Daniel Anthony Mitchell

Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR

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.

C1q Deficiency and Autoimmunity: The Effects of Genetic Background on Disease Expression

Gene-targeted C1q-deficient mice have been shown to develop a syndrome reminiscent of human systemic lupus erythematosus with antinuclear Abs and proliferative glomerulonephritis. Initial phenotypic analysis conducted in (129 × C57BL/6) hybrid mice showed that background genes were a significant factor for the full expression of the autoimmune disease. To assess the contribution of background genes in the expression of the autoimmune phenotype, the disrupted C1qa gene was backcrossed for seven generations onto C57BL/6 and MRL/Mp+/+ strains. These were intercrossed with C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains to generate C1q-deficient substrains. In C1q-deficient C57BL/6 mice, no evidence of an autoimmune phenotype was found, and C1q deficiency in both the C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains did not modify the autoimmune phenotype observed in wild-type controls. However, in C1q-deficient MRL/Mp+/+ animals an acceleration of both the onset and the severity of antinuclear Abs and glomerulonephritis was seen. Disease was particularly pronounced in females, which developed severe crescentic glomerulonephritis accompanied by heavy proteinuria. In addition, the C1q-deficient MRL/Mp+/+ mice had an impairment in the phagocytic clearance of apoptotic cells in vivo. These data demonstrate that the expression of autoimmunity in C1q-deficient mice is strongly influenced by other background genes. The work also highlights the potential value of the C1q-deficient MRL/Mp+/+ strain as a tool with which to dissect further the underlying mechanisms of the autoimmune syndrome associated with C1q deficiency.

Extended Neck Regions Stabilize Tetramers of the Receptors DC-SIGN and DC-SIGNR

The human cell surface receptors DC-SIGN (dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin) and DC-SIGNR (DC-SIGN-related) bind to oligosaccharide ligands found on human tissues as well as on pathogens including viruses, bacteria, and parasites. The extracellular portion of each receptor contains a membrane-distal carbohydrate-recognition domain (CRD) and forms tetramers stabilized by an extended neck region consisting of 23 amino acid repeats. Cross-linking analysis of full-length receptors expressed in fibroblasts confirms the tetrameric state of the intact receptors. Hydrodynamic studies on truncated receptors demonstrate that the portion of the neck of each protein adjacent to the CRD is sufficient to mediate the formation of dimers, whereas regions near the N terminus are needed to stabilize the tetramers. Some of the intervening repeats are missing from polymorphic forms of DC-SIGNR. Two different crystal forms of truncated DC-SIGNR comprising two neck repeats and the CRD reveal that the CRDs are flexibly linked to the neck, which contains α-helical segments interspersed with non-helical regions. Differential scanning calorimetry measurements indicate that the neck and CRDs are independently folded domains. Based on the crystal structures and hydrodynamic data, models for the full extracellular domains of the receptors have been generated. The observed flexibility of the CRDs in the tetramer, combined with previous data on the specificity of these receptors, suggests an important role for oligomerization in the recognition of endogenous glycans, in particular those present on the surfaces of enveloped viruses recognized by these proteins.

Sequence-controlled multi-block glycopolymers to inhibit DC-SIGN-gp120 binding.

