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1.
Glycobiology ; 32(12): 1101-1115, 2022 11 22.
Article in English | MEDLINE | ID: mdl-36048714

ABSTRACT

Vertebrate sialic acids (Sias) display much diversity in modifications, linkages, and underlying glycans. Slide microarrays allow high-throughput explorations of sialoglycan-protein interactions. A microarray presenting ~150 structurally defined sialyltrisaccharides with various Sias linkages and modifications still poses challenges in planning, data sorting, visualization, and analysis. To address these issues, we devised a simple 9-digit code for sialyltrisaccharides with terminal Sias and underlying two monosaccharides assigned from the nonreducing end, with 3 digits assigning a monosaccharide, its modifications, and linkage. Calculations based on the encoding system reveal >113,000 likely linear sialyltrisaccharides in nature. Notably, a biantennary N-glycan with 2 terminal sialyltrisaccharides could thus have >1010 potential combinations and a triantennary N-glycan with 3 terminal sequences, >1015 potential combinations. While all possibilities likely do not exist in nature, sialoglycans encode enormous diversity. While glycomic approaches are used to probe such diverse sialomes, naturally occurring bacterial AB5 toxin B subunits are simpler tools to track the dynamic sialome in biological systems. Sialoglycan microarray was utilized to compare sialoglycan-recognizing bacterial toxin B subunits. Unlike the poor correlation between B subunits and species phylogeny, there is stronger correlation with Sia-epitope preferences. Further supporting this pattern, we report a B subunit (YenB) from Yersinia enterocolitica (broad host range) recognizing almost all sialoglycans in the microarray, including 4-O-acetylated-Sias not recognized by a Yersinia pestis orthologue (YpeB). Differential Sia-binding patterns were also observed with phylogenetically related B subunits from Escherichia coli (SubB), Salmonella Typhi (PltB), Salmonella Typhimurium (ArtB), extra-intestinal E.coli (EcPltB), Vibrio cholera (CtxB), and cholera family homologue of E. coli (EcxB).


Subject(s)
Bacterial Toxins , Escherichia coli , Salmonella typhi/chemistry , Sialic Acids , Bacterial Toxins/chemistry , Polysaccharides , Cholera Toxin
2.
Glycobiology ; 32(12): 1116-1136, 2022 11 22.
Article in English | MEDLINE | ID: mdl-35926090

ABSTRACT

Glycans that are abundantly displayed on vertebrate cell surface and secreted molecules are often capped with terminal sialic acids (Sias). These diverse 9-carbon-backbone monosaccharides are involved in numerous intrinsic biological processes. They also interact with commensals and pathogens, while undergoing dynamic changes in time and space, often influenced by environmental conditions. However, most of this sialoglycan complexity and variation remains poorly characterized by conventional techniques, which often tend to destroy or overlook crucial aspects of Sia diversity and/or fail to elucidate native structures in biological systems, i.e. in the intact sialome. To date, in situ detection and analysis of sialoglycans has largely relied on the use of plant lectins, sialidases, or antibodies, whose preferences (with certain exceptions) are limited and/or uncertain. We took advantage of naturally evolved microbial molecules (bacterial adhesins, toxin subunits, and viral hemagglutinin-esterases) that recognize sialoglycans with defined specificity to delineate 9 classes of sialoglycan recognizing probes (SGRPs: SGRP1-SGRP9) that can be used to explore mammalian sialome changes in a simple and systematic manner, using techniques common in most laboratories. SGRP candidates with specificity defined by sialoglycan microarray studies were engineered as tagged probes, each with a corresponding nonbinding mutant probe as a simple and reliable negative control. The optimized panel of SGRPs can be used in methods commonly available in most bioscience labs, such as ELISA, western blot, flow cytometry, and histochemistry. To demonstrate the utility of this approach, we provide examples of sialoglycome differences in tissues from C57BL/6 wild-type mice and human-like Cmah-/- mice.


Subject(s)
Hemagglutinins, Viral , Sialic Acids , Humans , Mice , Animals , Mice, Inbred C57BL , Sialic Acids/chemistry , Mammals/metabolism , Polysaccharides
3.
J Biol Chem ; 298(5): 101900, 2022 05.
Article in English | MEDLINE | ID: mdl-35398357

ABSTRACT

Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y. pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y. pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.


