Publications by Year: 2017

Pantophlet R, Trattnig N, Murrell S, Lu N, Chau D, Rempel C, Wilson IA, Kosma P. Bacterially derived synthetic mimetics of mammalian oligomannose prime antibody responses that neutralize HIV infectivity. Nat Commun. 2017;8 (1) :1601.Abstract
Oligomannose-type glycans are among the major targets on the gp120 component of the HIV envelope protein (Env) for broadly neutralizing antibodies (bnAbs). However, attempts to elicit oligomannose-specific nAbs by immunizing with natural or synthetic oligomannose have so far not been successful, possibly due to B cell tolerance checkpoints. Here we design and synthesize oligomannose mimetics, based on the unique chemical structure of a recently identified bacterial lipooligosaccharide, to appear foreign to the immune system. One of these mimetics is bound avidly by members of a family of oligomannose-specific bnAbs and their putative common germline precursor when presented as a glycoconjugate. The crystal structure of one of the mimetics bound to a member of this bnAb family confirms the antigenic resemblance. Lastly, immunization of human-antibody transgenic animals with a lead mimetic evokes nAbs with specificities approaching those of existing bnAbs. These results provide evidence for utilizing antigenic mimicry to elicit oligomannose-specific bnAbs to HIV-1.
Collins BC, Nakahara H, Acharya S, Cooper MD, Herrin BR, Wilson IA. Crystal structure of an anti-idiotype variable lymphocyte receptor. Acta Crystallogr F Struct Biol Commun. 2017;73 (Pt 12) :682-687.Abstract
Variable lymphocyte receptors (VLRs), the leucine-rich repeat (LRR)-based antigen receptors of jawless fish, have great utility in a wide variety of biochemical and biological applications, similar to classical Ig-based antibodies. VLR-based reagents may be particularly useful when traditional antibodies are not available. An anti-idiotype lamprey VLR, VLR39, has previously been identified that recognizes the heavy-chain CDR3 of the B-cell receptor (BCR) of a leukemic clone from a patient with chronic lymphocytic leukemia (CLL). VLR39 was used successfully to track the re-emergence of this clone in the patient following chemotherapy. Here, the crystal structure of VLR39 is presented at 1.5 Å resolution and compared with those of other protein-specific VLRs. VLR39 adopts a curved solenoid fold and exhibits substantial structural similarity to other protein-binding VLRs. VLR39 has a short LRRCT loop that protrudes outwards away from the concave face and is similar to those of its protein-specific VLR counterparts. Analysis of the VLR39-BCR interaction by size-exclusion chromatography and biolayer interferometry using the scFv version of the BCR confirms that VLR39 recognizes the BCR Fv region. Such VLR-based reagents may be useful for identifying and monitoring leukemia in CLL patients and in other clinical diagnostic assays.
Zhang P, Li K, Yang G, Xia C, Polston JE, Li G, Li S, Lin Z, Yang L-J, Bruner SD, et al. Cytotoxic protein from the mushroom possesses a unique mode for glycan binding and specificity. Proc Natl Acad Sci U S A. 2017;114 (34) :8980-8985.Abstract
Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan-GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcβ1-4(Fucα1-3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αβα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel β-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment.
Zhang W, van Eijk M, Guo H, van Dijk A, Bleijerveld OB, Verheije HM, Wang G, Haagsman HP, Veldhuizen EJA. Expression and characterization of recombinant chicken mannose binding lectin. Immunobiology. 2017;222 (3) :518-528.Abstract
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.
Tati S, Fisk JC, Abdullah J, Karacosta L, Chrisikos T, Philbin P, Morey S, Ghazal D, Zazala F, Jessee J, et al. Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and InVitro Efficacy Analysis. Neoplasia. 2017;19 (9) :716-733.Abstract
JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag) which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11). Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1) suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.
Mahajan S, Khairnar A, Bishnoi R, Ramya TNC. Microbial F-type lectin domains with affinity for blood group antigens. Biochem Biophys Res Commun. 2017;491 (3) :708-713.Abstract
F-type lectins are fucose binding lectins with characteristic fucose binding and calcium binding motifs. Although they occur with a selective distribution in viruses, prokaryotes and eukaryotes, most biochemical studies have focused on vertebrate F-type lectins. Recently, using sensitive bioinformatics search techniques on the non-redundant database, we had identified many microbial F-type lectin domains with diverse domain organizations. We report here the biochemical characterization of F-type lectin domains from Cyanobium sp. PCC 7001, Myxococcus hansupus and Leucothrix mucor. We demonstrate that while all these three microbial F-type lectin domains bind to the blood group H antigen epitope on fucosylated glycans, there are fine differences in their glycan binding specificity. Cyanobium sp. PCC 7001 F-type lectin domain binds exclusively to extended H type-2 motif, Myxococcus hansupus F-type lectin domain binds to B, H type-1 and Lewis motifs, and Leucothrix mucor F-type lectin domain binds to a wide range of fucosylated glycans, including A, B, H and Lewis antigens. We believe that these microbial lectins will be useful additions to the glycobiologist's toolbox for labeling, isolating and visualizing glycans.
