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      Binding Sites for Acylated Trehalose Analogs of Glycolipid Ligands on an Extended Carbohydrate Recognition Domain of the Macrophage Receptor Mincle*

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          Abstract

          The macrophage receptor mincle binds to trehalose dimycolate on the surface of Mycobacterium tuberculosis. Signaling initiated by this interaction leads to cytokine production, which underlies the ability of mycobacteria to evade the immune system and also to function as adjuvants. In previous work the mechanism for binding of the sugar headgroup of trehalose dimycolate to mincle has been elucidated, but the basis for enhanced binding to glycolipid ligands, in which hydrophobic substituents are attached to the 6-hydroxyl groups, has been the subject of speculation. In the work reported here, the interaction of trehalose derivatives with bovine mincle has been probed with a series of synthetic mimics of trehalose dimycolate in binding assays, in structural studies by x-ray crystallography, and by site-directed mutagenesis. Binding studies reveal that, rather than reflecting specific structural preference, the apparent affinity of mincle for ligands with hydrophobic substituents correlates with their overall size. Structural and mutagenesis analysis provides evidence for interaction of the hydrophobic substituents with multiple different portions of the surface of mincle and confirms the presence of three Ca 2+-binding sites. The structure of an extended portion of the extracellular domain of mincle, beyond the minimal C-type carbohydrate recognition domain, also constrains the way the binding domains may interact on the surface of macrophages.

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          Most cited references30

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          The integration of macromolecular diffraction data.

          The objective of any modern data-processing program is to produce from a set of diffraction images a set of indices (hkls) with their associated intensities (and estimates of their uncertainties), together with an accurate estimate of the crystal unit-cell parameters. This procedure should not only be reliable, but should involve an absolute minimum of user intervention. The process can be conveniently divided into three stages. The first (autoindexing) determines the unit-cell parameters and the orientation of the crystal. The unit-cell parameters may indicate the likely Laue group of the crystal. The second step is to refine the initial estimate of the unit-cell parameters and also the crystal mosaicity using a procedure known as post-refinement. The third step is to integrate the images, which consists of predicting the positions of the Bragg reflections on each image and obtaining an estimate of the intensity of each reflection and its uncertainty. This is carried out while simultaneously refining various detector and crystal parameters. Basic features of the algorithms employed for each of these three separate steps are described, principally with reference to the program MOSFLM.
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            Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle

            Tuberculosis remains a fatal disease caused by Mycobacterium tuberculosis, which contains various unique components that affect the host immune system. Trehalose-6,6′-dimycolate (TDM; also called cord factor) is a mycobacterial cell wall glycolipid that is the most studied immunostimulatory component of M. tuberculosis. Despite five decades of research on TDM, its host receptor has not been clearly identified. Here, we demonstrate that macrophage inducible C-type lectin (Mincle) is an essential receptor for TDM. Heat-killed mycobacteria activated Mincle-expressing cells, but the activity was lost upon delipidation of the bacteria; analysis of the lipid extracts identified TDM as a Mincle ligand. TDM activated macrophages to produce inflammatory cytokines and nitric oxide, which are completely suppressed in Mincle-deficient macrophages. In vivo TDM administration induced a robust elevation of inflammatory cytokines in sera and characteristic lung inflammation, such as granuloma formation. However, no TDM-induced lung granuloma was formed in Mincle-deficient mice. Whole mycobacteria were able to activate macrophages even in MyD88-deficient background, but the activation was significantly diminished in Mincle/MyD88 double-deficient macrophages. These results demonstrate that Mincle is an essential receptor for the mycobacterial glycolipid, TDM.
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              Mincle is an ITAM-coupled activating receptor that senses damaged cells.

              Macrophage-inducible C-type lectin (Mincle) is expressed mainly in macrophages and is induced after exposure to various stimuli and stresses. Here we show that Mincle selectively associated with the Fc receptor common gamma-chain and activated macrophages to produce inflammatory cytokines and chemokines. Mincle-expressing cells were activated in the presence of dead cells, and we identified SAP130, a component of small nuclear ribonucloprotein, as a Mincle ligand that is released from dead cells. To investigate whether Mincle is required for normal responses to cell death in vivo, we induced thymocyte death by irradiating mice and found that transient infiltration of neutrophils into the thymus could be blocked by injection of Mincle-specific antibody. Our results suggest that Mincle is a receptor that senses nonhomeostatic cell death and thereby induces the production of inflammatory cytokines to drive the infiltration of neutrophils into damaged tissue.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A. )
                0021-9258
                1083-351X
                30 September 2016
                19 August 2016
                19 August 2016
                : 291
                : 40
                : 21222-21233
                Affiliations
                From the []Departments of Structural Biology and Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305,
                the [§ ]Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom, and
                []Chemical Biology Laboratory, Department of Chemistry, Aarhus University, DK-8000 Aarhus, Denmark
                Author notes
                [2 ] To whom correspondence should be addressed: Dept. of Life Sciences, Sir Ernst Chain Bldg., Imperial College, London SW7 2AZ, UK. Tel.: 44-20-7594-5282; Fax: 44-20-7594-3057; E-mail: k.drickamer@ 123456imperial.ac.uk .
                [1]

                Both authors contributed equally to this work.

                Article
                M116.749515
                10.1074/jbc.M116.749515
                5076529
                27542410
                c757a507-860c-442b-842c-7fc8025da719
                © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version free via Creative Commons CC-BY license.

                History
                : 20 July 2016
                : 18 August 2016
                Funding
                Funded by: Biotechnology and Biological Sciences Research Council http://dx.doi.org/10.13039/501100000268
                Award ID: BB/K007718/1
                Categories
                Glycobiology and Extracellular Matrices

                Biochemistry
                carbohydrate-binding protein,glycolipid,lectin,mycobacteria,tuberculosis
                Biochemistry
                carbohydrate-binding protein, glycolipid, lectin, mycobacteria, tuberculosis

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