Subject(s)
Bacteria , Bacterial Toxins , Host Specificity , Polysaccharides , Animals , Bacteria/classification , Bacteria/metabolism , Bacterial Toxins/metabolism , Bacterial Toxins/toxicity , Evolution, Molecular , Mammals/metabolism , N-Acetylneuraminic Acid/metabolism , Phylogeny , Plague/microbiology , Polysaccharides/metabolism , Protein Binding , Protein Subunits/metabolism , Yersinia pestis/metabolism
4.
Angew Chem Int Ed Engl ; 58(17): 5592-5597, 2019 04 16.
Article in English | MEDLINE | ID: mdl-30854738

ABSTRACT

The need for atom-precise biomolecule modification, and particularly the irreversible formation of covalent bonds to specific amino acids in proteins, has become an essential issue in the fields of pharmaceuticals and chemical biology. For example, antibody-drug conjugates (ADCs) are increasingly common entries into the clinical oncology pipeline. Herein, we report a new method of affinity peptide mediated regiodivergent functionalization (AJICAP™) that enables the synthesis of ADCs from native IgG antibodies. We succeeded in introducing thiol functional groups onto three lysine residues in IgGs using Fc affinity peptide reagents without antibody engineering. A cytotoxic molecule was then connected to the newly introduced thiol group, and both a surface plasmon resonance binding assay and in vivo xenograft mouse model results showed that the resulting ADC could selectively target and kill HER2-positive cells. Our strategy provides a new approach for constructing complex antibody-derived biomolecules.


Subject(s)
Immunoconjugates/chemistry , Peptides/metabolism , Humans
5.
Bioorg Med Chem ; 26(21): 5751-5757, 2018 11 15.
Article in English | MEDLINE | ID: mdl-30389408

ABSTRACT

Sialidases or neuraminidases are enzymes that catalyze the cleavage of terminal sialic acids from oligosaccharides and glycoconjugates. They play important roles in bacterial and viral infection and have been attractive targets for drug development. Structure-based drug design has led to potent inhibitors against neuraminidases of influenza A viruses that have been used successfully as approved therapeutics. However, selective and effective inhibitors against bacterial and human sialidases are still being actively pursued. Guided by crystal structural analysis, several derivatives of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en or DANA) were designed and synthesized as triazole-linked transition state analogs. Inhibition studies revealed that glycopeptide analog E-(TriazoleNeu5Ac2en)-AKE and compound (TriazoleNeu5Ac2en)-A were selective inhibitors against Vibrio cholerae sialidase, while glycopeptide analog (TriazoleNeu5Ac2en)-AdE selectively inhibited Vibrio cholerae and A. ureafaciens sialidases.


Subject(s)
Enzyme Inhibitors/chemistry , Glycopeptides/chemistry , Neuraminidase/antagonists & inhibitors , Triazoles/chemistry , Vibrio cholerae/enzymology , Catalytic Domain , Enzyme Assays , Enzyme Inhibitors/chemical synthesis , Glycopeptides/chemical synthesis , Humans , Molecular Docking Simulation , Neuraminidase/chemistry , Triazoles/chemical synthesis
6.
PLoS One ; 13(6): e0197464, 2018.
Article in English | MEDLINE | ID: mdl-29912879

