluble precipitate. The supernatants were harvested and supplemented with protease inhibitors according to the manufacturer’s instructions. This fraction was adjusted to a Triton-X-100 concentration of 0.3% (v/v) by the addition of a 5% aqueous solution of this detergent in PBS. The detergent solubilized fraction was incubated at 37°C for 30 min. A small column containing 1 ml packed volume of Lotus lectin-agarose was equilibrated in PBS containing 0.3% Triton-X-100. A solubilized seminal plasma sample (2 ml) was applied to this column and eluted under gravity. The flow through was collected and reapplied to the column. After elution, this process was repeated again. The column was eluted under gravity, and fractions (2 ml) were collected and monitored at 280 nm until the absorbance dropped below 0.01. The column was stopped, and the matrix was eluted with 1 ml of PBS containing 0.1 m fucose. The eluant was collected, and the column was capped and incubated for 30 min. The column was opened, and 2 one ml aliquots of buffer containing 0.1 m fucose were applied to the column and collected separately. The column was eluted with PBS, collecting 1-ml fractions. Fractions were analyzed by absorbance at 280 nm to detect the eluted glycoproteins. Fractions containing the bound glycoproteins were pooled, concentrated on Ultrafree cartridges against PBS to remove fucose, and stored frozen at −20°C until analyzed. Proteins from individual fractions eluted from Lotus lectin-agarose were precipitated in 10% trichloroacetic acid and analyzed by SDS-gel electrophoresis as described previously (21Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar). To perform proteomic analysis of the Coomassie blue stained bands, they were excised, extracted with acetonitrile to remove the stain, and dried in a vacuum centrifuge. Proteins were reduced with 10 mm dithiothreitol, alkylated with 55 mm iodoacetic acid, digested with 20 μl of trypsin working solution (Promega sequencing grade modified trypsin, prepared according to manufacturer’s instructions, Promega, Madison, WI) and incubated overnight at 37°C. Peptides were released from gel slices by incubation with 0.1% trifluoroacetic acid for 10 min, followed by the addition of acetonitrile for 15 min at 37°C. This extraction process was repeated once more. Tryptic peptides were then dried on a vacuum centrifuge and resuspended in 80 μl 0.1% trifluoroacetic acid prior to mass spectrometry. For offline liquid chromatography matrix-assisted laser desorption ionization/time of flight (MALDI TOF/TOF)-MS analysis, tryptic peptides were separated by using the Ultimate 3000 LC system (Dionex, Sunnyvale CA), fitted with a Pepmap analytical C-18 nanocapillary (75 μm internal diameter ×15 cm length; Dionex). The digest was loaded onto the column and eluted using solvent A (0.1% (v/v) formic acid in 2% (v/v) acetonitrile) and solvent B (0.1% (v/v) formic acid in 90% (v/v) acetonitrile), in the following gradient: 0–60% solvent B (0–60 min), 60–90% solvent B (60–61 min), 90% solvent B (61–66 min) and 100% solvent A (66–67 min). Eluting fractions were mixed with α -cyano-4hydroxy cinnamic acid matrix and spotted onto a metal MALDI target plate using a Probot (LC Packings, Dionex, Sunnyvale, CA). Peptides were analyzed by MALDI-MS and MS-MS profiling on an Applied Biosystems 4800 MALDITOF/TOF mass spectrometer. The ten most abundant ions in each sample were sequenced. Peak picking was conducted using GPS Explorer software version 3.6 (Applied Biosystems, Foster City, CA). A signal-to-noise threshold of 10 was used. Sequazyme peptide mass standards were used as external standards for calibration purposes and no contaminant ions were excluded. For MS/MS experiments, peak list generation and database searching were conducted using GPS Explorer software version 3.6 (Applied Biosystems) with the default parameters. Both LC-MS and LC-MS/MS data were used to search 283,454 entries in release 54.2 of the SwissProt database with version 2.2 of the Mascot database search algorithm (www.matrixscience.com). The following parameters were implemented for this analysis: (1) peptide masses were fixed as monoisotopic; (2) partial oxidation of methionine residues was considered; (3) partial carboxymethylation of cysteine residues was considered; (4) the mass tolerance was set at 75 ppm for precursor ions and 0.1 Da for fragment ions; and (5) tryptic digests were assumed to have no more than one missed cleavage. Peptide matches from both MS and MS/MS data were used to generate probability-based Mowse protein scores. Scores greater than 55 were considered significant (p < 0.05) (22Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6842) Google Scholar). The search for other potential proteins in the Lotus lectin-binding fractions from seminal plasma was broadened by increasing the mass tolerance for precursor ions to 100 ppm, enlarging the taxonomy to include all species, and allowing the possibility of two missed cleavages. Each sample was subjected to reduction, carboxymethylation, and tryptic digestion as described previously (23Sutton-Smith M. Wong N.K. Khoo K.H. Wu S.W. Yu S.Y. Patankar M.S. Easton R. Lattanzio F.A. Morris H.R. Dell A. Clark G.F. Analysis of protein-linked glycosylation in a sperm-somatic cell adhesion system.Glycobiology. 