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1.
Chembiochem ; 21(22): 3212-3215, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-32597008

RESUMO

In human serum immunoglobulin G (IgG), a rare modification of biantennary complex N-glycans lead to a ß1,4-galactosylated bisecting GlcNAc branch. We found that the bisecting GlcNAc on a biantennary core-fucosylated N-glycan was enzymatically galactosylated under stringent reaction conditions. Further optimizations led to an efficient enzymatic approach to this particular modification for biantennary substrates. Notably, tri- and tetra-antennary complex N-glycans were not converted by bovine galactosyltransferase. An N-glycan with a galactosylated bisecting GlcNAc was linked to a lanthanide binding tag. The pseudo-contact shifts (PCS) obtained from the corresponding Dy-complex were used to calculate the conformational preferences of the rare N-glycan. Besides two extended conformations only a single folded conformation was found.


Assuntos
Acetilglucosamina/metabolismo , Galactose/metabolismo , Polissacarídeos/biossíntese , Acetilglucosamina/química , Configuração de Carboidratos , Galactose/química , Glicosilação , Humanos , Polissacarídeos/química
2.
Cell Host Microbe ; 21(1): 23-34, 2017 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-28017661

RESUMO

Human and avian influenza viruses recognize different sialic acid-containing receptors, referred to as human-type (NeuAcα2-6Gal) and avian-type (NeuAcα2-3Gal), respectively. This presents a species barrier for aerosol droplet transmission of avian viruses in humans and ferrets. Recent reports have suggested that current human H3N2 viruses no longer have strict specificity toward human-type receptors. Using an influenza receptor glycan microarray with extended airway glycans, we find that H3N2 viruses have in fact maintained human-type specificity, but they have evolved preference for a subset of receptors comprising branched glycans with extended poly-N-acetyl-lactosamine (poly-LacNAc) chains, a specificity shared with the 2009 pandemic H1N1 (Cal/04) hemagglutinin. Lipid-linked versions of extended sialoside receptors can restore susceptibility of sialidase-treated MDCK cells to infection by both recent (A/Victoria/361/11) and historical (A/Hong Kong/8/1968) H3N2 viruses. Remarkably, these human-type receptors with elongated branches have the potential to increase avidity by simultaneously binding to two subunits of a single hemagglutinin trimer.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H3N2/metabolismo , Receptores Virais/metabolismo , Ligação Viral , Animais , Linhagem Celular , Cães , Galactanos/metabolismo , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N8/metabolismo , Virus da Influenza A Subtipo H5N1/metabolismo , Células Madin Darby de Rim Canino , Simulação de Dinâmica Molecular , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Especificidade da Espécie
3.
Nat Chem Biol ; 10(6): 470-6, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24814672

RESUMO

Genomic approaches continue to provide unprecedented insight into the microbiome, yet host immune interactions with diverse microbiota can be difficult to study. We therefore generated a microbial microarray containing defined antigens isolated from a broad range of microbial flora to examine adaptive and innate immunity. Serological studies with this microarray show that immunoglobulins from multiple mammalian species have unique patterns of reactivity, whereas exposure of animals to distinct microbes induces specific serological recognition. Although adaptive immunity exhibited plasticity toward microbial antigens, immunological tolerance limits reactivity toward self. We discovered that several innate immune galectins show specific recognition of microbes that express self-like antigens, leading to direct killing of a broad range of Gram-negative and Gram-positive microbes. Thus, host protection against microbes seems to represent a balance between adaptive and innate immunity to defend against evolving antigenic determinants while protecting against molecular mimicry.


Assuntos
Antígenos de Bactérias/imunologia , Galectinas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Análise em Microsséries/métodos , Polissacarídeos/imunologia , Imunidade Adaptativa , Animais , Antígenos de Bactérias/sangue , Antígenos de Bactérias/metabolismo , Sítios de Ligação , Células CHO , Sobrevivência Celular/imunologia , Cricetinae , Cricetulus , Fluorometria/métodos , Galectinas/sangue , Galectinas/metabolismo , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Positivas/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Camundongos , Polissacarídeos/sangue , Polissacarídeos/metabolismo , Coelhos
4.
Methods Mol Biol ; 1022: 1-14, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23765649

RESUMO

Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.