ABSTRACT

BACKGROUND: N-glycolylneuraminic acid (Neu5Gc) is a non-human red-meat-derived sialic acid immunogenic to humans. Neu5Gc can be metabolically incorporated into glycan chains on human endothelial and epithelial surfaces. This represents the first example of a "xeno-autoantigen", against which circulating human "xeno-autoantibodies" can react. The resulting inflammation ("xenosialitis") has been demonstrated in human-like Neu5Gc-deficient mice and contributed to carcinoma progression via antibody-mediated inflammation. Anti-Neu5Gc antibodies have potential as biomarkers for diseases associated with red meat consumption such as carcinomas, atherosclerosis, and type 2 diabetes. METHODS: ELISA assays measured antibodies against Neu5Gc or Neu5Gc-glycans in plasma or serum samples from the Nurses' Health Studies, the Health Professionals Follow-up Study, and the European Prospective Investigation into Cancer and Nutrition, including inter-assay reproducibility, stability with delayed sample processing, and within-person reproducibility over 1-3 years in archived samples. We also assessed associations between antibody levels and coronary artery disease risk (CAD) or red meat intake. A glycan microarray was used to detected antibodies against multiple Neu5Gc-glycan epitopes. A nested case-control study design assessed the association between total anti-Neu5Gc antibodies detected in the glycan array assay and the risk of colorectal cancer (CRC). RESULTS: ELISA assays showed a wide range of anti-Neu5Gc responses and good inter-assay reproducibility, stability with delayed sample processing, and within-person reproducibility over time, but these antibody levels did not correlate with CAD risk or red meat intake. Antibodies against Neu5Gc alone or against individual Neu5Gc-bearing epitopes were also not associated with colorectal cancer (CRC) risk. However, a sialoglycan microarray study demonstrated positive association with CRC risk when the total antibody responses against all Neu5Gc-glycans were combined. Individuals in the top quartile of total anti-Neu5Gc IgG antibody concentrations had nearly three times the risk compared to those in the bottom quartile (Multivariate Odds Ratio comparing top to bottom quartile: 2.98, 95% CI: 0.80, 11.1; P for trend = 0.02). CONCLUSIONS: Further work harnessing the utility of these anti-Neu5Gc antibodies as biomarkers in red meat-associated diseases must consider diversity in individual antibody profiles against different Neu5Gc-bearing glycans. Traditional ELISA assays for antibodies directed against Neu5Gc alone, or against specific Neu5Gc-glycans may not be adequate to define risk associations. Our finding of a positive association of total anti-Neu5Gc antibodies with CRC risk also warrants confirmation in larger prospective studies.


Subject(s)
Antibodies/immunology , Colorectal Neoplasms/immunology , Neuraminic Acids/immunology , Polysaccharides/immunology , Adult , Aged , Atherosclerosis/blood , Atherosclerosis/immunology , Atherosclerosis/pathology , Autoantigens/immunology , Colorectal Neoplasms/blood , Colorectal Neoplasms/epidemiology , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/epidemiology , Diabetes Mellitus, Type 2/immunology , Epitopes/immunology , Female , Humans , Middle Aged , N-Acetylneuraminic Acid/immunology , Neuraminic Acids/isolation & purification , Polysaccharides/isolation & purification , Red Meat/adverse effects , Risk Factors
7.
Nat Commun ; 8(1): 2196, 2017 12 19.
Article in English | MEDLINE | ID: mdl-29259165

ABSTRACT

Ruminococcus gnavus is a human gut symbiont wherein the ability to degrade mucins is mediated by an intramolecular trans-sialidase (RgNanH). RgNanH comprises a GH33 catalytic domain and a sialic acid-binding carbohydrate-binding module (CBM40). Here we used glycan arrays, STD NMR, X-ray crystallography, mutagenesis and binding assays to determine the structure and function of RgNanH_CBM40 (RgCBM40). RgCBM40 displays the canonical CBM40 ß-sandwich fold and broad specificity towards sialoglycans with millimolar binding affinity towards α2,3- or α2,6-sialyllactose. RgCBM40 binds to mucus produced by goblet cells and to purified mucins, providing direct evidence for a CBM40 as a novel bacterial mucus adhesin. Bioinformatics data show that RgCBM40 canonical type domains are widespread among Firmicutes. Furthermore, binding of R. gnavus ATCC 29149 to intestinal mucus is sialic acid mediated. Together, this study reveals novel features of CBMs which may contribute to the biogeography of symbiotic bacteria in the gut.