Glycan–protein interactions are essential for many physiological processes including cell–cell recognition, cell adhesion, cell signalling, pathogen identification, and differentiation. Dendritic cell-specific intercellular adhesion molecule3-grabbing non-integrin (DC-SIGN; CD209) is a C-type lectin (carbohydrate-binding protein) present on both macrophages and dendritic cell subpopulations and plays a critical role in many cell interactions. DC-SIGN binds to microorganisms and host molecules by recognizing surface-rich mannose-containing glycans through multivalent glycan– protein interactions and serves as a target for several viruses, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Carbohydrate-binding proteins (CBP) have been suggested as potential microbicides for the prevention of HIV infection. However, the isolation of natural CBPs is relatively difficult because of their hydrophilic nature and low affinity for the virus. 4] Thus, synthetic lectins are of interest for carbohydrate recognition studies. Alternatively, noncarbohydrate inhibitors of mammalian lectins can be used to prevent the interaction between DC-SIGN and gp120. The structures of these multivalent ligands have a great effect on carbohydrate binding to lectins, and the use of linear polymers to effectively inhibit lectin binding has been demonstrated by several research groups. Synthetic polymer chemistry has developed rapidly in recent years. Currently, polymerization of functional monomers with the desired chain length, structure, and composition is straightforward; whereas producing polymers with monomer sequence control remains challenging, which has implications for the controlled folding of synthetic macromolecules. There are a few recent reports where sufficient control has been achieved in controlling the monomer sequence along the polymer chain. To the best of our knowledge, this is the first report where some control over the relative position of the sugars is exhibited and their binding to the human lectin DC-SIGN is demonstrated. We have used a controlled polymerization technique, single-electron transfer living radical polymerization (SET-LRP), to polymerize glycomonomers, which are prepared by copper catalyzed azide–alkyne click (CuAAC) reaction prior to polymerization. A series of glycomonomers were prepared by reaction of 3-azidopropylacrylate (APA) and alkylated mannose, glucose, and fucose using a Fischer–Helferich glycosylation. This was performed using CuSO4 and sodium ascorbate in a methanol/water mixture (see the Supporting Information). SET-LRP of the glucose monomer (GluA) was performed in dimethylsulfoxide (DMSO) using a copper(0)/copper(II) and tris[2-(dimethylamino)ethyl]amine (Me6TREN)-derived catalyst. Polymerization reached over 90 % monomer conversion in six hours whilst maintaining a narrow molecular weight distribution with increasing molecular weight. (Supporting Information, Figure S4). The obtained polymers were characterized by size exclusion chromatography (SEC) and MALDI-TOF mass spectroscopy (MS) or high-resolution electrospray ionization mass spectroscopy (HR-ESI MS), which indicated very high chain-end fidelity allowing for sequential monomer addition. We designed a polymerization reaction starting with one equivalent of initiator (I) and two equivalents of mannose glycomonomer (ManA; Figure 1a). ManA was fully consumed after 12 hours; then two equivalents of GluA in DMSO were added to the reaction mixture and GluA was consumed in 16 hours. Two equivalents of ManA in DMSO were subsequently added to the reaction mixture, and this cycle was continued until six short blocks of glycopolymers were produced (the degree of polymerization (DP) = 2 for each block, (mannose)2-(glucose)2-(mannose)2-(glucose)2(mannose)2-(glucose)2). No purification steps were required prior to addition of the subsequent monomer. The conversion of the first four blocks, as analyzed by H NMR spectroscopy, reached 100 %, shown by the complete disappearance of vinyl groups at 5.7–6.5 ppm. The glycomonomers were dissolved in purged DMSO prior to their addition and this resulted in further dilution of the reaction mixture upon each monomer addition. Traces of vinyl groups could still be detected after [*] Q. Zhang, J. Collins, A. Anastasaki, Dr. C. R. Becer, Prof. D. M. Haddleton Department of Chemistry, University of Warwick Gibbet Hill Road, Coventry, CV4 7AL (UK) E-mail: d.m.haddleton@warwick.ac.uk Homepage: http://www.warwick.ac.uk/go/polymers Dr. R. Wallis Department of Biochemistry, University of Leicester Leicester, LE1 9HN (UK) Dr. D. A. Mitchell Clinical Sciences Research Institute, Warwick Medical School, University of Warwick Coventry, CV2 2DX (UK) [**] We acknowledge financial support from the University of Warwick and the China Scholarship Council. Equipment used in this research was funded by the Innovative Uses for Advanced Materials in the Modern World (AM2) with support from AWM and ERDF. D.M.H. is a Royal Society/Wolfson Fellow and C.R.B. is a Science City Senior Research Fellow. Dr. Christopher N. Scanlan has provided the gp120. Supporting information for this article (syntheses of all materials and details of the characterization methods) is available on the WWW under http://dx.doi.org/10.1002/anie.201300068. Angewandte Chemie

High-Affinity Glycopolymer Binding to Human DC-SIGN and Disruption of DC-SIGN Interactions with HIV Envelope Glycoprotein

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.

Paths reunited: Initiation of the classical and lectin pathways of complement activation.

Understanding the structural organisation and mode of action of the initiating complex of the classical pathway of complement activation (C1) has been a central goal in complement biology since its isolation almost 50 years ago. Nevertheless, knowledge is still incomplete, especially with regard to the interactions between its subcomponents C1q, C1r and C1s that trigger activation upon binding to a microbial target. Recent studies have provided new insights into these interactions, and have revealed unexpected parallels with initiating complexes of the lectin pathway of complement: MBL-MASP and ficolin-MASP. Here, we develop and expand these concepts and delineate their implications towards the key aspects of complement activation via the classical and lectin pathways.

Dendritic cell lectin-targeting sentinel-like unimolecular glycoconjugates to release an anti-HIV drug.

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.

Dissecting the molecular mechanism of IVIg therapy: the interaction between serum IgG and DC-SIGN is independent of antibody glycoform or Fc domain.

