Publications

Diagnostic methods for parasite infections still highly depend on the identification of the parasites by direct methods such as microscopic examination of blood, stool and tissue biopsies. Serodiagnosis is often carried out to complement the direct methods; however, few synthetic antigens with sufficient sensitivity and specificity are available. Here we evaluated a glycan microarray approach to select for synthetic glycan antigens that could be used for serodiagnosis of parasitic infections. Using a glycan array containing over 250 different glycan antigens, we identified GalNAcβ1-4(Fucα1-3)GlcNAc-R (LDNF) as a glycan antigen that is recognized by antibodies from Trichinella-infected individuals. We synthesized a neoglycoconjugate, consisting of five LDNF molecules covalently coupled to bovine serum albumin (BSA), and used this neoglycoconjugate as an antigen to develop a highly sensitive total-Ig ELISA for serological screening of trichinellosis. The results indicate that glycan microarrays constitute a promising technology for fast and specific identification of parasite glycan antigens to improve serodiagnosis of different parasitic infections, either using an ELISA format, or parasite-specific glycan arrays.

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2-3- and α2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2-6- and α2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2-6-linked Neu5Ac9Lt or α2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.

Determination of the binding specificity of glycan-binding proteins (GBPs), such as lectins, antibodies, and receptors, has traditionally been difficult and laborious. The advent of glycan microarrays has revolutionized the field of glycobiology by allowing simultaneous screening of a GBP for interactions with a large set of glycans in a single format. This unit describes the theory and method for production of two types of glycan microarrays (chemo/enzymatically synthesized and naturally derived), and their application to functional glycomics to explore glycan recognition by GBPs. These procedures are amenable to various types of arrays and a wide range of GBP samples.

Parasitic helminths (worms) co-evolved with vertebrate immune systems to enable long-term survival of worms in infected hosts. Among their survival strategies, worms use their glycans within glycoproteins and glycolipids, which are abundant on helminth surfaces and in their excretory/ secretory products, to regulate and suppress host immune responses. Many helminths express unusual and antigenic (nonhost-like) glycans, including those containing polyfucose, tyvelose, terminal GalNAc, phosphorylcholine, methyl groups, and sugars in unusual linkages. In addition, some glycan antigens are expressed that share structural features with those in their intermediate and vertebrate hosts (host-like glycans), including Le(X) (Galbeta1-4[Fucalpha1-3]GlcNAc-), LDNF (GalNAcbeta1-4[Fucalpha1-3]GlcNAc-), LDN (GalNAcbeta1-4GlcNAc-), and Tn (GalNAcalpha1-O-Thr/Ser) antigens. The expression of host-like glycan determinants is remarkable and suggests that helminths may gain advantages by synthesizing such glycans. The expression of host-like glycans by parasites previously led to the concept of "molecular mimicry," in which molecules are either derived from the pathogen or acquired from the host to evade recognition by the host immune system. However, recent discoveries into the potential of host glycan-binding proteins (GBPs), such as C-type lectin receptors and galectins, to functionally interact with various host-like helminth glycans provide new insights. Host GBPs through their interactions with worm-derived glycans participate in shaping innate and adaptive immune responses upon infection. We thus propose an alternative concept termed "glycan gimmickry," which is defined as an active strategy of parasites to use their glycans to target GBPs within the host to promote their survival.

Lambs vaccinated with Haemonchus contortus excretory/secretory (ES) glycoproteins in combination with the adjuvant Alhydrogel are protected against H. contortus challenge infection. Using glycan micro-array analysis we showed that serum from such vaccinated lambs contains IgG antibodies that recognise the glycan antigen Galalpha1-3GalNAc-R and GalNAcbeta1-4(Fucalpha1-3)GlcNAc-R. Our studies revealed that H. contortus glycoproteins contain Galalpha1-3Gal-R as well as significant levels of Galalpha1-3GalNAc-R, which has not been previously reported. Extracts from H. contortus adult worms contain a galactosyltransferase acting on glycan substrates with a terminal GalNAc, indicating that the worms possess the enzymatic potential to synthesise terminal Gal-GalNAc moieties. These data illustrate that glycan micro-arrays constitute a promising technology for fast and specific analysis of serum anti-glycan antibodies in vaccination studies. In addition, this approach facilitates the discovery of novel, antigenic parasite glycan antigens that may have potential for developing glycoconjugate vaccines or utilization in diagnostics.