Subject(s)
Adhesins, Bacterial/chemistry , Glycoproteins/chemistry , Mucins/metabolism , N-Acetylneuraminic Acid/chemistry , Neuraminidase/chemistry , Ruminococcus/enzymology , Adhesins, Bacterial/genetics , Adhesins, Bacterial/metabolism , Animals , Catalytic Domain/genetics , Cell Line , Colon/cytology , Colon/metabolism , Computational Biology , Crystallography, X-Ray , Glycoproteins/genetics , Glycoproteins/metabolism , Goblet Cells/metabolism , Humans , Lactose/analogs & derivatives , Lactose/chemistry , Lactose/metabolism , Mice, Inbred C57BL , Mutagenesis, Site-Directed , N-Acetylneuraminic Acid/metabolism , Neuraminidase/genetics , Neuraminidase/metabolism , Protein Binding , Substrate Specificity , Symbiosis
8.
BMC Evol Biol ; 17(1): 228, 2017 11 23.
Article in English | MEDLINE | ID: mdl-29169316

ABSTRACT

BACKGROUND: Siglecs-11 and -16 are members of the sialic acid recognizing Ig-like lectin family, and expressed in same cells. Siglec-11 functions as an inhibitory receptor, whereas Siglec-16 exhibits activating properties. In humans, SIGLEC11 and SIGLEC16 gene sequences are extremely similar in the region encoding the extracellular domain due to gene conversions. Human SIGLEC11 was converted by the nonfunctional SIGLEC16P allele, and the converted SIGLEC11 allele became fixed in humans, possibly because it provides novel neuroprotective functions in brain microglia. However, the detailed evolutionary history of SIGLEC11 and SIGLEC16 in other primates remains unclear. RESULTS: We analyzed SIGLEC11 and SIGLEC16 gene sequences of multiple primate species, and examined glycan binding profiles of these Siglecs. The phylogenetic tree demonstrated that gene conversions between SIGLEC11 and SIGLEC16 occurred in the region including the exon encoding the sialic acid binding domain in every primate examined. Functional assays showed that glycan binding preference is similar between Siglec-11 and Siglec-16 in all analyzed hominid species. Taken together with the fact that Siglec-11 and Siglec-16 are expressed in the same cells, Siglec-11 and Siglec-16 are regarded as paired receptors that have maintained similar ligand binding preferences via gene conversions. Relaxed functional constraints were detected on the SIGLEC11 and SIGLEC16 exons that underwent gene conversions, possibly contributing to the evolutionary acceptance of repeated gene conversions. The frequency of nonfunctional SIGLEC16P alleles is much higher than that of SIGLEC16 alleles in every human population. CONCLUSIONS: Our findings indicate that Siglec-11 and Siglec-16 have been maintained as paired receptors by repeated gene conversions under relaxed functional constraints in the primate lineage. The high prevalence of the nonfunctional SIGLEC16P allele and the fixation of the converted SIGLEC11 imply that the loss of Siglec-16 and the gain of Siglec-11 in microglia might have been favored during the evolution of human lineage.


Subject(s)
Evolution, Molecular , Gene Conversion , Primates/genetics , Sialic Acid Binding Immunoglobulin-like Lectins/genetics , Animals , Humans , Phylogeny , Polysaccharides/metabolism , Receptors, Cell Surface/metabolism , Recombinant Proteins/metabolism , Time Factors
9.
mSphere ; 2(5)2017.
Article in English | MEDLINE | ID: mdl-28904995

