•Sugar-binding C-type carbohydrate-recognition domains fall in five structural groups.•Structures for many of these domains, covering all of the groups, have been obtained.•Not all human C-type ...lectins have clear orthologues in other mammals such as mice.•Different mechanisms by which C-type lectins initiate signalling remain to be defined.•Hetero-oligomeric receptors add to the complexity of overlapping specificities.
The majority of the C-type lectin-like domains in the human genome likely to bind sugars have been investigated structurally, although novel mechanisms of sugar binding are still being discovered. In the immune system, adhesion and endocytic receptors that bind endogenous mammalian glycans are often conserved, while pathogen-binding C-type lectins on cells of the innate immune system are more divergent. Lack of orthology between some human and mouse receptors, as well as overlapping specificities of many receptors and formation of receptor hetero-oligomers, can make it difficult to define the roles of individual receptors. There is good evidence that C-type lectins initiate signalling pathways in several different ways, but this function remains the least well understood from a mechanistic perspective.
Mammalian glycan‐binding receptors, sometimes known as lectins, interact with glycans, the oligosaccharide portions of endogenous mammalian glycoproteins and glycolipids as well as sugars on the ...surfaces of microbes. These receptors guide glycoproteins out of and back into cells, facilitate communication between cells through both adhesion and signaling, and allow the innate immune system to respond quickly to viral, fungal, bacterial, and parasitic pathogens. For many of the roughly 100 glycan‐binding receptors that are known in humans, there are good descriptions of what types of glycans they bind and how selectivity for these ligands is achieved at the molecular level. In some cases, there is also comprehensive evidence for the roles that the receptors play at the cellular and organismal levels. In addition to highlighting these well‐understood paradigms for glycan‐binding receptors, this review will suggest where gaps remain in our understanding of the physiological functions that they can serve.
Mammalian sugar‐binding receptors recognize oligosaccharides on cell surfaces and secreted glycoproteins, facilitating communication between cells, trafficking of glycoproteins into and out of cells, and innate immunity against pathogens. About 100 such proteins have been identified in humans and the physiological functions of some are well understood. This review summarizes the current state of knowledge and highlights gaps in our understanding.
The human mannose receptor expressed on macrophages and hepatic endothelial cells scavenges released lysosomal enzymes, glycopeptide fragments of collagen, and pathogenic microorganisms and thus ...reduces damage following tissue injury. The receptor binds mannose, fucose, or N-acetylglucosamine (GlcNAc) residues on these targets. C-type carbohydrate-recognition domain 4 (CRD4) of the receptor contains the site for Ca2+-dependent interaction with sugars. To investigate the details of CRD4 binding, glycan array screening was used to identify oligosaccharide ligands. The strongest signals were for glycans that contain either Manα1-2Man constituents or fucose in various linkages. The mechanisms of binding to monosaccharides and oligosaccharide substructures present in many of these ligands were examined in multiple crystal structures of CRD4. Binding of mannose residues to CRD4 results primarily from interaction of the equatorial 3- and 4-OH groups with a conserved principal Ca2+ common to almost all sugar-binding C-type CRDs. In the Manα1-2Man complex, supplementary interactions with the reducing mannose residue explain the enhanced affinity for this disaccharide. Bound GlcNAc also interacts with the principal Ca2+ through equatorial 3- and 4-OH groups, whereas fucose residues can bind in several orientations, through either the 2- and 3-OH groups or the 3- and 4-OH groups. Secondary contacts with additional sugars in fucose-containing oligosaccharides, such as the Lewis-a trisaccharide, provide enhanced affinity for these glycans. These results explain many of the biologically important interactions of the mannose receptor with both mammalian glycoproteins and microbes such as yeast and suggest additional classes of ligands that have not been previously identified.
•Carbohydrate-recognition domains fall into multiple fold families.•Many of these domains are in multidomain proteins that also have other activities.•Convergent evolution has led to shared features ...in domains with different folds.•Families found across the kingdoms of life reflect extensive divergent evolution.•Polymorphisms that affect sugar binding reflect recent evolutionary pressure.
Protein modules that bind specific oligosaccharides are found across all kingdoms of life from single-celled organisms to man. Different, overlapping and evolving designations for sugar-binding domains in proteins can sometimes obscure common features that often reflect convergent solutions to the problem of distinguishing sugars with closely similar structures and binding them with sufficient affinity to achieve biologically meaningful results. Structural and functional analysis has revealed striking parallels between protein domains with widely different structures and evolutionary histories that employ common solutions to the sugar recognition problem. Recent studies also demonstrate that domains descended from common ancestors through divergent evolution appear more widely across the kingdoms of life than had previously been recognized.
