Human Group C Rotavirus VP8*s Recognize Type A Histo-Blood Group Antigens as Ligands.

Group/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[2] RVC VP8* that is responsible for the host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood group antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, the high-resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two ß-sheets but with significant differences from cogent proteins of group A rotaviruses (RVAs). The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with that used by RVAs. Our findings shed light on the host-virus interaction and the coevolution of RVCs and will facilitate the development of specific antivirals and vaccines.IMPORTANCE Group/species C rotaviruses (RVCs), members of Reoviridae family, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the rotavirus host ligand binding domain that shares only ∼15% homology with the VP8* domains of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a mechanism distinct from that used by RVAs. Our new advancements provide insights into RVC-cell attachment, the critical step of virus infection, which will in turn help the development of control and prevention strategies against RVs.

Development and validation of a Luminex assay for detection of a predictive biomarker for PROSTVAC-VF therapy.

Cancer therapies can provide substantially improved survival in some patients while other seemingly similar patients receive little or no benefit. Strategies to identify patients likely to respond well to a given therapy could significantly improve health care outcomes by maximizing clinical benefits while reducing toxicities and adverse effects. Using a glycan microarray assay, we recently reported that pretreatment serum levels of IgM specific to blood group A trisaccharide (BG-Atri) correlate positively with overall survival of cancer patients on PROSTVAC-VF therapy. The results suggested anti-BG-Atri IgM measured prior to treatment could serve as a biomarker for identifying patients likely to benefit from PROSTVAC-VF. For continued development and clinical application of serum IgM specific to BG-Atri as a predictive biomarker, a clinical assay was needed. In this study, we developed and validated a Luminex-based clinical assay for measuring serum IgM specific to BG-Atri. IgM levels were measured with the Luminex assay and compared to levels measured using the microarray for 126 healthy individuals and 77 prostate cancer patients. This assay provided reproducible and consistent results with low %CVs, and tolerance ranges were established for the assay. IgM levels measured using the Luminex assay were found to be highly correlated to the microarray results with R values of 0.93-0.95. This assay is a Laboratory Developed Test (LDT) and is suitable for evaluating thousands of serum samples in CLIA certified laboratories that have validated the assay. In addition, the study demonstrates that discoveries made using neoglycoprotein-based microarrays can be readily migrated to a clinical assay.

Highly Branched Pentasaccharide-Bearing Amphiphiles for Membrane Protein Studies.

Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA/Es with a novel highly branched pentasaccharide hydrophilic group. The PSA/Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA/Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure-function studies of membrane proteins.

Branched-chain sugar nucleosides: stereocontrolled synthesis and bioevaluation of novel 3'-C-trifluoromethyl and 3'-C-methyl pyranonucleosides.

A new series of 3'-C-trifluoromethyl- and 3'-C-methyl-ß-d-allopyranonucleosides of 5-fluorouracil and their deoxy derivatives has been designed and synthesized. Treatment of ketosugar 1 with trifluoromethyltrimethylsilane under catalytic fluoride activation and methyl magnesium bromide, gave 1,2:5,6-di-O-isopropylidene-3-C-trifluoromethyl (2a) and 3-C-methyl (2b)-α-D-allofuranose, respectively, in a virtually quantitative yield and with complete stereoselectivity. Hydrolysis followed by acetylation led to the 1,2,4,6-tetra-O-acetyl-3-C-trifluoromethyl (3a) and 3-C-methyl (3b)-ß-D-allopyranose. Compounds 3a,b were then condensed with silylated 5-fluorouracil and deacetylated to afford the target nucleosides 5a,b. Deoxygenation of the peracylated allopyranoses 3a,b followed by condensation with silylated 5-fluorouracil and subsequent deacetylation yielded the target 3'-deoxy-3'-C-trifluoromethyl and 3'-deoxy-3'-C-methyl-ß-d-glucopyranonucleosides 14a,b. The newly synthesized compounds were evaluated for their potential antiviral and cytostatic activities. The 3'-deoxy-3'-C-methyl- ribonucleoside 11b showed significant cytotoxic activity (∼7 µM) almost equally active against a variety of tumor cell lines.

Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH-Π interactions.

Engineering of novel carbohydrate-binding proteins that can be utilized in various biochemical and biotechnical applications would benefit from a deeper understanding of the biochemical interactions that determine protein-carbohydrate specificity. In an effort to understand further the basis for specificity we present the crystal structure of the multi-specific carbohydrate-binding module (CBM) X-2 L110F bound to a branched oligomer of xyloglucan (XXXG). X-2 L110F is an engineered CBM that can recognize xyloglucan, xylans and ß-glucans. The structural observations of the present study compared with previously reported structures of X-2 L110F in complex with linear oligomers, show that the π-surface of a phenylalanine, F110, allows for interactions with hydrogen atoms on both linear (xylopentaose and cellopentaose) and branched ligands (XXXG). Furthermore, X-2 L110F is shown to have a relatively flexible binding cleft, as illustrated in binding to XXXG. This branched ligand requires a set of reorientations of protein side chains Q72, N31, and R142, although these residues have previously been determined as important for binding to xylose oligomers by mediating polar contacts. The loss of these polar contacts is compensated for in binding to XXXG by polar interactions mediated by other protein residues, T74, R115, and Y149, which interact mainly with the branching xyloses of the xyloglucan oligomer. Taken together, the present study illustrates in structural detail how CH-π interactions can influence binding specificity and that flexibility is a key feature for the multi-specificity displayed by X-2 L110F, allowing for the accommodation of branched ligands.

α2,6 sialylation associated with increased beta 1,6-branched N-oligosaccharides influences cellular adhesion and invasion.

Expression of ß1,6-branched N-linked oligosaccharides have a definite association with invasion and metastasis of cancer cells. However, the mechanism by which these oligosaccharides regulate these processes is not well understood. Invasive variants of B16 murine melanoma, B16F10 (parent) and B16BL6 (highly invasive variant) cell lines have been used for these studies. We demonstrate that substitution of α2,6-linked sialic acids on multiantennary structures formed as a result of ß1,6-branching modulate cellular adhesion on both extracellular matrix (ECM) and basement membrane (BM) components. Removal of α2,6 sialic acids either by enzymatic desialylation or by stably down-regulating the ST6Gal-I (enzyme that catalyses the addition of α2,6-linked sialic acids on N-linked oligosaccharides) by lentiviral driven shRNA decreased the adhesion on both ECM and BM components and invasion through reconstituted BM matrigel.

Branching pattern of gluco-oligosaccharides and 1.5kDa dextran grafted by the α-1,2 branching sucrase GBD-CD2.

GBD-CD2, an engineered sucrose-acting enzyme of glycoside hydrolase family 70, transfers D-glucopyranosyl (D-Glcp) units from sucrose onto dextrans or gluco-oligosaccharides (GOS) through the formation of α-(1→2) linkages leading to branched products of interest for health, food and cosmetic applications. Structural characterization of the branched products obtained from sucrose and pure GOS of degree of polymerization (DP) 4 or DP 5 revealed that highly α-(1→2) branched and new molecular structures can be synthesized by GBD-CD2. The formation of α-(1→2) branching is kinetically controlled and can occur onto vicinal α-(1→6)-linked D-Glcp residues. To investigate the mode of branching of 1.5 kDa dextran, simulations of various branching scenarios and resistance to glucoamylase degradation were performed. Analysis of the simulation results suggests that the branching process is stochastic and indicates that the enzyme acceptor site can accommodate both linear and poly-branched acceptors. This opens the way to the design of novel enzyme-based processes yielding carbohydrate structures varying in size and resistance to hydrolytic enzymes.

Surface-exposed glycoproteins of hyperthermophilic Sulfolobus solfataricus P2 show a common N-glycosylation profile.

Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the N-glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of S. solfataricus, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex4(GlcNAc)2 plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of S. acidocaldarius, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of S. solfataricus are N-glycosylated by the same sulfated oligosaccharide and we identified 56 N-glycosylation sites in this subproteome.

Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity.