ABSTRACT

Sialic acids (Sias) are important glycans displayed on the cells and tissues of many different animals and are frequent targets for binding and modification by pathogens, including influenza viruses. Influenza virus hemagglutinins bind Sias during the infection of their normal hosts, while the encoded neuraminidases and/or esterases remove or modify the Sia to allow virion release or to prevent rebinding. Sias naturally occur in a variety of modified forms, and modified Sias can alter influenza virus host tropisms through their altered interactions with the viral glycoproteins. However, the distribution of modified Sia forms and their effects on pathogen-host interactions are still poorly understood. Here we used probes developed from viral Sia-binding proteins to detect O-acetylated (4-O-acetyl, 9-O-acetyl, and 7,9-O-acetyl) Sias displayed on the tissues of some natural or experimental hosts for influenza viruses. These modified Sias showed highly variable displays between the hosts and tissues examined. The 9-O-acetyl (and 7,9-) modified Sia forms were found on cells and tissues of many hosts, including mice, humans, ferrets, guinea pigs, pigs, horses, dogs, as well as in those of ducks and embryonated chicken egg tissues and membranes, although in variable amounts. The 4-O-acetyl Sias were found in the respiratory tissues of fewer animals, being primarily displayed in the horse and guinea pig, but were not detected in humans or pigs. The results suggest that these Sia variants may influence virus tropisms by altering and selecting their cell interactions. IMPORTANCE Sialic acids (Sias) are key glycans that control or modulate many normal cell and tissue functions while also interacting with a variety of pathogens, including many different viruses. Sias are naturally displayed in a variety of different forms, with modifications at several positions that can alter their functional interactions with pathogens. In addition, Sias are often modified or removed by enzymes such as host or pathogen esterases or sialidases (neuraminidases), and Sia modifications can alter those enzymatic activities to impact pathogen infections. Sia chemical diversity in different hosts and tissues likely alters the pathogen-host interactions and influences the outcome of infection. Here we explored the display of 4-O-acetyl, 9-O-acetyl, and 7,9-O-acetyl modified Sia forms in some target tissues for influenza virus infection in mice, humans, birds, guinea pigs, ferrets, swine, horses, and dogs, which encompass many natural and laboratory hosts of those viruses.

10.
Bioconjug Chem ; 28(7): 1867-1877, 2017 07 19.
Article in English | MEDLINE | ID: mdl-28581731

ABSTRACT

Multiple sclerosis (MS) is a neurodegenerative disease that is estimated to affect over 2.3 million people worldwide. The exact cause for this disease is unknown but involves immune system attack and destruction of the myelin protein surrounding the neurons in the central nervous system. One promising class of compounds that selectively prevent the activation of immune cells involved in the pathway leading to myelin destruction are bifunctional peptide inhibitors (BPIs). Treatment with BPIs reduces neurodegenerative symptoms in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. In this work, as an effort to further improve the bioactivity of BPIs, BPI peptides were conjugated to the N- and C-termini of the fragment crystallizable (Fc) region of the human IgG1 antibody. Initially, the two peptides were conjugated to IgG1 Fc using recombinant DNA technology. However, expression in yeast resulted in low yields and one of the peptides being heavily proteolyzed. To circumvent this problem, the poorly expressed peptide was instead produced by solid phase peptide synthesis and conjugated enzymatically using a sortase-mediated ligation. The sortase-mediated method showed near-complete conjugation yield as observed by SDS-PAGE and mass spectrometry in small-scale reactions. This method was scaled up to obtain sufficient quantities for testing the BPI-Fc fusion in mice induced with EAE. Compared to the PBS-treated control, mice treated with the BPI-Fc fusion showed significantly reduced disease symptoms, did not experience weight loss, and showed reduced de-myelination. These results demonstrate that the BPI peptides were highly active at suppressing EAE when conjugated to the large Fc scaffold in this manner.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/prevention & control , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Peptides/chemistry , Animals , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Mice , Solid-Phase Synthesis Techniques , Treatment Outcome
11.
ACS Chem Biol ; 12(1): 214-224, 2017 01 20.
Article in English | MEDLINE | ID: mdl-27936566

ABSTRACT

9-O-Acetylation is a common natural modification on sialic acids (Sias) that terminate many vertebrate glycan chains. This ester group has striking effects on many biological phenomena, including microbe-host interactions, complement action, regulation of immune responses, sialidase action, cellular apoptosis, and tumor immunology. Despite such findings, 9-O-acetyl sialoglycoconjugates have remained largely understudied, primarily because of marked lability of the 9-O-acetyl group to even small pH variations and/or the action of mammalian or microbial esterases. Our current studies involving 9-O-acetylated sialoglycans on glycan microarrays revealed that even the most careful precautions cannot ensure complete stability of the 9-O-acetyl group. We now demonstrate a simple chemical biology solution to many of these problems by substituting the oxygen atom in the ester with a nitrogen atom, resulting in sialic acids with a chemically and biologically stable 9-N-acetyl group. We present an efficient one-pot multienzyme method to synthesize a sialoglycan containing 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc) and compare it to the one with naturally occurring 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). Conformational resemblance of the two molecules was confirmed by computational molecular dynamics simulations. Microarray studies showed that the Neu5Ac9NAc-sialoglycan is a ligand for viruses naturally recognizing Neu5,9Ac2, with a similar affinity but with much improved stability in handling and study. Feeding of Neu5Ac9NAc or Neu5,9Ac2 to mammalian cells resulted in comparable incorporation and surface expression as well as binding to 9-O-acetyl-Sia-specific viruses. However, cells fed with Neu5Ac9NAc remained resistant to viral esterases and showed a slower turnover. This simple approach opens numerous research opportunities that have heretofore proved intractable.


