Publications

Mannose binding lectin (MBL) is a serum collagenous C-type lectin that plays an important role in the innate immune protection against pathogens. Previously, human and mouse studies have demonstrated that MBL binds a broad range of pathogens that results in their neutralization through agglutination, enhanced phagocytosis, and/or complement activation via the lectin pathway. The role of MBL in chicken is not well understood although the MBL concentration in serum seems to correlate with protection against infections. To investigate the role of MBL in chicken further, recombinant chicken MBL (RcMBL) was produced in HeLa R19 cells and purified using mannan affinity chromatography followed by gel filtration. RcMBL was shown to be structurally and functionally similar to native chicken MBL (NcMBL) isolated from serum. RcMBL is expressed as an oligomeric protein (mixture of trimers and oligomerized trimers) with a monomeric mass of 26kDa as determined by mass spectrometry, corresponding to the predicted mass. Glycan array analysis indicated that RcMBL bound most strongly to high-mannose glycans but also glycans with terminal fucose and GlcNac residues. The biological activity of RcMBL was demonstrated via its capacity to agglutinate Salmonella Typhimurium and to inhibit the hemagglutination activity of influenza A virus. The production of a structurally well-characterized and functionally active RcMBL will facilitate detailed studies into the protective role of MBL in innate defense against pathogens in chicken and other avian species.

Infection with parasitic helminths affects humanity and animal welfare. Parasitic helminths have the capacity to modulate host immune responses to promote their survival in infected hosts, often for a long time leading to chronic infections. In contrast to many infectious microbes, however, the helminths are able to induce immune responses that show positive bystander effects such as the protection to several immune disorders, including multiple sclerosis, inflammatory bowel disease, and allergies. They generally promote the generation of a tolerogenic immune microenvironment including the induction of type 2 (Th2) responses and a sub-population of alternatively activated macrophages. It is proposed that this anti-inflammatory response enables helminths to survive in their hosts and protects the host from excessive pathology arising from infection with these large pathogens. In any case, there is an urgent need to enhance understanding of how helminths beneficially modulate inflammatory reactions, to identify the molecules involved and to promote approaches to exploit this knowledge for future therapeutic interventions. Evidence is increasing that C-type lectins play an important role in driving helminth-mediated immune responses. C-type lectins belong to a large family of calcium-dependent receptors with broad glycan specificity. They are abundantly present on immune cells, such as dendritic cells and macrophages, which are essential in shaping host immune responses. Here, we will focus on the role of the C-type lectin macrophage mannose receptor (MR) in helminth-host interactions, which is a critically understudied area in the field of helminth immunobiology. We give an overview of the structural aspects of the MR including its glycan specificity, and the functional implications of the MR in helminth-host interactions focusing on a few selected helminth species.

The recent paper by Stadlmann et al. (2017) provides a novel algorithm for glycoproteomics in which complex glycopeptides can be identified in complex mixtures to aid in characterizing both the site of glycosylation and the glycan structure.

Secretory granules released by cytotoxic T lymphocytes (CTLs) are powerful weapons against intracellular microbes and tumor cells. Despite significant progress, there is still limited information on the molecular mechanisms implicated in target-driven degranulation, effector cell survival and composition and structure of the lytic granules. Here, using a proteomic approach we identified a panel of putative cytotoxic granule proteins, including some already known granule constituents and novel proteins that contribute to regulate the CTL lytic machinery. Particularly, we identified galectin-1 (Gal1), an endogenous immune regulatory lectin, as an integral component of the secretory granule machinery and unveil the unexpected function of this lectin in regulating CTL killing activity. Mechanistic studies revealed the ability of Gal1 to control the non-secretory lytic pathway by influencing Fas-Fas ligand interactions. This study offers new insights on the composition of the cytotoxic granule machinery, highlighting the dynamic cross talk between secretory and non-secretory pathways in controlling CTL lytic function.

Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fluorescence-based screening of glycan-binding proteins. Taken together, these results confirm our combined synthesis strategy as a new, user-friendly route for supplying chemically defined human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.

Lewis^X (Le^X) is a branched trisaccharide Galβ1→4(Fucα1→3)GlcNAc that is expressed on many cell surface glycoproteins and plays critical roles in innate and adaptive immune responses. However, efficient synthesis of glycopeptides bearing Le^X remains a major limitation for structure-function studies of the Le^X determinant. Here we report a total synthesis of a Le^X pentasaccharide 1 using a regioselective 1-benzenesulfinyl piperidine/triflic anhydride promoted [3 + 2] glycosylation. The presence of an Fmoc-threonine amino acid facilitates incorporation of the pentasaccharide in solid phase peptide synthesis, providing a route to diverse O-linked Le^X glycopeptides. The described approach is broadly applicable to the synthesis of a variety of complex glycopeptides containing O-linked Le^X or sialyl Lewis^X (sLe^X).

High-quality reagents to study and detect glycans with high specificity for research and clinical applications are severely lacking. Here, we structurally and functionally characterize several variable lymphocyte receptor (VLR)-based antibodies from lampreys immunized with O erythrocytes that specifically recognize the blood group H-trisaccharide type II antigen. Glycan microarray analysis and biophysical data reveal that these VLRs exhibit greater specificity for H-trisaccharide compared with the plant lectin UEA-1, which is widely used in blood typing. Among these antibodies, O13 exhibits superior specificity for H-trisaccharide, the basis for which is revealed by comparative analysis of high-resolution VLR:glycan crystal structures. Using a structure-guided approach, we designed an O13 mutant with further enhanced specificity for H-trisaccharide. These insights into glycan recognition by VLRs suggest that lampreys can produce highly specific glycan antibodies, and are a valuable resource for the production of next-generation glycan reagents for biological and biomedical research and as diagnostics and therapeutics.

Glycans interact with many types of proteins including enzymes, antibodies, and lectins. Protein recognition of glycans represents a major way in which the information contained in glycan structures is deciphered and promotes biological activities. This chapter describes approaches to study the kinetics and thermodynamics of interactions between glycans and glycan-binding proteins (GBPs).

Fungi are a fascinating group of predominantly multicellular organisms. Fungal species, such as Saccharomyces cerevisiae, have been instrumental in defining the fundamental processes of glycosylation, but their glycobiology is significantly different from animal or plant systems. This chapter describes the glycan structures that compose the fungal cell wall, offers some insights into novel glycobiology revealed through studying fungal systems, addresses the use of fungi as experimental and synthetic systems, and delineates the relationships of several important glycoconjugates to fungal biology and pathogenesis.

Parasitic protozoans and helminths (worms) synthesize glycans with structures often different from those typically found in vertebrates and are typically antigenic. Parasites also express glycan-binding proteins (GBPs) involved in host invasion and parasitism. As part of the disease process, parasite glycans can trigger the host's innate immune system, which can lead to the induction of adaptive immune responses. This chapter discusses the major roles of glycoconjugates in parasitic infections.