Intravenous immunoglobulin (IVIg) therapy is used to treat a wide range of autoimmune conditions and consists of pooled immunoglobulin G (IgG) from healthy donors. The immunosuppressive effects of IVIg are, in part, attributed to terminal α2,6-linked sialic acid residues on the N-linked glycans of the IgG Fc (fragment crystallizable) domain. This α2,6-sialylated Fc (sFc) has been reported to bind to the carbohydrate recognition domain (CRD) of the cell-surface lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) and its murine orthologue SIGN-R1 (specific intracellular adhesion molecule-grabbing non-integrin R1) and, via this interaction, to signal the downstream expression of immunosuppressive cytokines and receptors. Consistent with this model, the antiinflammatory effect of IVIg treatment is abolished in a murine knock-out of SIGN-R1 and can be restored by a knock-in with human DC-SIGN. In contrast, however, existing glycan array and X-ray crystallographic studies indicate that the CRDs of both SIGN-R1 and DC-SIGN bind to a restricted set of primarily oligomannose-type glycans that does not include the glycans found on sFc. We attempted to reconcile these immunological and biophysical observations. We first generated hypersialylated, desialylated, deglycosylated and untreated serum IgG and found that the affinity for the complete extracellular region of the DC-SIGN tetramer was similar for all antibody glycoforms. Moreover, the binding could be attributed to cross-reactive, polyclonal Fab (fragment antigen-binding) specificities in serum as neither recombinant Fc nor sFc bound to DC-SIGN. In addition, serum IgG exhibited no competition against known ligands of the DC-SIGN CRD. These findings lead us to suggest that IVIg therapy does not involve binding of IgG Fc to DC-SIGN and that alternative cell-surface lectins are required for the antiinflammatory activity of sFc.

Metformin decreases angiogenesis via NF-κB and Erk1/2/Erk5 pathways by increasing the antiangiogenic thrombospondin-1

AIMS Polycystic ovary syndrome (PCOS) is associated with insulin resistance (IR), obesity, and cardiovascular complications. Thrombospondin-1 (TSP-1) is a novel antiangiogenic adipokine highly expressed in obese insulin-resistant subjects. We sought to assess TSP-1 levels in adipose tissue (AT) from PCOS women and matched controls. The effects of metformin treatment on circulating TSP-1 levels in PCOS subjects, the effects of serum from normal and PCOS women on in vitro migration and angiogenesis before and after metformin treatment, and ex vivo regulation of AT TSP-1 by D-glucose were also studied. METHODS AND RESULTS Serum TSP-1 (ELISA), subcutaneous and omental AT TSP-1 mRNA (reverse transcriptase-polymerase chain reaction), and protein (western blotting) were significantly lower in PCOS women (P < 0.05). Corresponding plasminogen activator inhibitor-1 (PAI-1) and PAI-1 activity were significantly higher (P < 0.01). After 6 months of metformin treatment, there was a significant increase in serum TSP-1 (P < 0.05) and a corresponding decrease in PAI-1 and PAI-1 activity (P < 0.01). In vitro migration and angiogenesis were significantly increased in serum from PCOS women (P < 0.01); these effects were significantly attenuated by metformin treatment (P < 0.01) through the regulation of TSP-1 levels via nuclear factor-kappaB (NF-kappaB), extracellular regulated-signal kinase 1/2 (Erk1/2) and Erk5 pathways. Importantly, changes in the intima media thickness were predictive of changes in serum TSP-1 (P = 0.049). In AT explants, glucose significantly decreased TSP-1 protein production and secretion into conditioned media (ELISA) (P < 0.05, P < 0.001). CONCLUSION TSP-1 levels are lower in PCOS women. Metformin treatment increases serum TSP-1 in these women. Our findings provide novel insights into the relationship between obesity, IR, and angiogenesis.

Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing

The cryopreservation of cells, tissue and organs is fundamental to modern biotechnology, transplantation medicine and chemical biology. The current state-of-the-art method of cryopreservation is the addition of large amounts of organic solvents such as glycerol or dimethyl sulfoxide, to promote vitrification and prevent ice formation. Here we employ a synthetic, biomimetic, polymer, which is capable of slowing the growth of ice crystals in a manner similar to antifreeze (glyco)proteins to enhance the cryopreservation of sheep and human red blood cells. We find that only 0.1 wt% of the polymer is required to attain significant cell recovery post freezing, compared with over 20 wt% required for solvent-based strategies. These results demonstrate that synthetic antifreeze (glyco)protein mimics could have a crucial role in modern regenerative medicine to improve the storage and distribution of biological material for transplantation.