A minor xylanase, named XYN IV, was purified from the cellulolytic system of the fungus Trichoderma reesei Rut C30. The enzyme was discovered on the basis of its ability to attack aldotetraohexenuronic acid (HexA-2Xyl-4Xyl-4Xyl, HexA(3)Xyl(3)), releasing the reducing-end xylose residue. XYN IV exhibited catalytic properties incompatible with previously described endo-ß-1,4-xylanases of this fungus, XYN I, XYN II and XYN III, and the xylan-hydrolyzing endo-ß-1,4-glucanase EG I. XYN IV was able to degrade several different ß-1,4-xylans, but was inactive on ß-1,4-mannans and ß-1,4-glucans. It showed both exo-and endo-xylanase activity. Rhodymenan, a linear soluble ß-1,3-ß-1,4-xylan, was as the best substrate. Linear xylooligosaccharides were attacked exclusively at the first glycosidic linkage from the reducing end. The gene xyn4, encoding XYN IV, was also isolated. It showed clear homology with xylanases classified in glycoside hydrolase family 30, which also includes glucanases and mannanases. The xyn4 gene was expressed slightly when grown on xylose and xylitol, clearly on arabinose, arabitol, sophorose, xylobiose, xylan and cellulose, but not on glucose or sorbitol, resembling induction of other xylanolytic enzymes from T. reesei. A recombinant enzyme prepared in a Pichia pastoris expression system exhibited identical catalytic properties to the enzyme isolated from the T. reesei culture medium. The physiological role of this unique enzyme remains unknown, but it may involve liberation of xylose from the reducing end of branched oligosaccharides that are resistant toward ß-xylosidase and other types of endoxylanases. In terms of its catalytic properties, XYN IV differs from bacterial GH family 30 glucuronoxylanases that recognize 4-O-methyl-D-glucuronic acid (MeGlcA) substituents as substrate specificity determinants.

Synthesis of branched arabinofuranose pentasaccharide fragment of mycobacterial arabinans as 2-azidoethyl glycoside.

Branched arabinofuranose pentasaccharide with 2-azidoethyl aglycon was prepared for the first time by [3+1+1] bis-(1,2-cis)-glycosylation of trisaccharide diol with silyl-protected thioglycoside glycosyl donor. The presence of 2-azidoethyl aglycon would enable the preparation of neoglycoconjugates using the click chemistry approaches.

Binding of N-acetylglucosamine (GlcNAc) ß1-6-branched oligosaccharide acceptors to ß4-galactosyltransferase I reveals a new ligand binding mode.

N-acetyllactosamine is the most prevalent disaccharide moiety in the glycans on the surface of mammalian cells and often found as repeat units in the linear and branched polylactosamines, known as i- and I-antigen, respectively. The ß1-4-galactosyltransferase-I (ß4Gal-T1) enzyme is responsible for the synthesis of the N-acetyllactosamine moiety. To understand its oligosaccharide acceptor specificity, we have previously investigated the binding of tri- and pentasaccharides of N-glycan with a GlcNAc at their nonreducing end and found that the extended sugar moiety in these acceptor substrates binds to the crevice present at the acceptor substrate binding site of the ß4Gal-T1 molecule. Here we report seven crystal structures of ß4Gal-T1 in complex with an oligosaccharide acceptor with a nonreducing end GlcNAc that has a ß1-6-glycosidic link and that are analogous to either N-glycan or i/I-antigen. In the crystal structure of the complex of ß4Gal-T1 with I-antigen analog pentasaccharide, the ß1-6-branched GlcNAc moiety is bound to the sugar acceptor binding site of the ß4Gal-T1 molecule in a way similar to the crystal structures described previously; however, the extended linear tetrasaccharide moiety does not interact with the previously found extended sugar binding site on the ß4Gal-T1 molecule. Instead, it interacts with the different hydrophobic surface of the protein molecule formed by the residues Tyr-276, Trp-310, and Phe-356. Results from the present and previous studies suggest that ß4Gal-T1 molecule has two different oligosaccharide binding regions for the binding of the extended oligosaccharide moiety of the acceptor substrate.

Toward creating cell membrane glyco-landscapes with glycan lipid constructs.

