Publications

Recombinant antibodies are of profound clinical significance; yet, anti-carbohydrate antibodies are prone to undesirable cross-reactivity with structurally related-glycans. Here we introduce a new technology called Computational Carbohydrate Grafting (CCG), which enables a virtual library of glycans to be assessed for protein binding specificity, and employ it to define the scope and structural origin of the binding specificity of antibody JAA-F11 for glycans containing the Thomsen-Friedenreich (TF) human tumor antigen. A virtual library of the entire human glycome (GLibrary-3D) was constructed, from which 1,182 TF-containing human glycans were identified and assessed for their ability to fit into the antibody combining site. The glycans were categorized into putative binders, or non-binders, on the basis of steric clashes with the antibody surface. The analysis employed a structure of the immune complex, generated by docking the TF-disaccharide (Galβ1-3GalNAcα) into a crystal structure of the JAA-F11 antigen binding fragment, which was shown to be consistent with saturation transfer difference (STD) NMR data. The specificities predicted by CCG were fully consistent with data from experimental glycan array screening, and confirmed that the antibody is selective for the TF-antigen and certain extended core-2 type mucins. Additionally, the CCG analysis identified a limited number of related putative binding motifs, and provided a structural basis for interpreting the specificity. CCG can be utilized to facilitate clinical applications through the determination of the three-dimensional interaction of glycans with proteins, thus augmenting drug and vaccine development techniques that seek to optimize the specificity and affinity of neutralizing proteins, which target glycans associated with diseases including cancer and HIV.

Ahead of Print article withdrawn by publisher. At request of the authors, this article will be published in the journal Cancer Biomarkers (ISSN 1574-0153).

It is generally accepted that human influenza viruses bind glycans containing sialic acid linked α2-6 to the next sugar, that avian influenza viruses bind glycans containing the α2-3 linkage, and that mutations that change the binding specificity might change the host tropism. We noted that human H3N2 viruses showed dramatic differences in their binding specificity, and so we embarked on a study of representative human H3N2 influenza viruses, isolated from 1968 to 2012, that had been isolated and minimally passaged only in mammalian cells, never in eggs. The 45 viruses were grown in MDCK cells, purified, fluorescently labeled and screened on the Consortium for Functional Glycomics Glycan Array. Viruses isolated in the same season have similar binding specificity profiles but the profiles show marked year-to-year variation. None of the 610 glycans on the array (166 sialylated glycans) bound to all viruses; the closest was Neu5Acα2-6(Galβ1-4GlcNAc)3 in either a linear or biantennary form, that bound 42 of the 45 viruses. The earliest human H3N2 viruses preferentially bound short, branched sialylated glycans while recent viruses bind better to long polylactosamine chains terminating in sialic acid. Viruses isolated in 1996, 2006, 2010 and 2012 bind glycans with α2-3 linked sialic acid; for 2006, 2010 and 2012 viruses this binding was inhibited by oseltamivir, indicating binding of α2-3 sialylated glycans by neuraminidase. More significantly, oseltamivir inhibited virus entry of 2010 and 2012 viruses into MDCK cells. All of these viruses were representative of epidemic strains that spread around the world, so all could infect and transmit between humans with high efficiency. We conclude that the year-to-year variation in receptor binding specificity is a consequence of amino acid sequence changes driven by antigenic drift, and that viruses with quite different binding specificity and avidity are equally fit to infect and transmit in the human population.

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.

