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Mechanism for Recognition of an Unusual Mycobacterial Glycolipid by the Macrophage Receptor Mincle*

Open AccessPublished:August 19, 2013DOI:https://doi.org/10.1074/jbc.M113.497149

      Background:

      Mincle facilitates establishment of persistent infections of macrophages by Mycobacterium tuberculosis.
      Results: The mechanism of mincle binding to mycobacterial glycolipids has been defined, and inhibitors have been synthesized.
      Conclusion: Mincle binds to both the sugar portion of the glycolipid and the hydrocarbon tail.
      Significance: The results suggest ways to manipulate the response to mycobacteria and to improve adjuvants that stimulate the immune system.
      Binding of the macrophage lectin mincle to trehalose dimycolate, a key glycolipid virulence factor on the surface of Mycobacterium tuberculosis and Mycobacterium bovis, initiates responses that can lead both to toxicity and to protection of these pathogens from destruction. Crystallographic structural analysis, site-directed mutagenesis, and binding studies with glycolipid mimics have been used to define an extended binding site in the C-type carbohydrate recognition domain (CRD) of bovine mincle that encompasses both the headgroup and a portion of the attached acyl chains. One glucose residue of the trehalose Glcα1–1Glcα headgroup is liganded to a Ca2+ in a manner common to many C-type CRDs, whereas the second glucose residue is accommodated in a novel secondary binding site. The additional contacts in the secondary site lead to a 36-fold higher affinity for trehalose compared with glucose. An adjacent hydrophobic groove, not seen in other C-type CRDs, provides a docking site for one of the acyl chains attached to the trehalose, which can be targeted with small molecule analogs of trehalose dimycolate that bind with 52-fold higher affinity than trehalose. The data demonstrate how mincle bridges between the surfaces of the macrophage and the mycobacterium and suggest the possibility of disrupting this interaction. In addition, the results may provide a basis for design of adjuvants that mimic the ability of mycobacteria to stimulate a response to immunization that can be employed in vaccine development.