Mucin type O-glycosylation involves sequential actions of several glycosyltransferases in the Golgi apparatus. Among those enzymes, a single gene product termed core 1 beta3-galactosyltransferase (T-synthase) in vertebrates is the key enzyme that converts the precursor Tn antigen GalNAcalpha1-Ser/Thr to the core 1 structure, Galbeta1-3GalNAcalpha1-Ser/Thr, also known as T antigen. This represents the most common structure within typical O-glycans of membrane and secreted glycoproteins. Formation of the active T-synthase requires that it interacts with Core 1 beta3Gal-T Specific Molecular Chaperone (Cosmc), which is a specific molecular chaperone in the endoplasmic reticulum (ER). T-synthase activity is commonly measured by its ability to transfer [3H]Gal from UDP-[3H]Gal to an artificial acceptor GalNAcalpha-1-O-phenyl to form [3H]Galbeta1-3GalNAcalpha-1-O-phenyl, which can then be isolated and quantified. Because the primary function of Cosmc is to form active T-synthase, the activity of Cosmc is assessed indirectly by its ability to promote formation of active T-synthase when it is coexpressed with T-synthase in cells lacking functional Cosmc. Such cells include insect cells, which constitutively lack Cosmc, and Cosmc-deficient mammalian cell lines. Cosmc is encoded by the X-linked Cosmc gene (Xq24 in human, Xc3 in mice), thus, acquired mutations in Cosmc, which have been observed in several human diseases, such as Tn syndrome and cancers, cause a loss of T-synthase, and expression of the Tn antigen. The methods described here allow the functional activities of such mutated Cosmc (mCosmc) to be measured and compared to wild-type (wtCosmc).

Incomplete or aberrant glycosylation leading to Tn antigen (GalNAcalpha1-Ser/Thr) expression on human glycoproteins is strongly associated with human pathological conditions, including tumors, certain autoimmune diseases, such as the idiopathic IgA nephropathy, and may modulate immune homeostasis. In addition, the Tn antigen is highly expressed by certain pathogens and plays a role in host-pathogen interactions. To enable experimental approaches to study interactions of the Tn antigen with the immune system and analyze anti-Tn antibody responses in infection or disorders, we generated a Tn-expressing resource that can be used for high-throughput screening. In consideration of IgA nephropathy in which the hinge region is incompletely glycosylated, we used this hinge sequence that encodes five potential glycosylation sites as the ideal template for the synthesis of a Tn antigen-expressing glycopeptide. Inclusion of an N-terminal biotin in the peptide enabled binding to streptavidin-coated ELISA plates as monitored using Helix pomatia agglutinin or anti-Tn monoclonal antibody. We also found that the biotinylated IgA-Tn peptide is a functional acceptor for beta1-3-galactosylation using recombinant T-synthase (beta1-3-galactosyltransferase). Besides its immunochemical functionality as a possible diagnostic tool for IgA nephropathy, the peptide is an excellent substrate for glycan elongation and represents a novel template applicable for glycan-antigen-associated diseases.

Selectins (L, E, and P) are vascular endothelial molecules that play an important role in the recruitment of leukocytes to inflamed tissue. In this regard, P-Selectin glycoprotein-1 (PSGL-1) has been identified as a ligand for P-Selectin. PSGL-1 binds to P-Selectin through the interaction of core-2 O-glycan expressing sialyl Lewis(x) oligosaccharide and the three tyrosine sulfate residues. Herein, we report the synthesis of threonine-linked core-2 O-glycan as an amino acid building block for the synthesis of PSGL-1. This building block was further incorporated in the Fmoc-assisted solid-phase peptide synthesis to provide a portion of the PSGL-1 glycopeptide.

Endoglycan is a mucin-like glycoprotein expressed by endothelial cells and some leukocytes and is recognized by L-selectin, a C-type lectin important in leukocyte trafficking and extravasation during inflammation. Here, we show that recombinant L-selectin and human T lymphocytes expressing L-selectin bind to synthetic glycosulfopeptides (GSPs). These synthetic glycosulfopeptides contain 37 amino acid residues modeled after the N-terminus of human endoglycan and contain one or two tyrosine sulfates (TyrSO(3)) along with a nearby core-2-based Thr-linked O-glycan with sialyl Lewis x (C2-SLe(x)). TyrSO(3) at position Y118 was more critical for binding than at Y97. C2-SLe(x) at T124 was required for L-selectin recognition. Interestingly, under similar conditions, neither L-selectin nor T lymphocytes showed appreciable binding to the sulfated carbohydrate epitope 6-sulfo-SLe(x). P-selectin also bound to endoglycan-based GSPs but with lower affinity than toward GSPs modeled after PSGL-1, the physiological ligand for P- and L-selectin that is expressed on leukocytes. These results demonstrate that TyrSO(3) residues in association with a C2-SLe(x) moiety within endoglycan and PSGL-1 are preferentially recognized by L-selectin.

We report herein a new and enabling approach for decorating both abiotic and cell surfaces with the extracellular matrix IKVAV peptide in a site-specific manner using strain promoted azide-alkyne cycloaddition. A cyclooctyne-derivatized IKVAV peptide was synthesized and immobilized on the surface of pancreatic islets through strain-promoted azide-alkyne cycloaddition with cell surface azides generated by the electrostatic adsorption of a cytocompatible poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) copolymer bearing azido groups (PP-N(3)). Both "one-pot" and sequential addition of PP-N(3) and a cyclooctyne-derivatized IKVAV peptide conjugate enabled efficient modification of the pancreatic islet surface in less than 60 min. The ability to bind peptides at controlled surface densities was demonstrated in a quantitative manner using microarrays. Additionally, the technique is remarkably rapid and highly efficient, opening new avenues for the molecular engineering of cellular interfaces and protein and peptide microarrays.