C-type lectins (CTLs) represent a large family of soluble and membrane-bound proteins which bind calcium dependently via carbohydrate recognition domains (CRDs) to glycan residues presented on the surface of a variety of pathogens. The deconvolution of a cell’s glycan code by CTLs underpins several important physiological processes in mammals such as pathogen neutralization and opsonization, leukocyte trafficking, and the inflammatory response. However, as our knowledge of CTLs has developed it has become apparent that the role of this innate immune family of proteins can be double-edged, where some pathogens have developed approaches to subvert and exploit CTL interactions to promote infection and sustain the pathological state. Equally, CTL interactions with host glycoproteins can contribute to inflammatory diseases such as arthritis and cancer whereby, in certain contexts, they exacerbate inflammation and drive malignant progression. This review discusses the ‘dual agent’ roles of some of the major mammalian CTLs in both resolving and promoting infection, inflammation and inflammatory disease and highlights opportunities and emerging approaches for their therapeutic modulation.

Binding of host immunoglobulin is a common immune evasion mechanism demonstrated by microbial pathogens. Previous work showed that the malaria parasite Plasmodium falciparum binds the Fc-region of human IgM molecules, resulting in a coating of IgM on the surface of infected erythrocytes. IgM binding is a property of P. falciparum strains showing virulence-related phenotypes such as erythrocyte rosetting. The parasite ligands for IgM binding are members of the diverse P. falciparum Erythrocyte Membrane Protein One (PfEMP1) family. However, little is known about the amino acid sequence requirements for IgM binding. Here we studied an IgM binding domain from a rosette-mediating PfEMP1 variant, DBL4ζ of TM284var1, and found that the minimal IgM binding region mapped to the central region of the DBL domain, comprising all of subdomain 2 and adjoining parts of subdomains 1 and 3. Site-directed mutagenesis of charged amino acids within subdomain 2, predicted by molecular modelling to form the IgM binding site, showed no marked effect on IgM binding properties. Overall, this study identifies the minimal IgM binding region of a PfEMP1 domain, and indicates that the existing homology model of PfEMP1-IgM interaction is incorrect. Further work is needed to identify the specific interaction site for IgM within the minimal binding region of PfEMP1.

Synthetic glycopolymers with pendant saccharides bind to mammalian carbohydrate-recognition proteins with high affinity due to their multivalency and primary structure. However, glycoproteins function in nature via an exquisitely tuned "glycocode" and mimicking this code remains an interesting challenge in polymer chemistry. In order to address this we have synthesized glycomonomers via a [3+2] cycloaddition reaction between sugar-alkyne and azido-acrylates and these monomers were used to synthesize a series of sequence controlled glycopolymers (SCGP) using single electron transfer living radical polymerization (SET-LRP).

Dendritic cell‐specific intercellular adhesion molecule 3‐grabbing non‐integrin‐related ( DC ‐ SIGNR ), also known as liver/lymph node‐specific intercellular adhesion molecule 3‐grabbing non‐integrin, CLEC 4M, CD 209L, and CD 299, is a Ca 2+ ‐dependent lectin that has been implicated in increasing the infection rates of several viruses, including HIV , but the physiological role of DC ‐ SIGNR in healthy cells is currently not known with certainty. A close homologue of DC ‐ SIGNR , dendritic‐cell specific intercellular adhesion molecule 3‐grabbing non‐integrin, has been shown to act as a recycling endocytic receptor, which binds pathogens at the cell's surface and then releases them in the low pH environment of endosomal compartments. However, it is currently under debate in the literature as to whether DC ‐ SIGNR plays a similar role. In this work, we used NMR to explore whether the DC ‐ SIGNR carbohydrate recognition domain ( CRD ) shows any pH dependence in its ability to bind carbohydrates and Ca 2+ . We found clear evidence of reduced or abolished CRD ‐binding affinities for three different glycans at low pH (4.2) as compared to neutral pH (6.8). We also report the assignment of the DC ‐ SIGNR CRD in the apo form, and use these new results to characterize the degree of structural rearrangement upon binding (or release) of Ca 2+ . Finally, we report a differential effect of pH on the affinities of glycans containing mannose exclusively versus glycans containing GlcNAc moieties. Our results lead us to propose that the DC ‐ SIGNR CRD rapidly and reversibly releases glycan ligands and Ca 2+ at reduced pH (behaviour that would be expected for an endocytic receptor), and that the binding of mannose‐containing oligosaccharides is more strongly affected by pH than the binding of GlcNAc‐containing oligosaccharides.

A series of cyclodextrin-based glycoconjugates, including glycoclusters and star glycopolymers, were synthesized via combination of CuAAC Huisgen coupling and copper-mediated living radical polymerization. These glycoconjugates showed high affinity binding to the human transmembrane lectin DC-SIGN and act as inhibitors to prevent the binding of HIV envelope protein gp120 to DC-SIGN at nanomolar concentrations. The star block glycopolymers showed high loading capacity of hydrophobic anticancer and anti-HIV drugs, indicating promising applications in HIV-therapeutic and smart drug delivery.

Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca2+-coordinating residues. The data explain the Ca2+-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway.

Surfactant Protein D (SP-D) is an oligomerized C-type lectin molecule with immunomodulatory properties and involvement in lung surfactant homeostasis in the respiratory tract. SP-D binds to the enveloped viruses, influenza A virus and respiratory syncytial virus and inhibits their replication in vitro and in vivo. SP-D has been shown to bind to HIV via the HIV envelope protein gp120 and inhibit infectivity in vitro. Here we show that SP-D binds to different strains of HIV (BaL and IIIB) and the binding occurs at both pH 7.4 and 5.0 resembling physiological relevant pH values found in the body and the female urogenital tract, respectively. The binding of SP-D to HIV particles and gp120 was inhibited by the presence of several hexoses with mannose found to be the strongest inhibitor. Competition studies showed that soluble CD4 and CVN did not interfere with the interaction between SP-D and gp120. However, soluble recombinant DC-SIGN was shown to inhibit the binding between SP-D and gp120. SP-D agglutinated HIV and gp120 in a calcium dependent manner. SP-D inhibited the infectivity of HIV strains at both pH values of 7.4 and 5.0 in a concentration dependent manner. The inhibition of the infectivity was abolished by the presence of mannose. SP-D enhanced the binding of HIV to immature monocyte derived dendritic cells (iMDDCs) and was also found to enhance HIV capture and transfer to the T-cell like line PM1. These results suggest that SP-D can bind to and inhibit direct infection of T-cells by HIV but also enhance the transfer of infectious HIV particles from DCs to T-cells in vivo.

Multivalent binding is a key for many critical biological processes and unique recognition and specificity in binding enables many of different glycans and proteins to work in a great harmony within the human body. In this study, the binding kinetics of synthetic glycopolypeptides to the dendritic cell lectin DC-SIGN and their inhibition potential for DC-SIGN interactions with the gp120 envelope glycoprotein of HIV-1 (gp120) are investigated.

Bildet eine Kette! Vielblock-Glykopolymere aus Monomeren mit Mannose- (M, siehe Schema), Glucose- und Diethylenglycolethylether(D)-Substituenten wurden mit kontrollierten Sequenzen synthetisiert. Die hoch monodispersen Glykopolymere wurden auf die Bindung und Inhibition von DC-SIGN, einem für die HIV-Infektion bedeutsamen Protein, hin getestet.

The C-type lectin DC-SIGNR (dendritic cell-specific ICAM-3-grabbing non-integrin-related; also known as L-SIGN or CD299) is a promising drug target due to its ability to promote infection and/or within-host survival of several dangerous pathogens (e.g. HIV and severe acute respiratory syndrome coronavirus (SARS)) via interactions with their surface glycans. Crystallography has provided excellent insight into the mechanism by which DC-SIGNR interacts with small glycans, such as (GlcNAc)(2)Man(3); however, direct observation of complexes with larger, physiological oligosaccharides, such as Man(9)GlcNAc(2), remains elusive. We have utilized solution-state nuclear magnetic resonance spectroscopy to investigate DC-SIGNR binding and herein report the first backbone assignment of its active, calcium-bound carbohydrate recognition domain. Direct interactions with the small sugar fragments Man(3), Man(5), and (GlcNAc)(2)Man(3) were investigated alongside Man(9)GlcNAc derived from recombinant gp120 (present on the HIV viral envelope), providing the first structural data for DC-SIGNR in complex with a virus-associated ligand, and unique binding modes were observed for each glycan. In particular, our data show that DC-SIGNR has a different binding mode for glycans on the HIV viral envelope compared with the smaller glycans previously observed in the crystalline state. This suggests that using the binding mode of Man(9)GlcNAc, instead of those of small glycans, may provide a platform for the design of DC-SIGNR inhibitors selective for high mannose glycans (like those on HIV). N-15 relaxation measurements provided the first information on the dynamics of the carbohydrate recognition domain, demonstrating that it is a highly flexible domain that undergoes ligand-induced conformational and dynamic changes that may explain the ability of DC-SIGNR to accommodate a range of glycans on viral surfaces.

Background. The role of the complement system in antibody-mediated rejection has been investigated in relation to circulating complement interacting with renal microvascular endothelium, resulting in the formation of peritubular capillary C4d. However, the possible importance of local complement synthesis is less clear. The aim of this study was to determine whether human vascular endothelium could produce C4 in response to stimulation in vitro.Methods. Human microvascular endothelial cells and glomerular endothelial cells were stimulated with endotoxins, cytokines, and human leukocyte antigen-specific antibodies. Synthesis of complement was investigated using western blotting and indirect immunofluorescence. De novo C4 synthesis was confirmed by using C4 small interfering RNA.Results. Glomerular and microvascular endothelium, both produce C3 and C4 complement protein. Complement synthesis was stimulant-specific-C3 was produced mainly after stimulation with lipopolysaccharide whereas C4 synthesis occurred on treatment with gamma interferon. Culture with human leukocyte antigen-specific antibodies resulted in a significant increase of C4 protein synthesis by both cell lines.Conclusions. We have shown for the first time that human microvascular endothelium can be stimulated to synthesize C4 in vitro. The implications of this for clinical transplantation, especially in the context of antibody-mediated rejection, its histological interpretation and as a potential target for therapy would have to be determined by further studies.