Human monocyte-derived dendritic cells (DCs) show remarkable phenotypic changes upon direct contact with soluble products (SPs) of Trichuris suis, a pig whipworm that is experimentally used in therapies to ameliorate inflammation in patients with Crohn's disease and multiple sclerosis. These changes may contribute to the observed induction of a T helper 2 (Th2) response and the suppression of Toll-like receptor (TLR)-induced Th1 and Th17 responses by human DCs primed with T. suis SPs. Here it is demonstrated that glycans of T. suis SPs contribute significantly to the suppression of the lipopolysaccharide (LPS)-induced expression in DCs of a broad variety of cytokines and chemokines, including important pro-inflammatory mediators such as TNF-α, IL-6, IL-12, lymphotoxin α (LTA), C-C Motif Ligand (CCL)2, C-X-C Motif Ligands (CXCL)9 and CXCL10. In addition, the data show that human DCs strongly bind T. suis SP-glycans via the C-type lectin receptors (CLRs) mannose receptor (MR) and DC-specific ICAM-3-grabbing non-integrin (DC-SIGN). The interaction of DCs with T. suis glycans likely involves mannose-type glycans, rather than fucosylated glycans, which differs from DC binding to soluble egg antigens of the human worm parasite, Schistosoma mansoni. In addition, macrophage galactose-type lectin (MGL) recognises T. suis SPs, which may contribute to the interaction with immature DCs or other MGL-expressing immune cells such as macrophages. The interaction of T. suis glycans with CLRs of human DCs may be essential for the ability of T. suis to suppress a pro-inflammatory phenotype of human DCs. The finding that the T. suis-induced modulation of human DC function is glycan-mediated is novel and indicates that helminth glycans contribute to the dampening of inflammation in a wide range of human inflammatory diseases.

Glycan structures were defined historically using multiple methods to determine composition, sequence, linkage, and anomericity of component monosaccharides. Such approaches have been replaced by more sensitive MS methods to profile or predict glycan structures, but these methods are limited in their ability to completely define glycan structures. Glycan-binding proteins, including lectins and antibodies, have been found to have exquisite binding specificities that can provide information about glycan structures. Here, we show glycan-binding proteins can be used along with MS to help define glycan linkages and other determinants in unknown glycans printed as shotgun glycan microarrays.

BACKGROUND: Modifications of proteins by O-glycosylation determine many of the properties and functions of proteins. We wish to understand the mechanisms of O-glycosylation and develop inhibitors that could affect glycoprotein functions and alter cellular behavior. METHODS: We expressed recombinant soluble human Gal- and GlcNAc-transferases that synthesize the O-glycan cores 1 to 4 and are critical for the overall structures of O-glycans. We determined the properties and substrate specificities of these enzymes using synthetic acceptor substrate analogs. Compounds that were inactive as substrates were tested as inhibitors. RESULTS: Enzymes significantly differed in their recognition of the sugar moieties and aglycone groups of substrates. Core 1 synthase was active with glycopeptide substrates but GlcNAc-transferases preferred substrates with hydrophobic aglycone groups. Chemical modifications of the acceptors shed light on enzyme-substrate interactions. Core 1 synthase was weakly inhibited by its substrate analog benzyl 2-butanamido-2-deoxy-α-d-galactoside while two of the three GlcNAc-transferases were selectively and potently inhibited by bis-imidazolium salts which are not substrate analogs. CONCLUSIONS: This work delineates the distinct specificities and properties of the enzymes that synthesize the common O-glycan core structures 1 to 4. New inhibitors were found that could selectively inhibit the synthesis of cores 1, 2 and 3 but not core 4. GENERAL SIGNIFICANCE: These studies help our understanding of the mechanisms of action of enzymes critical for O-glycosylation. The results may be useful for the re-engineering of O-glycosylation to determine the roles of O-glycans and the enzymes critical for O-glycosylation, and for biotechnology with potential therapeutic applications.