Subject(s)
Sialic Acids/metabolism , Acetylation , Antigens, CD/metabolism , Cell Line , Cell Membrane/metabolism , Glycosylation , Hemagglutinins, Viral/metabolism , Humans , Ligands , Microarray Analysis , Molecular Conformation , Molecular Dynamics Simulation , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Sialic Acid Binding Ig-like Lectin 2/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Sialic Acids/chemical synthesis , Sialic Acids/chemistry , Torovirus/enzymology , Viral Fusion Proteins/metabolism , Viral Proteins/metabolism
12.
J Biol Chem ; 292(3): 1029-1037, 2017 01 20.
Article in English | MEDLINE | ID: mdl-27920204

ABSTRACT

CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer.


Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/metabolism , Gene Expression Regulation/physiology , Protein Multimerization/physiology , Amino Acid Substitution , Animals , Antibodies/chemistry , Antigens, CD/chemistry , Antigens, CD/genetics , Antigens, Differentiation, B-Lymphocyte/chemistry , Antigens, Differentiation, B-Lymphocyte/genetics , Dendritic Cells/cytology , Dendritic Cells/metabolism , Glycosylation , Humans , Macrophages/cytology , Macrophages/metabolism , Mice , Mice, Knockout , Monocytes/cytology , Monocytes/metabolism , Mutagenesis , Mutation, Missense , Neutrophils/cytology , Neutrophils/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Rats , Rats, Inbred Lew , Sialic Acid Binding Immunoglobulin-like Lectins/chemistry , Sialic Acid Binding Immunoglobulin-like Lectins/genetics , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism
13.
Glycobiology ; 26(11): 1222-1234, 2016 11.
Article in English | MEDLINE | ID: mdl-27037304

ABSTRACT

Serine-rich repeat glycoproteins are adhesins expressed by commensal and pathogenic Gram-positive bacteria. A subset of these adhesins, expressed by oral streptococci, binds sialylated glycans decorating human salivary mucin MG2/MUC7, and platelet glycoprotein GPIb. Specific sialoglycan targets were previously identified for the ligand-binding regions (BRs) of GspB and Hsa, two serine-rich repeat glycoproteins expressed by Streptococcus gordonii While GspB selectively binds sialyl-T antigen, Hsa displays broader specificity. Here we examine the binding properties of four additional BRs from Streptococcus sanguinis or Streptococcus mitis and characterize the molecular determinants of ligand selectivity and affinity. Each BR has two domains that are essential for sialoglycan binding by GspB. One domain is structurally similar to the glycan-binding module of mammalian Siglecs (sialic acid-binding immunoglobulin-like lectins), including an arginine residue that is critical for glycan recognition, and that resides within a novel, conserved YTRY motif. Despite low sequence similarity to GspB, one of the BRs selectively binds sialyl-T antigen. Although the other three BRs are highly similar to Hsa, each displayed a unique ligand repertoire, including differential recognition of sialyl Lewis antigens and sulfated glycans. These differences in glycan selectivity were closely associated with differential binding to salivary and platelet glycoproteins. Specificity of sialoglycan adherence is likely an evolving trait that may influence the propensity of streptococci expressing Siglec-like adhesins to cause infective endocarditis.


