No abstract

Both the dendritic cell receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR bind human immunodeficiency virus and enhance infection. However, biochemical and structural comparison of these receptors now reveals that they have very different physiological functions. By screening an extensive glycan array, we demonstrated that DC-SIGN and DC-SIGNR have distinct ligand-binding properties. Our structural and mutagenesis data explain how both receptors bind high-mannose oligosaccharides on enveloped viruses and why only DC-SIGN binds blood group antigens, including those present on microorganisms. DC-SIGN mediates endocytosis, trafficking as a recycling receptor and releasing ligand at endosomal pH, whereas DC-SIGNR does not release ligand at low pH or mediate endocytosis. Thus, whereas DC-SIGN has dual ligand-binding properties and functions both in adhesion and in endocytosis of pathogens, DC-SIGNR binds a restricted set of ligands and has only the properties of an adhesion receptor.

C-type (Ca(2+)-dependent) animal lectins such as mannose-binding proteins mediate many cell-surface carbohydrate-recognition events. The crystal structure at 1.7 A resolution of the carbohydrate-recognition domain of rat mannose-binding protein complexed with an oligomannose asparaginyl-oligosaccharide reveals that Ca2+ forms coordination bonds with the carbohydrate ligand. Carbohydrate specificity is determined by a network of coordination and hydrogen bonds that stabilizes the ternary complex of protein, Ca2+ and sugar. Two branches of the oligosaccharide crosslink neighbouring carbohydrate-recognition domains in the crystal, enabling multivalent binding to a single oligosaccharide chain to be visualized directly.

Lectins are responsible for cell surface sugar recognition in bacteria, animals, and plants. Examples include bacterial toxins; animal receptors that mediate cell-cell interactions, uptake of glycoconjugates, and pathogen neutralization; and plant toxins and mitogens. The structural basis for selective sugar recognition by members of all of these groups has been investigated by x-ray crystallography. Mechanisms for sugar recognition have evolved independently in diverse protein structural frameworks, but share some key features. Relatively low affinity binding sites for monosaccharides are formed at shallow indentations on protein surfaces. Selectivity is achieved through a combination of hydrogen bonding to the sugar hydroxyl groups with van der Waals packing, often including packing of a hydrophobic sugar face against aromatic amino acid side chains. Higher selectivity of binding is achieved by extending binding sites through additional direct and water-mediated contacts between oligosaccharides and the protein surface. Dramatically increased affinity for oligosaccharides results from clustering of simple binding sites in oligomers of the lectin polypeptides. The geometry of such oligomers helps to establish the ability of the lectins to distinguish surface arrays of polysaccharides in some instances and to crosslink glycoconjugates in others.

Protein-carbohydrate interactions serve multiple functions in the immune system. Many animal lectins (sugar-binding proteins) mediate both pathogen recognition and cell-cell interactions using structurally related Ca(2+)-dependent carbohydrate-recognition domains (C-type CRDs). Pathogen recognition by soluble collections such as serum mannose-binding protein and pulmonary surfactant proteins, and also the macrophage cell-surface mannose receptor, is effected by binding of terminal monosaccharide residues characteristic of bacterial and fungal cell surfaces. The broad selectivity of the monosaccharide-binding site and the geometrical arrangement of multiple CRDs in the intact lectins explains the ability of the proteins to mediate discrimination between self and non-self. In contrast, the much narrower binding specificity of selectin cell adhesion molecules results from an extended binding site within a single CRD. Other proteins, particularly receptors on the surface of natural killer cells, contain C-type lectin-like domains (CTLDs) that are evolutionarily divergent from the C-type lectins and which would be predicted to function through different mechanisms.

Dendritic cell specific intracellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin (DC-SIGN), a C-type lectin present on the surface of dendritic cells, mediates the initial interaction of dendritic cells with T cells by binding to ICAM-3. DC-SIGN and DC-SIGNR, a related receptor found on the endothelium of liver sinusoids, placental capillaries, and lymph nodes, bind to oligosaccharides that are present on the envelope of human immunodeficiency virus (HIV), an interaction that strongly promotes viral infection of T cells. Crystal structures of carbohydrate-recognition domains of DC-SIGN and of DC-SIGNR bound to oligosaccharide, in combination with binding studies, reveal that these receptors selectively recognize endogenous high-mannose oligosaccharides and may represent a new avenue for developing HIV prophylactics.