Assuntos
Ensaios Enzimáticos/métodos , Glicosiltransferases/metabolismo , Análise em Microsséries/métodos , Polissacarídeos/metabolismo , Biotinilação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Polissacarídeos/química , Proteínas Recombinantes/metabolismo , Sialiltransferases/metabolismo , Especificidade por Substrato , beta-D-Galactosídeo alfa 2-6-Sialiltransferase
5.
Glycobiology ; 22(11): 1453-64, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22786570

RESUMO

Poly-N-acetyllactosamine extensions on N- and O-linked glycans are increasingly recognized as biologically important structural features, but access to these structures has not been widely available. Here, we report a detailed substrate specificity and catalytic efficiency of the bacterial ß3-N-acetylglucosaminyltransferase (ß3GlcNAcT) from Helicobacter pylori that can be adapted to the synthesis of a rich diversity of glycans with poly-LacNAc extensions. This glycosyltransferase has surprisingly broad acceptor specificity toward type-1, -2, -3 and -4 galactoside motifs on both linear and branched glycans, found commonly on N-linked, O-linked and I-antigen glycans. This finding enables the production of complex ligands for glycan-binding studies. Although the enzyme shows preferential activity for type 2 (Galß1-4GlcNAc) acceptors, it is capable of transferring N-acetylglucosamine (GlcNAc) in ß1-3 linkage to type-1 (Galß1-3GlcNAc) or type-3/4 (Galß1-3GalNAcα/ß) sequences. Thus, by alternating the use of the H. pylori ß3GlcNAcT with galactosyltransferases that make the ß1-4 or ß1-3 linkages, various N-linked, O-linked and I-antigen acceptors could be elongated with type-2 and type-1 LacNAc repeats. Finally, one-pot incubation of di-LacNAc biantennary N-glycopeptide with the ß3GlcNAcT and GalT-1 in the presence of uridine diphosphate (UDP)-GlcNAc and UDP-Gal, yielded products with 15 additional LacNAc units on the precursor, which was seen as a series of sequential ion peaks representing alternative additions of GlcNAc and Gal residues, on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Overall, our data demonstrate a broader substrate specificity for the H. pylori ß3GlcNAcT than previously recognized and demonstrate its ability as a potent resource for preparative chemo-enzymatic synthesis of complex glycans.


Assuntos
Amino Açúcares/biossíntese , Helicobacter pylori/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Acetilglucosamina/química , Amino Açúcares/química , Glicoesfingolipídeos/química , Glicoesfingolipídeos/metabolismo , Glicosilação , Polissacarídeos/biossíntese
6.
Angew Chem Int Ed Engl ; 51(20): 4860-3, 2012 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-22505324

RESUMO

Human influenza viruses are proposed to recognize sialic acids (pink diamonds) on glycans extended with poly-LacNAc chains (LacNAc=(yellow circle+blue square)). N- and O-linked glycans were extended with different poly-LacNAc chains with α2-3- and α2-6-linked sialic acids recognized by human and avian influenza viruses, respectively. The specificity of recombinant hemagglutinins (receptors in green) was investigated by using glycan microarray technology.


Assuntos
Hemaglutininas/metabolismo , Vírus da Influenza A/metabolismo , Influenza Aviária/virologia , Influenza Humana/virologia , Polissacarídeos/metabolismo , Ácidos Siálicos/metabolismo , Animais , Aves , Sequência de Carboidratos , Hemaglutininas/química , Humanos , Vírus da Influenza A/química , Análise em Microsséries , Dados de Sequência Molecular , Polissacarídeos/química , Ácidos Siálicos/química
7.
Chem Commun (Camb) ; 47(45): 12397-9, 2011 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-22016886

RESUMO

The structure of the pentasaccharide S259-1 in the Consortium for Functional Glycomics was investigated using a variety of techniques. Surprisingly, the structure differs from the structure assumed from the previously established specificity of the human fucosyltransferase FUT-III used in the last step of chemoenzymatic synthesis. When presented with a tetrasaccharide substrate containing both type I and type II disaccharide moieties, the enzyme generates a pentasaccharide in which the type II moiety is preferentially fucosylated. The unexpected product generated by FUT-III in this case highlights the importance of performing detailed structural analysis on products generated by enzymes.