      Introduction

      The balance between latent and active forms of tuberculosis lies in a complex set of interactions between mycobacteria and the host immune system (
      • Maartens G.
      • Wilkinson R.J.
      Tuberculosis.
      ,
      • Schäfer G.
      • Jacobs M.
      • Wilkinson R.J.
      • Brown G.D.
      Non-opsonic recognition of Mycobacterium tuberculosis by phagocytes.
      ,
      • Cooper A.M.
      • Torrado E.
      Protection versus pathology in tuberculosis: recent insights.
      ). In addition to tuberculosis in humans, caused by the infectious agent Mycobacterium tuberculosis, a bovine form of tuberculosis resulting from infection with Mycobacterium bovis is of wide interest because of the agricultural impact of persistent infections in domestic herds of cattle (
      • Gilbert M.
      • Mitchell A.
      • Bourn D.
      • Mawdsley J.
      • Clifton-Hadley R.
      • Wint W.
      Cattle movements and bovine tuberculosis in Great Britain.
      ). Macrophages play a key role in sequestering the mycobacteria in granulomas, leading to persistent infections (
      • Schäfer G.
      • Jacobs M.
      • Wilkinson R.J.
      • Brown G.D.
      Non-opsonic recognition of Mycobacterium tuberculosis by phagocytes.
      ). Recent studies have revealed that the macrophage-inducible C-type lectin CLEC4E, called mincle, is an important component of communication between infecting mycobacteria and macrophages. Mincle was initially identified as a receptor in stimulated macrophages that induces cytokine release (
      • Matsumoto M.
      • Tanaka T.
      • Kaisho T.
      • Sanjo H.
      • Copeland N.G.
      • Gilbert D.J.
      • Jenkins N.A.
      • Akira S.
      A novel LPS-inducible C-type lectin is a transcriptional target of NF-IL6 in macrophages.
      ). It is a type II transmembrane protein, with a C-type carbohydrate recognition domain (CRD)
      The abbreviation used is: CRD
      carbohydrate recognition domain.
      at the C-terminal end of the extracellular domain (see Fig. 1A). Although the CRD contains all of the amino acid residues commonly required for sugar binding by C-type CRDs (
      • Weis W.I.
      • Drickamer K.
      Structural basis of lectin-carbohydrate interaction.
      ,
      • Weis W.I.
      • Taylor M.E.
      • Drickamer K.
      The C-type lectin superfamily in the immune system.
      ), ligands for mincle have only recently been described. The major sugar-containing ligands are trehalose dimycolate, a glycolipid found in the outer membrane of mycobacteria (
      • Ishikawa E.
      • Ishikawa T.
      • Morita Y.S.
      • Toyonaga K.
      • Yamada H.
      • Takeuchi O.
      • Kinoshita T.
      • Akira S.
      • Yoshikai Y.
      • Yamasaki S.
      Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle.
      ,
      • Schoenen H.
      • Bodendorfer B.
      • Hitchens K.
      • Manzanero S.
      • Werninghaus K.
      • Nimmerjahn F.
      • Agger E.M.
      • Stenger S.
      • Andersen P.
      • Ruland J.
      • Brown G.D.
      • Wells C.
      • Lang R.
      Mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog trehalose-dibehenate.
      ), and other mannose- or glucose-containing glycoconjugates in fungi and yeast (
      • Ishikawa T.
      • Itoh F.
      • Yoshida S.
      • Saijo S.
      • Matsuzawa T.
      • Gonoi T.
      • Saito T.
      • Okawa Y.
      • Shibata N.
      • Miyamoto T.
      • Yamasaki S.
      Identification of distinct ligands for the C-type lectin receptors Mincle and Dectin-2 in the pathogenic fungus Malassezia.
      ,
      • Wells C.A.