Subject(s)
Glycoproteins/chemistry , Polysaccharides/analysis , Sialic Acid Binding Immunoglobulin-like Lectins/chemistry , Sialic Acids/analysis , Streptococcus/chemistry , Humans , Ligands
15.
PLoS Pathog ; 12(1): e1005411, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26816272

ABSTRACT

Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Orthomyxoviridae Infections/virology , Thogotovirus/metabolism , Viral Fusion Proteins/metabolism , Animals , Binding Sites , Blotting, Western , Cattle , Crystallography, X-Ray , Dogs , Humans , Madin Darby Canine Kidney Cells , Protein Array Analysis , Protein Conformation , Swine , Thogotovirus/chemistry , Virus Internalization
16.
Elife ; 3: e04066, 2014 Sep 03.
Article in English | MEDLINE | ID: mdl-25187624

ABSTRACT

Both pathogen- and tissue damage-associated molecular patterns induce inflammation through toll-like receptors (TLRs), while sialic acid-binding immunoglobulin superfamily lectin receptors (Siglecs) provide negative regulation. Here we report extensive and direct interactions between these pattern recognition receptors. The promiscuous TLR binders were human SIGLEC-5/9 and mouse Siglec-3/E/F. Mouse Siglec-G did not show appreciable binding to any TLRs tested. Correspondingly, Siglece deletion enhanced dendritic cell responses to all microbial TLR ligands tested, while Siglecg deletion did not affect the responses to these ligands. TLR4 activation triggers Neu1 translocation to cell surface to disrupt TLR4:Siglec-E interaction. Conversely, sialidase inhibitor Neu5Gc2en prevented TLR4 ligand-induced disruption of TLR4:Siglec E/F interactions. Absence of Neu1 in hematopoietic cells or systematic treatment with sialidase inhibitor Neu5Gc2en protected mice against endotoxemia. Our data raised an intriguing possibility of a broad repression of TLR function by Siglecs and a sialidase-mediated de-repression that allows positive feedback of TLR activation during infection.


Subject(s)
Antigens, CD/immunology , Antigens, Differentiation, B-Lymphocyte/immunology , Neuraminidase/immunology , Receptors, Pattern Recognition/immunology , Toll-Like Receptor 4/immunology , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/genetics , Antigens, Differentiation, B-Lymphocyte/metabolism , Cell Line , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Endotoxemia/immunology , Endotoxemia/microbiology , Endotoxemia/prevention & control , Enzyme Inhibitors/immunology , Enzyme Inhibitors/pharmacology , Humans , Immunoblotting , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Neuraminidase/genetics , Neuraminidase/metabolism , Oligodeoxyribonucleotides/immunology , Oligodeoxyribonucleotides/pharmacology , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/metabolism , Receptors, Pattern Recognition/genetics , Receptors, Pattern Recognition/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism
17.
Carbohydr Res ; 389: 100-11, 2014 May 07.
Article in English | MEDLINE | ID: mdl-24680514

ABSTRACT

Modifications at the glycerol side chain of sialic acid in sialosides modulate their recognition by sialic acid-binding proteins and sialidases. However, limited work has been focused on the synthesis and functional studies of sialosides with C7-modified sialic acids. Here we report chemical synthesis of C4-modified ManNAc and mannose and their application as sialic acid precursors in a highly efficient one-pot three-enzyme system for chemoenzymatic synthesis of α2-3- and α2-6-linked sialyl para-nitrophenyl galactosides in which the C7-hydroxyl group in sialic acid (N-acetylneuraminic acid, Neu5Ac, or 2-keto-3-deoxynonulosonic acid, Kdn) was systematically substituted by -F, -OMe, -H, and -N3 groups. Substrate specificity study of bacterial and human sialidases using the obtained sialoside library containing C7-modified sialic acids showed that sialosides containing C7-deoxy Neu5Ac were selective substrates for all bacterial sialidases tested but not for human NEU2. The information obtained from sialidase substrate specificity can be used to guide the design of new inhibitors that are selective against bacterial sialidases.