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 µM 2′,3′-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.

We demonstrate the expected preference of an immobilised oligosaccharide Man9(GlcNAc)2 upon a 96-well photochemical array, for its known receptor, the cell-surface lectin Dendritic Cell-Specific ICAM3 Grabbing Nonintegrin (DC-SIGN) when compared to immobilised competing monosaccharides.

We report the synthesis of a nucleic acid-encoded carbohydrate library, its combinatorial self-assembly into 37 485 pairs and a screen against DC-SIGN leading to the identification of consensus ligand motifs. A prototypical example from the selected pairs was shown to have enhanced binding. A dendrimer incorporating the selected motifs inhibited gp120's binding to dendritic cells with higher efficiency than mannan.

Complement activation contributes directly to health and disease. It neutralizes pathogens and stimulates immune processes. Defects lead to immunodeficiency and autoimmune diseases, whereas inappropriate activation causes self-damage. In the lectin and classical pathways, complement is triggered upon recognition of a pathogen by an activating complex. Here we present the first structure of such a complex in the form of the collagen-like domain of mannan-binding lectin (MBL) and the binding domain of its associated protease (MASP-1/-3). The collagen binds within a groove using a pivotal lysine side chain that interacts with Ca(2+)-coordinating residues, revealing the essential role of Ca(2+). This mode of binding is prototypic for all activating complexes of the lectin and classical pathways, and suggests a general mechanism for the global changes that drive activation. The structural insights reveal a new focus for inhibitors and we have validated this concept by targeting the binding pocket of the MASP.

Noncovalent interactions between complex carbohydrates and proteins drive many fundamental processes within biological systems, including human immunity. In this report we aimed to investigate the potential of mannose-containing glycopolymers to interact with human DC-SIGN and the ability of these glycopolymers to inhibit the interactions between DC-SIGN and the HIV envelope glycoprotein gp120. We used a library of glycopolymers that are prepared via combination of copper-mediated living radical polymerization and azide-alkyne [3+2] Huisgen cycloaddition reaction. We demonstrate that a relatively simple glycopolymer can effectively prevent the interactions between a human dendritic cell associated lectin (DC-SIGN) and the viral envelope glycoprotein gp120. This approach may give rise to novel insights into the mechanisms of HIV infection and provide potential new therapeutics.

Mice lacking complement components show delayed development of prion disease following peripheral inoculation. The delay could relate to reduced scrapie prion protein (PrPSc) accumulation on follicular dendritic cells (DCs). However conventional DCs (cDCs) play a crucial role in the early pathogenesis of prion diseases and complement deficiency could result in decreased PrPSc uptake by cDCs in the periphery. To explore this possibility, we cultured murine splenic or gut-associated lymph node cDCs with scrapie-infected whole brain homogenate in the presence or absence of complement. Uptake decreased significantly if the serum in the cultures was heat-inactivated. Because heat inactivation primarily denatures C1q, we used serum from C1q(-/-) mice and showed that PrPSc uptake was markedly decreased. PrPSc internalization was saturable and temperature-dependent, suggesting receptor-mediated uptake. Furthermore, uptake characteristics differed from fluid-phase endocytosis. Immunofluorescence showed colocalization of C1q and PrPSc, suggesting interaction between these molecules. We evaluated the expression of several complement receptors on cDCs and confirmed that cDCs that take up PrPSc express one of the C1q receptors, calreticulin. Our results show that C1q participates in PrPSc uptake by cDCs, revealing a critical role for cDCs in initial prion capture, an event that takes place before the PrPSc accumulation within the follicular DC network. The Journal of Immunology, 2009, 182: 1305-1313.

Complement C3 is a central component of the humoral immune system. Upon triggering of the complement cascade, proteolytic fragments of C3 mediate important processes such as opsonization and lymphocyte activation. C3 possesses an internal thioester that mediates covalent attachment of proteolytically activated C3 to target surfaces. Treatment of native C3 with methylamine cleaves the thioester bond and exposes a free sulfhydryl group at the target-binding face of the protein. Through the use of sulfhydryl-reactive heterobifunctional cross-linking and biotinylation reagents, we demonstrate the capacity to form stable, multimeric whole human C3-protein conjugates in a fashion reflecting the orientation of physiologically-activated C3. We speculate that this C3 conjugation strategy presents a route for targeting dendritic cells and macrophages. In addition, manipulation of the thioester bond could enhance the study of biological roles of C3 and related proteins such as C4, and also of transmissible agents that exploit complement function such as prions.