Assuntos
Toxinas Bacterianas/química , Clostridioides difficile/metabolismo , Enterotoxinas/química , Oligossacarídeos/biossíntese , Cristalografia por Raios X , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Conformação Molecular , Oligossacarídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
8.
J Mol Biol ; 405(4): 1027-39, 2011 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-21112338

RESUMO

Langerin mediates the carbohydrate-dependent uptake of pathogens by Langerhans cells in the first step of antigen presentation to the adaptive immune system. Langerin binds to an unusually diverse number of endogenous and pathogenic cell surface carbohydrates, including mannose-containing O-specific polysaccharides derived from bacterial lipopolysaccharides identified here by probing a microarray of bacterial polysaccharides. Crystal structures of the carbohydrate-recognition domain from human langerin bound to a series of oligomannose compounds, the blood group B antigen, and a fragment of ß-glucan reveal binding to mannose, fucose, and glucose residues by Ca(2+) coordination of vicinal hydroxyl groups with similar stereochemistry. Oligomannose compounds bind through a single mannose residue, with no other mannose residues contacting the protein directly. There is no evidence for a second Ca(2+)-independent binding site. Likewise, a ß-glucan fragment, Glcß1-3Glcß1-3Glc, binds to langerin through the interaction of a single glucose residue with the Ca(2+) site. The fucose moiety of the blood group B trisaccharide Galα1-3(Fucα1-2)Gal also binds to the Ca(2+) site, and selective binding to this glycan compared to other fucose-containing oligosaccharides results from additional favorable interactions of the nonreducing terminal galactose, as well as of the fucose residue. Surprisingly, the equatorial 3-OH group and the axial 4-OH group of the galactose residue in 6SO(4)-Galß1-4GlcNAc also coordinate Ca(2+), a heretofore unobserved mode of galactose binding in a C-type carbohydrate-recognition domain bearing the Glu-Pro-Asn signature motif characteristic of mannose binding sites. Salt bridges between the sulfate group and two lysine residues appear to compensate for the nonoptimal binding of galactose at this site.


Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/metabolismo , Polissacarídeos/metabolismo , Antígenos CD/genética , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Cristalografia por Raios X , Humanos , Técnicas In Vitro , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Lectinas Tipo C/genética , Ligantes , Lectinas de Ligação a Manose/genética , Análise em Microsséries , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trissacarídeos/química , Trissacarídeos/metabolismo , beta-Glucanas/química , beta-Glucanas/metabolismo
9.
Glycoconj J ; 25(1): 59-68, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17914671

RESUMO

Here we demonstrate that glycan microarrays can be used for high-throughput acceptor specificity screening of various recombinant sialyltransferases. Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) was biotinylated at position 9 of N-acetylneuraminic acid (Neu5Ac) by chemoenzymatic synthesis generating CMP-9Biot-Neu5Ac. The activated sugar nucleotide was used as donor substrate for various mammalian sialyltranferases which transferred biotinylated sialic acids simultaneously onto glycan acceptors immobilized onto a microarray glass slide. Biotinylated glycans detected with fluorescein-streptavidin conjugate to generate a specificity profile for each enzyme both confirming previously known specificities and reveal additional specificity information. Human alpha2,6sialyltransferase-I (hST6Gal-I) also sialylates chitobiose structures (GlcNAcbeta1-4GlcNAc)(n) including N-glycans, rat alpha2,3sialyltransferase (rST3Gal-III) tolerates fucosylated acceptors such as Lewis(a), human alpha2,3sialyltransferase-IV (hST3Gal-IV) broadly sialylates oligosaccharides of types 1-4 and porcine alpha2,3sialyltransferase-I (pST3Gal-I) sialylates ganglio-oligosaccharides and core 2 O-glycans in our array system. Several of these sialyltransferases perform a substitution reaction and exchange a sialylated acceptor with a biotinylated sialic acid but are restricted to the most specific acceptor substrates. Thus, this method allows for a rapid generation of enzyme specificity information and can be used towards synthesis of new carbohydrate compounds and expand the glycan array compound library.