Bunker JJ, Erickson SA, Flynn TM, Henry C, Koval JC, Meisel M, Jabri B, Antonopoulos DA, Wilson PC, Bendelac A. Natural polyreactive IgA antibodies coat the intestinal microbiota. Science. 2017;358 (6361).Abstract
Large quantities of immunoglobulin A (IgA) are constitutively secreted by intestinal plasma cells to coat and contain the commensal microbiota, yet the specificity of these antibodies remains elusive. Here we profiled the reactivities of single murine IgA plasma cells by cloning and characterizing large numbers of monoclonal antibodies. IgAs were not specific to individual bacterial taxa but rather polyreactive, with broad reactivity to a diverse, but defined, subset of microbiota. These antibodies arose at low frequencies among naïve B cells and were selected into the IgA repertoire upon recirculation in Peyer's patches. This selection process occurred independent of microbiota or dietary antigens. Furthermore, although some IgAs acquired somatic mutations, these did not substantially influence their reactivity. These findings reveal an endogenous mechanism driving homeostatic production of polyreactive IgAs with innate specificity to microbiota.
van Die I, Cummings RD. The Mannose Receptor in Regulation of Helminth-Mediated Host Immunity. Front Immunol. 2017;8 (1677). Publisher's VersionAbstract
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.
Cummings RD. The Haystack Is Full of Needles: Technology Rescues Sugars!. Mol Cell. 2017;7 (68) :827-829. Publisher's VersionAbstract
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.
Clemente T, Vieira NJ, Cerliani JP, Adrain C, Luthi A, Dominguez MR, Yon M, Barrence FC, Riul TB, Cummings RD, et al. Proteomic and functional analysis identifies galectin-1 as a novel regulatory component of the cytotoxic granule machinery. Cell Death Dis. 2017;8 (12). Publisher's VersionAbstract
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.
Hamilton BS, Wilson JD, Shumakovich MA, Fisher AC, Brooks JC, Pontes A, Naran R, Heiss C, Gao C, Kardish R, et al. A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors. Sci Rep. 2017;7 (1). Publisher's VersionAbstract
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.
Sardar MYR, Krishnamurthy VR, Park S, Mandhapati AR, Wever WJ, Park D, Cummings RD, Chaikof EL. Synthesis of LewisX-O-Core-1 threonine: A building block for O-linked LewisX glycopeptides. Carbohydrate Research. 2017;452 (17) :47-53. Publisher's VersionAbstract
LewisX (LeX) 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 LeX remains a major limitation for structure-function studies of the LeX determinant. Here we report a total synthesis of a LeX 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 LeX glycopeptides. The described approach is broadly applicable to the synthesis of a variety of complex glycopeptides containing O-linked LeX or sialyl LewisX (sLeX).
Collins BC, Gunn RJ, McKitrick TR, Cummings RD, Cooper MD, Herrin BR, Wilson IA. Structural Insights into VLR Fine Specificity for Blood Group Carbohydrates. CellPress. 2017;25 (11) :1667-1678. Publisher's VersionAbstract
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.
Cummings RD, Schnaar RL, Esko JD, Drickamer K, Taylor ME. Principles of Glycan Recognition. In: ; 2017. Publisher's VersionAbstract
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).
Doering TL, Cummings RD, Aebi M. Fungi. In: Essentials of Glycobiology. 3rd edition. ; 2017. Publisher's VersionAbstract
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.
Cummings RD, van Die I. Parasitic Infections. In: Essentials of Glycobiology. 3rd edition. ; 2017. Publisher's VersionAbstract
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.
Cummings RD, Liu F-T, Vasta GR. Galectins. In: Essentials of Glycobiology. 3rd edition. ; 2017. Publisher's VersionAbstract
Galectins are among the most widely expressed class of lectins in all organisms. They typically bind β-galactose-containing glycoconjugates and share primary structural homology in their carbohydrate-recognition domains (CRDs). Galectins have many biological functions, including roles in development, regulation of immune cell activities, and microbial recognition as part of the innate immune system. This chapter describes the diversity of the galectin family and presents an overview of what is known about their biosynthesis, secretion, and biological roles.
Stanley P, Cummings RD. Structures Common to Different Glycans. In: Essentials of Glycobiology. 3rd edition. ; 2017. Publisher's VersionAbstract
This chapter describes the variable components of N-glycans, O-glycans, and glycolipids attached to the core of each glycan class and presented in Chapters 9, 10, and 11. The glycan extensions of these cores form the mature glycan and may include human blood group determinants. The terminal sugars of the mature glycan often regulate the function(s) or recognition properties of a glycoconjugate. Also discussed are milk oligosaccharides, that carry many of the same extensions on a lactose core.
Cummings RD, McEver RP. C-Type Lectins. In: Essentials of Glycobiology. 3rd edition. ; 2017. Publisher's VersionAbstract
C-type lectins (CTLs) are Ca++-dependent glycan-binding proteins (GBPs) that share primary and secondary structural homology in their carbohydrate-recognition domains (CRDs). The CRD of CTLs is more generally defined as the CTL domain (CTLD), because not all proteins with this domain bind either glycans or Ca++. CTLs include collectins, selectins, endocytic receptors, and proteoglycans, some of which are secreted and others are transmembrane proteins. They often oligomerize, which increases their avidity for multivalent ligands. CTLs differ significantly in the types of glycans that they recognize with high affinity. These proteins function as adhesion and signaling receptors in many pathways, including homeostasis and innate immunity, and are crucial in inflammatory responses and leukocyte and platelet trafficking.
Cummings RD, Schnaar R. R-Type Lectins. In: Essentials of Glycobiology. 3rd edition. ; 2017. Publisher's VersionAbstract
The R-type lectins are members of a superfamily of proteins that contain a carbohydrate-recognition domain (CRD) that is structurally similar to the one in ricin. Ricin is considered the first lectin to be discovered, and it is thus the prototypical lectin in this category. R-type lectins are present in plants, animals, and bacteria, and the lectin domain in some cases is associated with a separate subunit that is a potent toxin. The structure–function relationships of this group of proteins are discussed in this chapter.