2007; 17: 553-567Crossref PubMed Scopus (21) Google Scholar). Peptide N-glycosidase F digestion of the tryptic glycopeptides was carried out in 50 mm ammonium bicarbonate, pH 8.5, for 24 h at 37°C with 5 units of enzyme (Roche Applied Science, UK). The released N-glycans were purified from O-glycopeptides and peptides by chromatography on a Sep-Pak C18 cartridge (Waters Corp., Milford, MA) as described previously (24Jang-Lee J. North S.J. Sutton-Smith M. Goldberg D. Panico M. Morris H. Haslam S. Dell A. Glycomic profiling of cells and tissues by mass spectrometry: fingerprinting and sequencing methodologies.Methods Enzymol. 2006; 415: 59-86Crossref PubMed Scopus (137) Google Scholar). The purified native N-glycans were either subsequently derivatized or subjected to modifications before derivatization. They were subjected to the sodium hydroxide permethylation procedure as described previously before mass spectrometric analyses (24Jang-Lee J. North S.J. Sutton-Smith M. Goldberg D. Panico M. Morris H. Haslam S. Dell A. Glycomic profiling of cells and tissues by mass spectrometry: fingerprinting and sequencing methodologies.Methods Enzymol. 2006; 415: 59-86Crossref PubMed Scopus (137) Google Scholar). MALDI-TOF data were acquired on a Voyager-DE STR mass spectrometer (Applied Biosystems, Foster City, CA) in the reflectron mode with delayed extraction. Permethylated samples were dissolved in 10 μl of 70% (v/v) aqueous methanol, and 1 μl of dissolved sample was premixed with 1 μl of matrix (20 mg/ml 2,5-dihydroxybenzoic acid in 70% (v/v) aqueous methanol), spotted onto a target plate, and dried under vacuum. Further MS/MS analyses of peaks observed in the MS spectra were carried out by using a 4800 MALDI-TOF/TOF (Applied Biosystems) mass spectrometer in positive ion mode (M + Na)+. The collision energy was set to 1 kV, and argon was used as collision gas. Samples were dissolved in 10 μL of methanol, and 1 μL was mixed at a 1:1 ratio (v/v) with 2,5-dihydroxybenzoic acid (20 mg/ml in 70% methanol in water) as matrix. The MS and MS/MS data were processed by using Data Explorer 4.9 Software (Applied Biosystems, UK). The mass spectra were baseline corrected (default settings), noise filtered (with correction factor of 0.7), and then converted to ASCII format. The processed spectra were subjected to manual assignment and annotation with the aid of a glycobioinformatics tool known as GlycoWorkBench (25Ceroni A. Maass K. Geyer H. Geyer R. Dell A. Haslam S.M. GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans.J. Proteome Res. 2008; 7: 1650-1659Crossref PubMed Scopus (783) Google Scholar). Peak picking was done manually, and proposed assignments for the selected peaks were based on molecular mass composition of the 12C isotope together with knowledge of the biosynthetic pathways. The proposed structures were then confirmed by data obtained from MS/MS experiments. DC-SIGN-Fc consists of the extracellular portion of DC-SIGN (amino acid residues 64–404) fused at the COOH terminus to a human IgG1-Fc fragment (R&D Systems, Minneapolis, MN). The soluble DC-SIGN binding assay was performed as follows. Total seminal plasma proteins or fractions derived from the Lotus lectin-agarose column as indicated (∼20 μg total protein for each) were resolved by SDS-gel electrophoresis and transferred to Immobilon-P PVDF membranes as described previously (21Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar, 26Towbin H. Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 4350-4354Crossref PubMed Scopus (44969) Google Scholar). Following transfer, PVDF membranes were rinsed three times with Dulbecco’s PBS (DPBS) and then blocked for 30 min in StartingBlock Blocking Buffer (Thermo Fisher Scientific, Rockford, IL) at 23°C. Soluble DC-SIGN-Fc was diluted in StartingBlock Blocking Buffer to a final concentration of 15 nm and incubated with membranes for 2 h at RT. Unbound DC-SIGN-Fc was removed by washing membranes thrice with Dulbecco’s PBS for 5 min per wash. Membranes were then incubated with a goat anti-human IgG-HRP conjugate for 15 min, washed three times as described above, and incubated with chemiluminescent substrate for 5 min (SuperSignal West Pico, Thermo Fisher Scientific, Rockford, IL). Images were captured by using a Cell Biosciences AlphaImager system. The following antibodies were used in this study as indicated below: Goat anti-human galectin3-binding protein (1:500, R&D Systems, Minneapolis, MN), Mouse anti-human clusterin (1:250, Santa Cruz Biotechnology, Santa Cruz, CA), Mouse anti-human prostatic acid phosphatase (1:100, Santa Cruz Biotechnology), and rabbit anti-human protein C inhibitor (1:640, Abcam, Cambridge, MA). Seminal plasma proteins derived from the Lotus lectin-agarose column (Fraction 11, ∼50 μg total protein) were diluted in 500 μl of DPBS and incubated overnight at 4°C either alone or in the presence of 3.25 μg soluble DC-SIGN-Fc (Final concentration = 100 nm). Following incubation, samples were transferred to fresh tubes containing 20 μl of protein-A Sepharose beads which had been previously equilibrated in DPBS. Samples were incubated for a further 60 min at 4°C with continuous rotation. Beads were collected by brief centrifugation (5000 × g, 1 min) and subjected to five washes with DPBS. Bound proteins were released from the beads by boiling in 40 μl of Laemmli sample buffer, resolved by SDS-gel electrophoresis and transferred to Immobilon-P PVDF membranes as described previously (21Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar, 26Towbin H. Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 4350-4354Crossref PubMed Scopus (44969) Google Scholar). Following transfer, PVDF membranes were rinsed three times with DPBS and then blocked for 30 min in 3% w/v nonfat milk in DPBS at 23°C. The indicated antibodies were diluted in 3% w/v nonfat milk in DPBS and incubated with membranes for 2 h at room temperature. Unbound antibody was removed by washing membranes three times with DPBS for 5 min per wash. Membranes were then incubated with appropriate secondary antibody-HRP conjugates for 30 min, washed three times as described above, and incubated with chemiluminescent substrate for 5 min (SuperSignal West Dura, Thermo Fisher Scientific, Rockford, IL). Images were captured using a Cell Biosciences AlphaImager system. Human seminal plasma was isolated as previously described (17Pang P.C. Tissot B. Drobnis E.Z. Morris H.R. Dell A. Clark G.F. Analysis of the human seminal plasma glycome reveals the presence of immunomodulatory carbohydrate functional groups.J. Proteome Res. 2009; 8: 4906-4915Crossref PubMed Scopus (44) Google Scholar). This fraction was analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 1). The separated proteins were subjected to Western blot analysis with DC-SIGN-Fc. Only very weak binding of glycoproteins in the whole seminal plasma fraction was detected by using this approach, except for a minor band migrating at about 30–35 kDa (Fig. 1). This result indicated that there were very marginal amounts of glycoprotein ligands for DC-SIGN in human seminal plasma. This low level of DC-SIGN-Fc binding during Western blot analysis was inconsistent with previous results indicating substantial modification of seminal plasma glycoproteins with Lewisx and Lewisy type sequences (17Pang P.C. Tissot B. Drobnis E.Z. Morris H.R. Dell A. Clark G.F. Analysis of the human seminal plasma glycome reveals the presence of immunomodulatory carbohydrate functional groups.J. Proteome Res. 2009; 8: 4906-4915Crossref PubMed Scopus (44) Google Scholar). Two potential reasons for this lack of binding were considered. One hypothesis was that the amount of DC-SIGN binding glycoproteins was substantially lower than expected based upon the glycomic analysis of human seminal plasma (17Pang P.C. Tissot B. Drobnis E.Z. Morris H.R. Dell A. Clark G.F. Analysis of the human seminal plasma glycome reveals the presence of immunomodulatory carbohydrate functional groups.J. Proteome Res. 2009; 8: 4906-4915Crossref PubMed Scopus (44) Google Scholar). Another possibility was that other proteins present in seminal plasma were interfering with the interaction of DC-SIGN with its glycoprotein ligands during Western blot analyses. One approach to evaluate both of these possibilities was to selectively enrich for seminal plasma glycoproteins that bear Lewisx and Lewisy sequences. Lotus tetragonolobus agglutinin is a lectin that readily binds to glycans terminated with these sequences (19Yan L. Wilkins P.P. Alvarez Manilla G. Do S.I. Smith D.F. Cummings R.D. Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Le(x) determinant.Glycoconj. J. 1997; 14: 45-55Crossref PubMed Scopus (71) Google Scholar, 27Pereira M.E. Kabat E.A. Specificity of purified hemagglutinin (lectin) from Lotus tetragonolobus.Biochemistry. 1974; 13: 3184-3192Crossref PubMed Scopus (186) Google Scholar). Like DC-SIGN, this lectin also exists as a tetramer (5Mitchell D.A. Fadden A.J. Drickamer K. A novel mechanism of carbohydrate recognition by the C-type lectins DC- SIGN and DC-SIGNR: Subunit organization and binding to multivalent ligands.J. Biol. Chem. 2001; 276: 28939-28945Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 28Moreno F.B. de Oliveira T.M. Martil D.E. Viçoti M.M. Bezerra G.A. Abrego J.R. Cavada B.S. Filgueira de Azevedo Jr., W. Identification of a new quaternary association for legume lectins.J. Struct. Biol. 2008; 161: 133-143Crossref PubMed Scopus (26) Go
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