Assuntos
Análise em Microsséries/métodos , Polissacarídeos/análise , Polissacarídeos/metabolismo , Sialiltransferases/metabolismo , Animais , Linhagem Celular , Glicômica/métodos , Humanos , Ratos , Especificidade por Substrato , Suínos
10.
Methods Enzymol ; 415: 137-53, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17116472

RESUMO

The expanding interest for carbohydrates and glycoconjugates in cell communication has led to an increased demand of these structures for biological studies. Complicated chemical strategies in glycan synthesis are now more frequently replaced by regio- and stereo-specific enzymes. The exploration of microbial resources and improved production of mammalian enzymes have established glycosyltransferases as an efficient complementary tool for glycan synthesis. In this chapter, we demonstrate the feasibility of preparative enzymatic synthesis of different categories of glycans, such as blood group and tumor-associated poly-N-acetyllactosamines antigens, ganglio-oligosaccharides, N- and O-glycans. The enzymatic approach has generated over 100 novel oligosaccharides in amounts allowing milligram to gram distribution to many researchers in the field. Our diverse library has also formed the foundation for the successful developments of both the noncovalent enzyme-linked immunosorbent assay glycan array and the covalent printed glycan microarray.


Assuntos
Bases de Dados Factuais , Polissacarídeos , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Células Cultivadas , Humanos , Dados de Sequência Molecular , Polissacarídeos/síntese química , Polissacarídeos/química
11.
Carbohydr Res ; 341(10): 1447-57, 2006 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-16650392

RESUMO

Poly-N-acetyllactosamines (pLNs) are common terminal sugars of many N- and O-linked glycan structures present in glycoproteins and glycolipids. Utilizing various glycosyltransferases, we developed new and efficient chemoenzymatic methods for the synthesis of pLNs in gram-scale. Specifically, the use of sialyltransferases and fucosyltransferases enabled us to synthesize and purify 24 blood group and tumor-associated pLN derivatives with alpha-(2-->3)- and alpha-(2-->6)-linked sialic acid, as well as with alpha-(1-->2)- and alpha-(1-->3)-linked fucose. All synthesized derivatives were linked to a short 2-azidoethyl spacer for further modification.


Assuntos
Antígenos de Neoplasias/biossíntese , Antígenos de Grupos Sanguíneos/biossíntese , Polissacarídeos/síntese química , Fucosiltransferases/metabolismo , Polissacarídeos/biossíntese , Sialiltransferases/metabolismo
12.
Carbohydr Res ; 340(12): 1963-72, 2005 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-16005859

RESUMO

We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.


Assuntos
Gangliosídeos/síntese química , Escherichia coli/enzimologia , Gangliosídeo G(M2)/síntese química , Galactosiltransferases/metabolismo , Lactosilceramidas/síntese química , N-Acetilgalactosaminiltransferases/metabolismo , Sialiltransferases/metabolismo , Especificidade por Substrato
13.
Carbohydr Res ; 340(2): 221-33, 2005 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-15639242

RESUMO

Derivatives of lactose with the galactose ring substituents replaced by deoxy or acylamino functions were prepared. The 2'-, 3'-, 4'- and 6'-deoxy, 3'-acetamido and 3'-benzamido derivatives of phenyl 4-O-(beta-D-galactopyranosyl)-beta-D-glucopyranoside (phenyl beta-lactoside) were synthesized from disaccharide or monosaccharide precursors. The derivatives were tested as substrates for the N-acetylglucosaminyltransferase from Neisseria meningitidis, which uses lactosyl derivatives as acceptors and UDP-GlcNAc as the donor in a beta-(1-->3) glycosylation reaction. The 6'-deoxy derivative was nearly threefold as active as phenyl beta-lactoside, whereas the 2'- and 4'-deoxy derivatives were less active. The other derivatives were inactive, as expected.


Assuntos
Lactose/análogos & derivados , Lactose/síntese química , Sondas Moleculares/síntese química , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/metabolismo , Neisseria meningitidis/enzimologia , Sítios de Ligação , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Sondas Moleculares/química , Dados de Sequência Molecular , Estrutura Molecular
14.
Proc Natl Acad Sci U S A ; 101(49): 17033-8, 2004 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-15563589

RESUMO

Here we describe a glycan microarray constructed by using standard robotic microarray printing technology to couple amine functionalized glycans to an amino-reactive glass slide. The array comprises 200 synthetic and natural glycan sequences representing major glycan structures of glycoproteins and glycolipids. The array has remarkable utility for profiling the specificity of a diverse range of glycan binding proteins, including C-type lectins, siglecs, galectins, anticarbohydrate antibodies, lectins from plants and microbes, and intact viruses.


Assuntos
Polissacarídeos/química , Análise Serial de Proteínas/métodos , Proteínas/química , Sequência de Carboidratos , Reagentes de Ligações Cruzadas , Ligantes , Ligação Proteica , Robótica
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