      • Salvage-Jones J.A.
      • Li X.
      • Hitchens K.
      • Butcher S.
      • Murray R.Z.
      • Beckhouse A.G.
      • Lo Y.-L.-S.
      • Manzanero S.
      • Cobbold C.
      • Schroder K.
      • Ma B.
      • Orr S.
      • Stewart L.
      • Lebus D.
      • Sobieszczuk P.
      • Hume D.A.
      • Stow J.
      • Blanchard H.
      • Ashman R.B.
      The macrophage-inducible C-type lectin, Mincle, is an essential component of the innate immune response to Candida albicans.
      ).
      Figure thumbnail gr1
      FIGURE 1Organization of mincle. A, diagram of mincle showing the location of the CRD and association with the immunoreceptor tyrosine activation motif (ITAM) in the Fc receptor-γ (FcRγ) subunit through a charge-charge interaction in the membrane. B, comparison of the sequences of the predicted CRDs of bovine (Bo), human (Hu), and mouse (Mo) mincle. Conserved cysteine residues that form the three disulfide bonds characteristic of C-type CRDs are highlighted in yellow, and ligands for the conserved Ca2+ site are highlighted in green (
      • Weis W.I.
      • Taylor M.E.
      • Drickamer K.
      The C-type lectin superfamily in the immune system.
      ). Key residues in the secondary glucose-binding site are shaded pink, and residues that form the hydrophobic groove are shaded blue. Residues that chelate the supplemental Ca2+ are indicated in violet. The mouse and cow CRDs show sequence identities of 71 and 76% to the human protein, respectively.
      Trehalose dimycolate, which is a characteristic component of the mycobacterial surface, is also referred to as cord factor. It comprises a Glcα1–1Glcα headgroup and two complex branched and hydroxylated acyl chains attached to the 6-OH groups of each of the sugar residues. The importance of mincle in the interaction of macrophages with mycobacteria stems both from its ability to interact with trehalose dimycolate and from its association with the common Fc receptor-1γ subunit, which activates the Syk-CARD signaling pathway (
      • Yamasaki S.
      • Matsumoto M.
      • Takeuchi O.
      • Matsuzawa T.
      • Ishikawa E.
      • Sakuma M.
      • Tateno H.
      • Uno J.
      • Hirabayashi J.
      • Mikami Y.
      • Takeda K.
      • Akira S.
      • Saito T.
      C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia.
      ,
      • Werninghaus K.
      • Babiak A.
      • Gross O.
      • Hölscher C.
      • Dietrich H.
      • Agger E.M.
      • Mages J.
      • Mocsai A.
      • Schoenen H.
      • Finger K.
      • Nimmerjahn F.
      • Brown G.D.
      • Kirschning C.
      • Heit A.
      • Andersen P.
      • Wagner H.
      • Ruland J.
      • Lang R.
      Adjuvanticity of a synthetic cord factor analogue for subunit Mycobacterium tuberculosis vaccination requires FcRγ-Syk-Card9-dependent innate immune activation.
      ). This interaction leads to granuloma formation and may participate in directing the macrophages to tolerate rather than destroy internalized mycobacteria (
      • Lang R.
      Recognition of the mycobacterial cord factor by Mincle: relevance for granuloma formation and resistance to tuberculosis.
      ).
      The studies described here define the mechanism by which bovine mincle targets trehalose dimycolate. X-ray crystallography and mutagenesis studies have revealed an extended ligand-binding site in mincle that interacts with both the sugar headgroup and acyl portions of the glycolipid. Small molecule mimics of trehalose dimycolate have been generated to establish the importance of different parts of the glycolipid for recognition.