Subject(s)
Enzyme Assays , Neuraminidase/metabolism , Sialic Acids/chemical synthesis , Sialic Acids/metabolism , Bacteria/enzymology , Chemistry Techniques, Synthetic , Enzyme Inhibitors/pharmacology , Humans , Neuraminidase/antagonists & inhibitors , Sialic Acids/chemistry , Substrate Specificity
18.
Bioorg Med Chem ; 21(16): 4778-85, 2013 Aug 15.
Article in English | MEDLINE | ID: mdl-23535562

ABSTRACT

A series of STn-MUC1 and ST-MUC1 glycopeptides containing naturally occurring and non-natural sialic acids have been chemoenzymatically synthesized from Tn-MUC1 glycopeptide using one-pot multienzyme (OPME) approaches. In situ generation of the sialyltransferase donor cytidine 5'-monophosphate-sialic acid (CMP-Sia) using a CMP-sialic acid synthetase in the presence of an extra amount of cytidine 5'-triphosphate (CTP) and removal of CMP from the reaction mixture by flash C18 cartridge purification allow the complete consumption of Tn-MUC1 glycopeptide for quantitative synthesis of STn-MUC1. A Campylobacter jejuni ß1-3GalT (CjCgtBΔ30-His6) mutant has been found to catalyze the transfer of one or more galactose residues to Tn-MUC1 for the synthesis of T-MUC1 and galactosylated T-MUC1. Sialylation of T-MUC1 using Pasteurella multocida α2-3-sialyltransferase 3 (PmST3) with Neisseria meningitidis CMP-sialic acid synthetase (NmCSS) and Escherichia coli sialic acid aldolase in one pot produced ST-MUC1 efficiently. These glycopeptides are potential cancer vaccine candidates.


Subject(s)
Bacterial Proteins/metabolism , Glycopeptides/biosynthesis , Sialic Acids/chemistry , Antigens, Viral, Tumor/chemistry , Bacterial Proteins/genetics , Base Sequence , Campylobacter jejuni/enzymology , Cloning, Molecular , Escherichia coli/enzymology , Glycopeptides/chemistry , Molecular Sequence Data , Mutation , Neisseria meningitidis/enzymology , Oxo-Acid-Lyases/genetics , Oxo-Acid-Lyases/metabolism , Pasteurella multocida/enzymology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Sialyltransferases/genetics , Sialyltransferases/metabolism
19.
Org Biomol Chem ; 10(30): 6112-20, 2012 Aug 14.
Article in English | MEDLINE | ID: mdl-22641268

ABSTRACT

Sialidases or neuraminidases catalyze the hydrolysis of terminal sialic acid residues from sialyl oligosaccharides and glycoconjugates. Despite successes in developing potent inhibitors specifically against influenza virus neuraminidases, the progress in designing and synthesizing selective inhibitors against bacterial and human sialidases has been slow. Guided by sialidase substrate specificity studies and sialidase crystal structural analysis, a number of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA or Neu5Ac2en) analogues with modifications at C9 or at both C5 and C9 were synthesized. Inhibition studies of various bacterial sialidases and human cytosolic sialidase NEU2 revealed that Neu5Gc9N(3)2en and Neu5AcN(3)9N(3)2en are selective inhibitors against V. cholerae sialidase and human NEU2, respectively.


Subject(s)
Cytosol/enzymology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Neuraminidase/antagonists & inhibitors , Vibrio cholerae/enzymology , Chemistry Techniques, Synthetic , Drug Design , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Neuraminic Acids/chemical synthesis , Neuraminic Acids/chemistry , Neuraminic Acids/pharmacology , Neuraminidase/chemistry , Protein Conformation
20.
Chem Commun (Camb) ; 48(27): 3357-9, 2012 Apr 04.
Article in English | MEDLINE | ID: mdl-22361713

ABSTRACT

A library of α2-3- and α2-6-linked sialyl galactosides containing C9-modified sialic acids was synthesized from C6-modified mannose derivatives using an efficient one-pot three-enzyme system. These sialosides were used in a high-throughput sialidase substrate specificity assay to elucidate the importance of C9-OH in sialidase recognition.


Subject(s)
Bacterial Proteins/chemistry , Galactosides/chemistry , Neuraminidase/chemistry , Sialic Acids/chemistry , Sialyltransferases/chemistry , High-Throughput Screening Assays , Humans , Mannose/analogs & derivatives , Mannose/chemistry , Substrate Specificity
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