Dectin-2, a C-type lectin on macrophages and other cells of the innate immune system, functions in response to pathogens, particularly fungi. The carbohydrate-recognition domain (CRD) in dectin-2 is ...linked to a transmembrane sequence that interacts with the common Fc receptor γ subunit to initiate immune signaling. The molecular mechanism by which dectin-2 selectively binds to pathogens has been investigated by characterizing the CRD expressed in a bacterial system. Competition binding studies indicated that the CRD binds to monosaccharides with modest affinity and that affinity was greatly enhanced for mannose-linked α1–2 or α1–4 to a second mannose residue. Glycan array analysis confirmed selective binding of the CRD to glycans that contain Manα1–2Man epitopes. Crystals of the CRD in complex with a mammalian-type high-mannose Man9GlcNAc2 oligosaccharide exhibited interaction with Manα1–2Man on two different termini of the glycan, with the reducing-end mannose residue ligated to Ca2+ in a primary binding site and the nonreducing terminal mannose residue occupying an adjacent secondary site. Comparison of the binding sites in DC-SIGN and langerin, two other pathogen-binding receptors of the innate immune system, revealed why these two binding sites accommodate only terminal Manα1–2Man structures, whereas dectin-2 can bind Manα1–2Man in internal positions in mannans and other polysaccharides. The specificity and geometry of the dectin-2-binding site provide the molecular mechanism for binding of dectin-2 to fungal mannans and also to bacterial lipopolysaccharides, capsular polysaccharides, and lipoarabinomannans that contain the Manα1–2Man disaccharide unit.
To investigate patterns of prognostic associations over time of insulin- and obesity-related variables measured at diagnosis of early breast cancer (BC), focusing on whether the prognostic ...associations with distant recurrence and death changed over time.
Five hundred thirty-five nondiabetic women with T1-3, N0-1, M0 invasive BC diagnosed from 1989 to 1996 were included in the study. Insulin-related variables included fasting insulin, Homeostasis Model Assessment, C-peptide, and glucose. Obesity-related variables included weight, body mass index (BMI), waist and hip circumference, and leptin. Correlations were examined using the Pearson correlation coefficient and prognostic associations using the Cox model.
There was evidence that associations of baseline insulin-related variables with distant recurrence and death were not constant over time; univariable adverse prognostic associations were significant only during the first 5 years (eg, insulin quartile 4 v 1: hazard ratio HR, 2.32; 95% CI, 1.39 to 3.86; P < .001 for distant disease-free survival DDFS; and HR, 2.85; 95% CI, 1.48 to 5.50; P = .002 for overall survival OS, with little attenuation of this pattern in multivariable analyses). In contrast, obesity-related variables (BMI, weight, leptin) exerted significant adverse univariable associations that were constant over time (eg, BMI quartile 4 v 2: HR, 1.40; 95% CI, 1.07 to 1.82 for DDFS; P = .014; and HR, 1.50; 95% CI, 1.16 to 1.93; P < .001 for OS); prognostic associations of leptin remained significant in multivariable analyses.
Baseline insulin- and obesity-related variables exert different patterns of prognostic associations over time in early BC.
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 Ca2+-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.
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.
Screening of glycan arrays represents a powerful, high-throughput approach to defining oligosaccharide ligands for glycan-binding receptors, commonly referred to as lectins. Correlating results from ...such arrays with structural analysis of receptor-ligand complexes provide one way to validate the arrays. Using examples drawn from the family of proteins that contain C-type carbohydrate-recognition domains, this review illustrates how information from the arrays reflects the way that selectivity and affinity for glycan ligands is achieved. A range of binding profiles is observed, from very restricted binding to a small set of structurally similar ligands to binding of broad classes of ligands with related terminal sugars and even to failure to bind any of the glycans on an array. These outcomes provide insights into the importance of multiple factors in defining the selectivity of these receptors, including the presence of conformationally defined units in some oligosaccharide ligands, local and extended interactions between glycans and the surfaces of receptors, and steric factors that exclude binding of some ligands.
Abstract
The murine CLEC4f gene encodes the Kupffer cell receptor, a galactose-binding receptor containing a C-type carbohydrate-recognition domain. Orthologs have been identified in nearly 100 ...species. The receptors from rat and mouse have previously been characterized and data presented here show that functional CLEC4f protein is expressed in domestic cattle (Bos taurus). However, the human CLEC4f gene does not encode a functional receptor because a mutation in the splice acceptor site of the final exon prevents appropriate splicing and a missense mutation disrupts the sugar-binding site. Transcriptomic and PCR analysis of transcripts confirms the absence of a spliced transcript containing the final exon and only background levels of transcripts are detected in human tissues. These mutations are also present in the CLEC4f gene in Neanderthals. In contrast to humans, closely related species, including chimpanzees, do have CLEC4f genes that encode full-length receptors. Affinity chromatography and glycan array results demonstrate that the chimpanzee, bovine and murine proteins all bind to galactose, but they show preferences for different subsets of galactose-containing glycans. In non-human primates, the receptor is expressed in spleen rather than in liver. The results indicate that the CLEC4f protein probably has distinct functions in different species. Absence of the receptor precludes using it for targeting of glycoconjugates to cells in human liver. The fact that CLEC4f protein is expressed in spleen in non-human primates and the close evolutionary relationship of the CLEC4f protein to langerin (CD207) suggest that it may function in the immune system, possibly as a pathogen receptor.
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