      DISCUSSION

      The structural and binding data presented here support a model for binding of trehalose dimycolate in which one of the acyl chains of the lipid interacts directly with the CRD, providing specificity directed by both the sugar and lipid portions of the mycobacterial ligand (Fig. 10). The structural and biochemical description of the ligand-binding site in mincle presented here provides a novel paradigm for interaction of a glycan-binding receptor with a glycolipid target. The binding site consists of a canonical C-type primary binding site centered on Ca2+, supplemented on one side by a secondary binding site for the second glucose residue in the trehalose headgroup and on the other side by a hydrophobic channel that can bind acyl groups. In combination, these three sites provide an ideal mechanism for recognition of the unique features of a pathogen-specific glycolipid. The glucose diglycoside is essentially clamped between the primary and secondary sugar-binding sites, with stereospecificity derived from a network of hydrogen bonds to hydroxyl groups on each sugar, as well as non-polar contacts. In addition to allowing hydrophobic interactions with the linear hydrocarbon portion of mycolic acid, the open side of the hydrophobic channel accommodates the branching and hydroxylation of this mycobacterium-specific surface structure. The interaction of both the polar and non-polar portions of trehalose dimycolate with adjacent sites on the mincle CRD contrasts with the T-cell receptor-CD1 complex, in which the headgroup and acyl portions of a glycolipid are bound by separate receptors on two different cells (
      • Young D.C.
      • Moody D.B.
      T-cell recognition of glycolipid presented by CD1 proteins.
      ).
      Figure thumbnail gr10
      FIGURE 10Model of the interaction of mincle with mycobacteria. Mincle, on the surface of macrophages, is shown forming a bridge with trehalose dimycolate on the surface of mycobacteria. ITAM, immunoreceptor tyrosine activation motif; FcRγ, Fc receptor-γ subunit.
      C-type lectins (such as DC-SIGN) that are involved in pathogen recognition in innate immunity show high degrees of structural and functional divergence between species (
      • Powlesland A.S.
      • Ward E.M.
      • Sadhu S.K.
      • Guo Y.
      • Taylor M.E.
      • Drickamer K.
      Novel mouse homologs of human DC-SIGN. Widely divergent biochemical properties of the complete set of mouse DC-SIGN-related proteins.
      ). In contrast, sequence conservation (Fig. 1B) suggests that the trehalose dimycolate-binding site described here is conserved in human and mouse orthologs of bovine mincle. Conservation of the mechanism for interaction with trehalose dimycolate across mammalian species may reflect evolutionary coexistence of mammalian hosts and mycobacterial pathogens (
      • Gagneux S.
      Host-pathogen coevolution in human tuberculosis.
      ). The arrangement of a hydrophobic channel adjacent to the sugar-binding site in mincle suggests how other glycolipids with alternative headgroups, such as those found in the fungus Malassezia (
      • Ishikawa T.
      • Itoh F.
      • Yoshida S.
      • Saijo S.
      • Matsuzawa T.
      • Gonoi T.
      • Saito T.
      • Okawa Y.
      • Shibata N.
      • Miyamoto T.
      • Yamasaki S.
      Identification of distinct ligands for the C-type lectin receptors Mincle and Dectin-2 in the pathogenic fungus Malassezia.
      ), might bind to and activate mincle. On the other hand, the absence of the residues that form the hydrophobic channel from the related macrophage receptor C-type lectin (MCL) indicates that weak binding of this additional receptor to trehalose dimycolate (
      • Miyake Y.
      • Toyonaga K.
      • Mori D.
      • Kakuta S.
      • Hoshino Y.
      • Oyamada A.
      • Yamada H.
      • Ono K.
      • Suyama M.
      • Iwakura Y.
      • Yoshikai Y.
      • Yamasaki S.
      C-type lectin MCL is an FcRγ-coupled receptor that mediates the adjuvanticity of mycobacterial cord factor.
      ) probably involves the headgroup only.
      Formation of the hydrophobic groove depends on the loop between residues 170 and 177 assuming the conformation observed in the trehalose complex, so the different conformation of this region observed in the native crystals may contribute to the observed pH dependence of ligand binding. The finding that binding would be reversed under endosomal conditions in turn provides a mechanism for down-regulation of signaling following internalization of mycobacteria. Interaction of mincle with a portion of the acyl chains attached to trehalose may also result in disruption of the mycobacterial membrane organization.
      The action of trehalose dimycolate or cord factor as an adjuvant has been widely exploited (
      • Levitz S.M.
      • Golenbock D.T.
      Beyond empiricism: informing vaccine develop through innate immunity research.
      ,
      • Matsunaga I.
      • Moody D.B.
      Mincle is a long sought receptor for mycobacterial cord factor.
      ,
      • Lang R.
      • Schoenen H.
      • Desel C.
      Targeting Syk-Card9-activating C-type lectin receptors by vaccine adjuvants: findings, implications and open questions.
      ). In addition to yielding insights into mycobacterial interactions with host macrophages, this investigation reveals how ligands such as the synthetic adjuvant trehalose dibehenate interact with the CRD in mincle and provides a basis for further development of adjuvants.