Synthetic glycolipid-like constructs dispersible in biological media and capable of incorporating into cell membranes have the ability to create novel artificial glyco-landscapes on living cells. Using a variety of different glycans ranging from disaccharides to polysaccharides, together with different lengths and high hydrophilicity spacers, we created a series of synthetic glycolipid-like constructs. Contacting these constructs with live cells gave modified cells with controlled glycan density and/or altered biological function. The ability to also use these constructs as solutions to inhibit antibodies, toxins, and virions extends the potential diagnostic and therapeutic uses for these synthetic glycolipid-like constructs.

Purification and characterization of highly branched α-glucan-producing enzymes from Paenibacillus sp. PP710.

Highly branched α-glucan molecules exhibit low digestibility for α-amylase and glucoamylase, and abundant in α-(1→3)-, α-(1→6)-glucosidic linkages and α-(1→6)-linked branch points where another glucosyl chain is initiated through an α-(1→3)-linkage. From a culture supernatant of Paenibacillus sp. PP710, we purified α-glucosidase (AGL) and α-amylase (AMY), which were involved in the production of highly branched α-glucan from maltodextrin. AGL catalyzed the transglucosylation reaction of a glucosyl residue to a nonreducing-end glucosyl residue by α-1,6-, α-1,4-, and α-1,3-linkages. AMY catalyzed the hydrolysis of the α-1,4-linkage and the intermolecular or intramolecular transfer of maltooligosaccharide like cyclodextrin glucanotransferase (CGTase). It also catalyzed the transfer of an α-1,4-glucosyl chain to a C3- or C4-hydroxyl group in the α-1,4- or α-1,6-linked nonreducing-end residue or the α-1,6-linked residue located in the other chains. Hence AMY was regarded as a novel enzyme. We think that the mechanism of formation of highly branched α-glucan from maltodextrin is as follows: α-1,6- and α-1,3-linked residues are generated by the transglucosylation of AGL at the nonreducing ends of glucosyl chains. Then AMY catalyzes the transfer of α-1,4-chains to C3- or C4-hydroxyl groups in the α-1,4- or α-1,6-linked residues generated by AGL. Thus the concerted reactions of both AGL and AMY are necessary to produce the highly branched α-glucan from maltodextrin.

Human prostate cancer in a clinically relevant xenograft mouse model: identification of ß(1,6)-branched oligosaccharides as a marker of tumor progression.

PURPOSE: To establish xenograft mouse models of metastatic and nonmetastatic human prostate cancer and to apply these models to the search for aberrant glycosylation patterns associated with tumor progression in vivo and in patients. EXPERIMENTAL DESIGN: Prostate cancer cells (LNCaP, PC-3, LuCaP 23.1, and DU-145) were xenografted subcutaneously into immunodeficient pfp(-/-)/rag2(-/-) mice. Tumor growth and metastasis formation were quantified and as altered glycosylation patterns have been associated with metastasis formation in several other malignancies, prostate cancer cells were profiled by a quantitative real-time PCR (qRT-PCR) glycosylation array and compared with normal human prostate cells. The activity of upregulated glycosyltransferases was analyzed by their sugar residues end products using lectin histochemistry on primary tumors and metastases in the animal experiments and on 2,085 clinical samples. RESULTS: PC-3 cells produced the largest number of spontaneous lung metastases, followed by LNCaP and LuCaP 23.1, whereas DU-145 was nonmetastatic. qRT-PCR revealed an upregulation of ß1,6-N-acetylglucosaminyltransferase-5b (Mgat5b) in all prostate cancer cell lines. Mgat5b products [ß(1,6)-branched oligosaccharides] were predominantly detectable in metastatic xenografts as shown by increased binding of Phaseolus vulgaris leukoagglutinin (PHA-L). The percentage of prostate cancer patients who were PHA-L positive was 86.5. PHA-L intensity correlated with serum prostate-specific antigen and a cytoplasmic staining negatively affected disease-free survival. CONCLUSION: We show a novel xenograft mouse model for human prostate cancer respecting the complete metastatic cascade. Specific glycosylation patterns reveal Mgat5b products as relevant markers of both metastatic competence in mice and disease-free survival in patients. This is the first description of Mgat5b in prostate cancer indicating a significant biologic importance of ß(1,6)-branched oligosaccharides for prostate cancer progression.

