RESUMO
Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein-glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.
Assuntos
Proteínas de Transporte/química , Análise em Microsséries/métodos , Polissacarídeos/química , Polissacarídeos/imunologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Células CHO , Cricetulus , Glicômica , Humanos , Sistema Imunitário , Lectinas , Oligossacarídeos , Polissacarídeos/classificação , Ligação Proteica , Proteínas Recombinantes , Especificidade da EspécieRESUMO
Reliable and rapid access to defined biopolymers by automated DNA and peptide synthesis has fundamentally altered biological research and medical practice. Similarly, the procurement of defined glycans is key to establishing structure-activity relationships and thereby progress in the glycosciences. Here, we describe the rapid assembly of oligosaccharides using the commercially available Glyconeer 2.1 automated glycan synthesizer, monosaccharide building blocks, and a linker-functionalized polystyrene solid support. Purification and quality-control protocols for the oligosaccharide products have been standardized. Synthetic glycans prepared in this way are useful reagents as the basis for glycan arrays, diagnostics, and carbohydrate-based vaccines.
RESUMO
The mixed-linkage (1â3),(1â4)-d-glucan (MLG) specific glycosyl hydrolase lichenase is an important biochemical tool for the structural characterization of MLGs. It holds potential for application in the brewery, animal feed, and biofuel industries. Several defined MLG oligosaccharides obtained by automated glycan assembly are used to analyze the substrate specificities of Bacillus subtilis lichenase. Two glucose building blocks (BBs), equipped with a temporary fluorenylmethyloxycarbonyl chloride (Fmoc) protecting group in the C-3 or C-4 position, served to assemble different oligosaccharides by using an automated oligosaccharide synthesizer. Light-induced cleavage of the glycan products from the solid support followed by global deprotection provided seven MLG oligosaccharides of different length and connectivity. After incubation of the MLG oligosaccharides with lichenase, the digestion products were analyzed by HPLC-MS. These digestion experiments provided insights into the enzyme's active site that is in line with other recent evidence suggesting that the substrate specificity of lichenases has to be reconsidered. These results demonstrate that synthetic MLG oligosaccharides are useful tools to analyze mixed-linkage ß-glucanases.
Assuntos
Bacillus subtilis/enzimologia , Glucanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/metabolismo , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Glucanos/química , Glicosídeo Hidrolases/química , Espectrometria de Massas , Oligossacarídeos/química , Especificidade por SubstratoRESUMO
Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized resin. Arabinofuranose and glucuronic acid residues have been selectively attached to the backbone using fully orthogonal 2-(methyl)naphthyl (Nap) and 2-(azidomethyl)benzoyl (Azmb) protecting groups at the C2 and C3 hydroxyls of the xylose building blocks. The arabinoxylan oligosaccharides are excellent tools to map the active site of glycosyl hydrolases involved in xylan deconstruction. The substrate specificities of several xylanases and arabinofuranosidases were determined by analyzing the digestion products after incubation of the oligosaccharides with glycosyl hydrolases.
Assuntos
Bacteroides/enzimologia , Domínio Catalítico , Cellvibrio/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Bacteroides/química , Bacteroides/metabolismo , Cellvibrio/química , Cellvibrio/metabolismo , Hidrólise , Oligossacarídeos/síntese química , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Técnicas de Síntese em Fase Sólida , Especificidade por Substrato , Xilanos/síntese química , Xilanos/química , Xilanos/metabolismo , Xilosidases/química , Xilosidases/metabolismoRESUMO
Pectin is a structurally complex plant polysaccharide with many industrial applications in food products. The structural elucidation of pectin is aided by digestion assays with glycosyl hydrolases. We report the automated glycan assembly of oligosaccharides related to the arabinogalactan side chains of pectin as novel biochemical tools to determine the substrate specificities of endogalactanases. Analysis of the digestion products revealed different requirements for the lengths and arabinose substitution pattern of the oligosaccharides to be recognized and hydrolyzed by the galactanases.
Assuntos
Automação , Galactanos/química , Glicosídeo Hidrolases/metabolismo , Polissacarídeos/biossíntese , Estrutura Molecular , Polissacarídeos/química , Especificidade por SubstratoRESUMO
Lactotetraosyl (Lc4) and neo-lactotetraosyl (nLc4) are backbones that are common to many glycans. Using automated glycan assembly, these common core structures were constructed and elaborated to access synthetically challenging glycans of biological relevance. The incorporation of α-fucoses is demonstrated for H-type I and II; α(1,3)-galactose epitopes were prepared, and the pentasaccharide HNK-1 required incorporation of a 3-O-sulfate. In addition to preparing the target structures, essential insights were gained regarding the relationships of glycosylating agents and nucleophiles as well as the linker stability.
Assuntos
Antígenos de Grupos Sanguíneos/química , Glicosilação , Oligossacarídeos/química , Polissacarídeos/química , Química Orgânica , Cromatografia Líquida de Alta Pressão , Epitopos/química , Fucose/química , Humanos , Tioglicosídeos/químicaRESUMO
We report the automated glycan assembly of oligosaccharide fragments related to the hemicellulose xyloglucan (XG). Iterative addition of monosaccharide and disaccharide building blocks to a solid support provided seven cellulose and xyloglucan fragments including XXGG- and XXXG-type oligosaccharides.
Assuntos
Automação , Glucanos/síntese química , Oligossacarídeos/síntese química , Xilanos/síntese química , Glucanos/química , Estrutura Molecular , Oligossacarídeos/química , Xilanos/químicaRESUMO
Arabinogalactan proteins are heavily glycosylated proteoglycans in plants. Their glycan portion consists of type-II arabinogalactan polysaccharides whose heterogeneity hampers the assignment of the arabinogalactan protein function. Synthetic chemistry is key to the procurement of molecular probes for plant biologists. Described is the automated glycan assembly of 14 oligosaccharides from four monosaccharide building blocks. These linear and branched glycans represent key structural features of natural type-II arabinogalactans and will serve as tools for arabinogalactan biology.
Assuntos
Mucoproteínas/química , Oligossacarídeos/síntese química , Polissacarídeos/síntese química , Proteínas/metabolismo , Galactanos/química , Glicosilação , Estrutura Molecular , Oligossacarídeos/química , Proteínas de Plantas/química , Polissacarídeos/química , ProteoglicanasRESUMO
Monoclonal antibodies that recognize plant cell wall glycans are used for high-resolution imaging, providing important information about the structure and function of cell wall polysaccharides. To characterize the binding epitopes of these powerful molecular probes a library of eleven plant arabinoxylan oligosaccharides was produced by automated solid-phase synthesis. Modular assembly of oligoarabinoxylans from few building blocks was enabled by adding (2-naphthyl)methyl (Nap) to the toolbox of orthogonal protecting groups for solid-phase synthesis. Conjugation-ready oligosaccharides were obtained and the binding specificities of xylan-directed antibodies were determined on microarrays.
Assuntos
Anticorpos Monoclonais/imunologia , Parede Celular/imunologia , Células Vegetais/imunologia , Xilanos/síntese química , Análise em Microsséries , Polissacarídeos/imunologia , Técnicas de Síntese em Fase Sólida , Xilanos/imunologiaRESUMO
Dermatan sulfates are glycosaminoglycan polysaccharides that serve a multitude of biological roles as part of the extracellular matrix. Orthogonally protected D-galactosamine and L-iduronic acid building blocks and a photo-cleavable linker are instrumental for the automated synthesis of dermatan sulfate oligosaccharides. Conjugation-ready oligosaccharides were obtained in good yield.