      Acknowledgments

      We thank Yonek Hleba for assistance in acquiring NMR data and Paul Hitchen for performing mass spectrometry.

      REFERENCES

        • Maartens G.
        • Wilkinson R.J.
        Tuberculosis.
        Lancet. 2007; 370: 2030-2043
        • Schäfer G.
        • Jacobs M.
        • Wilkinson R.J.
        • Brown G.D.
        Non-opsonic recognition of Mycobacterium tuberculosis by phagocytes.
        J. Innate Immun. 2009; 1: 231-243
        • Cooper A.M.
        • Torrado E.
        Protection versus pathology in tuberculosis: recent insights.
        Curr. Opin. Immunol. 2012; 24: 431-437
        • Gilbert M.
        • Mitchell A.
        • Bourn D.
        • Mawdsley J.
        • Clifton-Hadley R.
        • Wint W.
        Cattle movements and bovine tuberculosis in Great Britain.
        Nature. 2005; 435: 491-496
        • Matsumoto M.
        • Tanaka T.
        • Kaisho T.
        • Sanjo H.
        • Copeland N.G.
        • Gilbert D.J.
        • Jenkins N.A.
        • Akira S.
        A novel LPS-inducible C-type lectin is a transcriptional target of NF-IL6 in macrophages.
        J. Immunol. 1999; 163: 5039-5048
        • Weis W.I.
        • Drickamer K.
        Structural basis of lectin-carbohydrate interaction.
        Annu. Rev. Biochem. 1996; 65: 441-473
        • Weis W.I.
        • Taylor M.E.
        • Drickamer K.
        The C-type lectin superfamily in the immune system.
        Immunol. Rev. 1998; 163: 19-34
        • Ishikawa E.
        • Ishikawa T.
        • Morita Y.S.
        • Toyonaga K.
        • Yamada H.
        • Takeuchi O.
        • Kinoshita T.
        • Akira S.
        • Yoshikai Y.
        • Yamasaki S.
        Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle.
        J. Exp. Med. 2009; 206: 2879-2888
        • Schoenen H.
        • Bodendorfer B.
        • Hitchens K.
        • Manzanero S.
        • Werninghaus K.
        • Nimmerjahn F.
        • Agger E.M.
        • Stenger S.
        • Andersen P.
        • Ruland J.
        • Brown G.D.
        • Wells C.
        • Lang R.
        Mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog trehalose-dibehenate.
        J. Immunol. 2010; 184: 2756-2760
        • Ishikawa T.
        • Itoh F.
        • Yoshida S.
        • Saijo S.
        • Matsuzawa T.
        • Gonoi T.
        • Saito T.
        • Okawa Y.
        • Shibata N.
        • Miyamoto T.
        • Yamasaki S.
        Identification of distinct ligands for the C-type lectin receptors Mincle and Dectin-2 in the pathogenic fungus Malassezia.
        Cell Host Microbe. 2013; 13: 477-488
        • Wells C.A.
        • Salvage-Jones J.A.
        • Li X.
        • Hitchens K.
        • Butcher S.
        • Murray R.Z.
        • Beckhouse A.G.
        • Lo Y.-L.-S.
        • Manzanero S.
        • Cobbold C.
        • Schroder K.
        • Ma B.
        • Orr S.
        • Stewart L.
        • Lebus D.
        • Sobieszczuk P.
        • Hume D.A.
        • Stow J.
        • Blanchard H.
        • Ashman R.B.
        The macrophage-inducible C-type lectin, Mincle, is an essential component of the innate immune response to Candida albicans.
        J. Immunol. 2008; 180: 7404-7413
        • Yamasaki S.
        • Matsumoto M.
        • Takeuchi O.
        • Matsuzawa T.
        • Ishikawa E.
        • Sakuma M.
        • Tateno H.
        • Uno J.
        • Hirabayashi J.
        • Mikami Y.
        • Takeda K.
        • Akira S.
        • Saito T.
        C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 1897-1902
        • Werninghaus K.
        • Babiak A.
        • Gross O.
        • Hölscher C.
        • Dietrich H.
        • Agger E.M.
        • Mages J.
        • Mocsai A.
        • Schoenen H.
        • Finger K.
        • Nimmerjahn F.
        • Brown G.D.
        • Kirschning C.
        • Heit A.
        • Andersen P.
        • Wagner H.
        • Ruland J.
        • Lang R.
        Adjuvanticity of a synthetic cord factor analogue for subunit Mycobacterium tuberculosis vaccination requires FcRγ-Syk-Card9-dependent innate immune activation.
        J. Exp. Med. 2009; 206: 89-97
        • Lang R.
        Recognition of the mycobacterial cord factor by Mincle: relevance for granuloma formation and resistance to tuberculosis.
        Front. Immunol. 2013; 4 (10.3389/fimmu.2013.00005)
        • Eisenberg S.P.
        • Evans R.J.
        • Arend W.P.
        • Verderber E.
        • Brewer M.T.
        • Hannum C.H.
        • Thompson R.C.
        Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist.
        Nature. 1990; 343: 341-346
        • Schatz P.J.
        Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli.
        Biotechnology. 1993; 11: 1138-1143
        • Cormack B.
        Site-directed mutagenesis by the polymerase chain reaction.
        in: Ausubel F. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology. 3rd Ed. John Wiley & Sons, Inc., New York1997: 8.16-18.22