DC-SIGN1 (dendritic cell-specific ICAM-3 grabbing nonintegrin; CD209), a novel cell-surface C-type lectin expressed on dendritic cells, has been shown to mediate interactions between dendritic cells and T-cells by binding ICAM-3 (1). These interactions are independent of lymphocyte function-associated antigen-1, which is the conventional ICAM-3 ligand. DC-SIGN also binds the gp120 envelope glycoprotein of human immunodeficiency virus-1 and facilitates viral infection in trans of target CD4ϩ T-cells (2, 3). The DC-SIGN gene is located on human chromosome 19p13, proximal to a gene encoding a closely related protein, termed DC-SIGNR (DC-SIGN-related) (4). Possible biological roles of DC-SIGNR have recently emerged, with reports demonstrating its capacity to bind ICAM-3, and also to gp120, mediating human immunodeficiency virus-1 infection in trans (5, 6). However, it is expressed on liver sinusoidal endothelium, the endothelium of lymph node sinuses, and placental capillary endothelium, rather than on dendritic cells. Low levels of DC-SIGN are co-expressed with DC-SIGNR on lymph node sinus endothelium.2 DC-SIGN and DC-SIGNR are type II transmembrane proteins that share 77% amino acid sequence identity (4). The extracellular domain of each consists of a series of seven and a half tandem repeats of a highly conserved sequence of 23 amino acids followed by a C-terminal C-type carbohydrate recognition domain (CRD). Both ICAM-3 and gp120 carry an abundance of N-linked high mannose oligosaccharides. Binding of ICAM-3 to DC-SIGN requires Ca 2ϩ , and interaction between DC-SIGN and gp120 is inhibited by mannan, mannose, and EGTA (1, 2). These findings indicate that ligand binding is probably mediated through binding of carbohydrates by the CRD in a Ca 2ϩ -dependent manner. This hypothesis is consistent with the presence of residues believed to be necessary for mannose-binding to a C-type CRD in both DC-SIGN and DC-SIGNR (7).It has been suggested that the repeating sequences between the transmembrane region and the CRDs mediate oligomer formation by forming an ␣-helical coiled-coil (2, 4). Similar structures mediate the oligomerization of other cell surface C-type lectins such as the mammalian hepatic asialoglycoprotein receptor, and the subunit organization of these oligomeric complexes is critical to their biological functions. Polymorphic variants of the DC-SIGNR cDNA have been identified in which the length of the encoded neck regions differ, indicating that the cell-surface role of DC-SIGNR could be influenced by its specific neck structure (5).In this study, soluble recombinant fragments of DC-SIGN and DC-SIGNR have been used to demonstrate that the extracellular domain of each molecule is a tetramer stabilized by an ␣-helical neck and that the individual CRDs possess high affinity for mannose-containing oligosaccharides. This information suggests that DC-SIGN and DC-SIGNR employ a novel mechanism of carbohydrate recognition to achieve specificity for their natural ligands by binding multiple high mannose oligosacchar...

Calcium-dependent (C-type) animal lectins participate in many cell surface recognition events mediated by protein-carbohydrate interactions. The C-type lectin family includes cell adhesion molecules, endocytic receptors, and extracellular matrix proteins. Mammalian mannose-binding proteins are C-type lectins that function in antibody-independent host defense against pathogens. The crystal structure of the carbohydrate-recognition domain of a rat mannose-binding protein, determined as the holmium-substituted complex by multiwavelength anomalous dispersion (MAD) phasing, reveals an unusual fold consisting of two distinct regions, one of which contains extensive nonregular secondary structure stabilized by two holmium ions. The structure explains the conservation of 32 residues in all C-type carbohydrate-recognition domains, suggesting that the fold seen here is common to these domains. The strong anomalous scattering observed at the Ho LIII edge demonstrates that traditional heavy atom complexes will be generally amenable to the MAD phasing method.