Anti-GM1/GD1a complex antibodies in GBS sera specifically recognize the hybrid dimer GM1-GD1a.

It is now emerging the new concept that the antibodies from some patients with Guillain-Barré syndrome (GBS) recognize an antigenic epitope formed by two different gangliosides, a ganglioside complex (GSC). We prepared the dimeric GM1-GD1a hybrid ganglioside derivative that contains two structurally different oligosaccharide chains to mimic the GSC. We use this compound to analyze sera from GBS patients by high-performance thin-layer chromatography immunostaining and enzyme-linked immunosorbent assay. We also synthesized the dimeric GM1-GM1 and GD1a-GD1a compounds that were used in control experiments together with natural gangliosides. The hybrid dimeric GM1-GD1a was specifically recognized by human sera from GBS patients that developed anti-oligosaccharide antibodies specific for grouped complex oligosaccharides, confirming the information that GBS patients developed antibodies against a GSC. High-resolution (1)H-(13)C heteronuclear single-quantum coherence-nuclear overhauser effect spectroscopy nuclear magnetic resonance experiments showed an interaction between the IV Gal-H1 of GM1 and the IV Gal-H2 of GD1a suggesting that the two oligosaccharide chains of the dimeric ganglioside form a single epitope recognized by a single-antibody domain. The availability of a method capable to prepare several hybrid gangliosides, and the availability of simple analytical approaches, opens new perspectives for the understanding and the therapy of several neuropathies.

Structure elucidation of native N- and O-linked glycans by tandem mass spectrometry (tutorial).

Oligosaccharides play important roles in many biological processes. However, the structural elucidation of oligosaccharides remains a major challenge due to the complexities of their structures. Mass spectrometry provides a powerful method for determining oligosaccharide composition. Tandem mass spectrometry (MS) provides structural information with high sensitivity. Oligosaccharide structures differ from other polymers such as peptides because of the large number of linkage combinations and branching. This complexity makes the analysis of oligosaccharide unique from that of peptides. This tutorial addresses the issue of spectral interpretation of tandem MS under conditions of collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). The proper interpretation of tandem MS data can provide important structural information on different types of oligosaccharides including O- and N-linked.

Changes in oligosaccharide chains of autoantibodies to GRP78 expressed during progression of malignant melanoma stimulate melanoma cell growth and survival.

A correlation between expression of the glucose-regulated protein of 78 kDa (GRP78) in malignant melanoma tumors and poor patient survival is well established. In this study, in addition to demonstrating the expression of GRP78 in tumor tissue, we investigated the immune response against GRP78 in a group of patients with different progression stages of malignant melanoma. Furthermore, we analyzed the glycosylation status of GRP78 immunoglobulin (Ig) G autoantibodies at these stages and evaluated their capacities to affect the protein B-dependent protein kinase signaling pathway and unfolded protein response signaling mechanisms, all known to promote malignant melanoma cell proliferation and survival. We found that progression of disease correlates not only with enhanced expression of GRP78 in the tumor but also with an increase in GRP78 autoantibody serum titers in these patients. We also found that the glycosylation status of anti-GRP78 IgG changes as the disease progresses. The anti-GRP78 IgG is abnormally glycosylated in the Fc region and asymmetrically glycosylated in the Fab region. We demonstrate that hyperglycosylated anti-GRP78 IgGs stimulate cell proliferation through protein B-dependent protein kinase signaling pathways. They also mimic the effects of α2-macroglobulin on the upregulation of GRP78 and X-box binding protein 1, activating transcription factor 6 α, and serine/threonine-protein kinase/endoribonuclease precursor α as endoplasmic reticulum stress biomarkers and show no effect on expression or activation of caspases 3, 9, or 12. In conclusion, the anti-GRP78 IgG autoantibodies downregulate apoptosis and activate unfolded protein response mechanisms, which are essential to promote melanoma cell growth and survival.