The parasitic blood fluke Schistosoma mansoni synthesizes immunogenic glycans containing the human Lewis x antigen (Le(x); Galactose-β1-4(Fucα1-3)N-acetylglucosamine-β-R, also called CD15), but the biological role(s) of this antigen in the parasites and in humans is poorly understood. To develop IgG-based monoclonal antibodies (mAbs) specific for Le(x), we harvested splenocytes from S. mansoni-infected Swiss Webster mice at Week 10 postinfection, when peak IgG responses to glycan antigens occur, and generated a panel of hybridomas secreting anti-glycan IgG that recognize periodate-sensitive epitopes in soluble egg antigens of the parasites, and also recognizes a neoglycoprotein containing a pentasaccharide with the Le(x) sequence. One murine mAb, an IgG3 designated F8A1.1, bound to glycoproteins and glycolipids from schistosome adults and human promyelocytic leukemic HL-60 cells that express Le(x) antigens, as assessed by a wide variety of approaches including immunofluorescence staining, confocal microscopy, flow cytometry and western blotting, as well as overlay assays of glycolipids after thin-layer chromatography. In contrast, F8A1.1 bound weakly to cercariae, 3-h schistosomula and human Jurkat cells. We also directly compared the glycan specificity of F8A1.1 with commercially available anti-CD15 IgG1 (clone W6D3) using a defined glycan microarray. The results demonstrated that F8A1.1 recognized glycans expressing Le(x) epitopes in a terminal nonreducing position, whereas anti-CD15 bound to glycans with multiple repeats of Le(x) epitopes, but not to glycans with a single, terminal Le(x) epitope. Our results show that F8A1.1 recognizes terminal Le(x) epitopes and can be used for identification, immunolocalization, immunoprecipitation and purification of Le(x)-containing glycoconjugates from schistosomes and mammalian cells.

Mucin-type O-glycans on glycoproteins in animal cells play important roles in many biological processes. Core 1 β3galactosyltransferase (Core 1 β3GalT, T-synthase) is a key enzyme in the O-glycan biosynthetic pathway. Emerging evidence has shown the importance of O-glycans and the absolute requirement of T-synthase in this pathway. The assessment of the T-synthase activity has historically been conducted using a radioactive method. Here we describe a fluorescence-based assay procedure for T-synthase activity. T-synthase utilizes the acceptor substrate 4-methylumbelliferone-α-GalNAc (GalNAcα-(4-MU)) and the donor substrate UDP-Gal to synthesize the disaccharide product Galβ1,3GalNAcα-(4-MU) structure. This product is specifically hydrolyzed by endo-α-N-acetylgalactosaminidase (O-glycosidase) releasing free 4-MU. Free 4-MU is highly fluorescent at pH 9.6-10 and can be easily measured by a fluorescent detector (Ex: 355 nm; Em: 460 nm). This fluorescence-based T-synthase assay is simple, sensitive, reproducible, not affected by enzyme source, and adaptable for high-throughput assays.

BACKGROUND: There are 45 known genetic diseases that impair the lysosomal degradation of macromolecules. The loss of a single lysosomal hydrolase leads to the accumulation of its undegraded substrates in tissues and increases of related glycoconjugates in urine, some of which can be detected by screening of free oligosaccharides (FOS) in urine. Traditional 1-dimensional TLC for urine oligosaccharide analysis has limited analytical specificity and sensitivity. We developed fast and robust urinary FOS and glycoaminoacid analyses by MALDI-time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry for the diagnosis of oligosaccharidoses and other lysosomal storage diseases. METHODS: The FOS in urine equivalent to 0.09 mg creatinine were purified through sequential passage over a Sep-Pak C18 column and a carbograph column and were then permethylated. MALDI-TOF/TOF was used to analyze the permethylated FOS. We studied urine samples from individuals in 7 different age groups ranging from 0-1 months to ≥ 17 years as well as urine from known patients with different lysosomal storage diseases. RESULTS: We identified diagnostic urinary FOS patterns for α-mannosidosis, galactosialidosis, mucolipidosis type II/III, sialidosis, α-fucosidosis, aspartylglucosaminuria (AGU), Pompe disease, Gaucher disease, and GM1 and GM2 gangliosidosis. Interestingly, the increase in urinary FOS characteristic of lysosomal storage diseases relative to normal FOS appeared to correlate with the disease severity. CONCLUSIONS: The analysis of urinary FOS by MALDI-TOF/TOF is a powerful tool for first-tier screening of oligosaccharidoses and lysosomal storage diseases.