        • Mitchell 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: 28939-28945
        • Fornstedt N.
        • Porath J.
        Characterization studies on a new lectin found in seed of Vicia ervilia.
        FEBS Lett. 1975; 57: 187-191
        • Stambach N.S.
        • Taylor M.E.
        Characterization of carbohydrate recognition by langerin, a C-type lectin of Langerhans cell.
        Glycobiology. 2003; 13: 401-410
        • Collaborative Computational Project
        The CCP4 suite: programs for protein crystallography.
        Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763
        • McCoy A.J.
        • Grosse-Kunstleve R.W.
        • Adams P.D.
        • Winn M.D.
        • Storoni L.C.
        • Read R.J.
        Phaser crystallographic software.
        J. Appl. Crystallogr. 2007; 40: 658-674
        • Emsley P.
        • Cowtan K.
        Coot: model-building tools for molecular graphics.
        Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132
        • Adams P.D.
        • Grosse-Kunstleve R.W.
        • Hung L.W.
        • Ioerger T.R.
        • McCoy A.J.
        • Moriarty N.W.
        • Read R.J.
        • Sacchettini J.C.
        • Sauter N.K.
        • Terwilliger T.C.
        PHENIX: building new software for automated crystallographic structure determination.
        Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 1948-1954
        • Kobayashi T.
        Lipase-catalyzed syntheses of sugar esters in non-aqueous media.
        Biotechnol. Lett. 2011; 33: 1911-1919
        • Skipski V.P.
        • Smolowe A.F.
        • Barclay M.
        Separation of neutral glycosphingolipids and sulfatides by thin-layer chromatography.
        J. Lipid Res. 1967; 8: 295-299
        • Spiro R.G.
        Analysis of sugars found in glycoproteins.
        Methods Enzymol. 1966; 8: 3-26
        • Weis W.I.
        • Drickamer K.
        • Hendrickson W.A.
        Structure of a C-type mannose-binding protein complexed with an oligosaccharide.
        Nature. 1992; 360: 127-134
        • Wragg S.
        • Drickamer K.
        Identification of amino acid residues that determine pH sensitivity of ligand binding to the asialoglycoprotein receptor.
        J. Biol. Chem. 1999; 274: 35400-35406
        • Feinberg H.
        • Torgersen D.
        • Drickamer K.
        • Weis W.I.
        Mechanism of pH-dependent N-acetylgalactosamine binding to a function mimic of the hepatic asialoglycoprotein receptor.
        J. Biol. Chem. 2000; 275: 35176-35184
        • Guo Y.
        • Feinberg H.
        • Conroy E.
        • Mitchell D.A.
        • Alvarez R.
        • Blixt O.
        • Taylor M.E.
        • Weis W.I.
        • Drickamer K.
        Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR.
        Nat. Struct. Mol. Biol. 2004; 11: 591-598
        • Bock K.
        • Defaye J.
        • Driguez H.
        • Bar-Guilloux E.
        Conformations in solution of α,α-trehalose, α-d-glucopyranosyl α-d-mannopyranoside, and their 1-thioglycosyl analogs, and a tentative correlation of their behaviour with respect to the enzyme trehalase.
        Eur. J. Biochem. 1983; 131: 595-600
        • Young D.C.
        • Moody D.B.
        T-cell recognition of glycolipid presented by CD1 proteins.
        Glycobiology. 2006; 16: 103R-112R
        • Powlesland A.S.
        • Ward E.M.
        • Sadhu S.K.
        • Guo Y.
        • Taylor M.E.
        • Drickamer K.
        Novel mouse homologs of human DC-SIGN. Widely divergent biochemical properties of the complete set of mouse DC-SIGN-related proteins.
        J. Biol. Chem. 2006; 281: 20440-20449
        • Gagneux S.
        Host-pathogen coevolution in human tuberculosis.
        Phil. Trans. R. Soc. B Biol. Sci. 2012; 367: 850-859
        • Miyake Y.
        • Toyonaga K.
        • Mori D.
        • Kakuta S.
        • Hoshino Y.
        • Oyamada A.
        • Yamada H.
        • Ono K.
        • Suyama M.
        • Iwakura Y.
        • Yoshikai Y.
        • Yamasaki S.
        C-type lectin MCL is an FcRγ-coupled receptor that mediates the adjuvanticity of mycobacterial cord factor.
        Immunity. 2013; 38: 1050-1062
        • Levitz S.M.
        • Golenbock D.T.
        Beyond empiricism: informing vaccine develop through innate immunity research.
        Cell. 2012; 148: 1284-1292
        • Matsunaga I.
        • Moody D.B.
        Mincle is a long sought receptor for mycobacterial cord factor.
        J. Exp. Med. 2009; 206: 2865-2868
        • Lang R.
        • Schoenen H.
        • Desel C.
        Targeting Syk-Card9-activating C-type lectin receptors by vaccine adjuvants: findings, implications and open questions.
        Immunobiology. 2011; 216: 1184-1191
        • Meier M.
        • Bider M.D.
        • Malashkevich V.N.
        • Spiess M.
        • Burkhard P.
        Crystal structure of the carbohydrate recognition domain of the H1 subunit of the asialoglycoprotein receptor.
        J. Mol. Biol. 2000; 300: 857-865