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1.
mBio ; 12(3): e0080021, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34061603

RESUMO

Capsular polysaccharides (CPSs) are major virulence factors that decorate the surfaces of many human bacterial pathogens. In their pure form or as glycoconjugate vaccines, CPSs are extensively used in vaccines deployed in clinical practice worldwide. However, our understanding of the structural requirements for interactions between CPSs and antibodies is limited. A longstanding model based on comprehensive observations of antibody repertoires binding to CPSs is that antibodies expressing heavy chain variable gene family 3 (VH3) predominate in these binding interactions in humans and VH3 homologs in mice. Toward understanding this highly conserved interaction, we generated a panel of mouse monoclonal antibodies (MAb) against Streptococcus pneumoniae serotype 3 CPS, determined an X-ray crystal structure of a protective MAb in complex with a hexasaccharide derived from enzymatic hydrolysis of the polysaccharide, and elucidated the structural requirements for this binding interaction. The crystal structure revealed a binding pocket containing aromatic side chains, suggesting the importance of hydrophobicity in the interaction. Through mutational analysis, we determined the amino acids that are critical in carbohydrate binding. Through elucidating the structural and functional properties of a panel of murine MAbs, we offer an explanation for the predominant use of the human VH3 gene family in antibodies against CPSs with implications in knowledge-based vaccine design. IMPORTANCE Infectious diseases caused by pathogenic bacteria are a major threat to human health. Capsular polysaccharides (CPSs) of many pathogenic bacteria have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide, with various degrees of success. Immunization with a glycoconjugate vaccine elicits T cell help for B cells that produce IgG antibodies to the CPS. Thus, it is important to develop an in-depth understanding of the interactions of carbohydrate epitopes with the antibodies. Structural characterization of the ligand binding of polysaccharide-specific antibodies laid out in this study may have fundamental biological implications for our comprehension of how the humoral immune system recognizes polysaccharide antigens, and in future knowledge-based vaccine design.


Assuntos
Anticorpos Antibacterianos/imunologia , Cápsulas Bacterianas/química , Polissacarídeos Bacterianos/imunologia , Polissacarídeos Bacterianos/metabolismo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Monoclonais , Cápsulas Bacterianas/classificação , Cápsulas Bacterianas/imunologia , Cristalização , Feminino , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Modelos Estruturais , Polissacarídeos Bacterianos/química , Sorogrupo , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/patogenicidade , Vacinação
2.
mBio ; 12(3)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006653

RESUMO

The mucophilic anaerobic bacterium Akkermansia muciniphila is a prominent member of the gastrointestinal (GI) microbiota and the only known species of the Verrucomicrobia phylum in the mammalian gut. A high prevalence of A. muciniphila in adult humans is associated with leanness and a lower risk for the development of obesity and diabetes. Four distinct A. muciniphila phylogenetic groups have been described, but little is known about their relative abundance in humans or how they impact human metabolic health. In this study, we isolated and characterized 71 new A. muciniphila strains from a cohort of children and adolescents undergoing treatment for obesity. Based on genomic and phenotypic analysis of these strains, we found several phylogroup-specific phenotypes that may impact the colonization of the GI tract or modulate host functions, such as oxygen tolerance, adherence to epithelial cells, iron and sulfur metabolism, and bacterial aggregation. In antibiotic-treated mice, phylogroups AmIV and AmII outcompeted AmI strains. In children and adolescents, AmI strains were most prominent, but we observed high variance in A. muciniphila abundance and single phylogroup dominance, with phylogroup switching occurring in a small subset of patients. Overall, these results highlight that the ecological principles determining which A. muciniphila phylogroup predominates in humans are complex and that A. muciniphila strain genetic and phenotypic diversity may represent an important variable that should be taken into account when making inferences as to this microbe's impact on its host's health.IMPORTANCE The abundance of Akkermansia muciniphila in the gastrointestinal (GI) tract is linked to multiple positive health outcomes. There are four known A. muciniphila phylogroups, yet the prevalence of these phylogroups and how they vary in their ability to influence human health is largely unknown. In this study, we performed a genomic and phenotypic analysis of 71 A. muciniphila strains and identified phylogroup-specific traits such as oxygen tolerance, adherence, and sulfur acquisition that likely influence colonization of the GI tract and differentially impact metabolic and immunological health. In humans, we observed that single Akkermansia phylogroups predominate at a given time but that the phylotype can switch in an individual. This collection of strains provides the foundation for the functional characterization of A. muciniphila phylogroup-specific effects on the multitude of host outcomes associated with Akkermansia colonization, including protection from obesity, diabetes, colitis, and neurological diseases, as well as enhanced responses to cancer immunotherapies.


Assuntos
Variação Genética , Genótipo , Fenótipo , Akkermansia/classificação , Akkermansia/genética , Akkermansia/isolamento & purificação , Animais , Estudos de Coortes , Feminino , Microbioma Gastrointestinal , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
3.
J Bacteriol ; 203(7)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33468592

RESUMO

The pneumococcal serine-rich repeat protein (PsrP) is a high-molecular-weight, glycosylated adhesin that promotes the attachment of Streptococcus pneumoniae to host cells. PsrP, its associated glycosyltransferases (GTs), and dedicated secretion machinery are encoded in a 37-kb genomic island that is present in many invasive clinical isolates of S. pneumoniae PsrP has been implicated in establishment of lung infection in murine models, although specific roles of the PsrP glycans in disease progression or bacterial physiology have not been elucidated. Moreover, enzymatic specificities of associated glycosyltransferases are yet to be fully characterized. We hypothesized that the glycosyltransferases that modify PsrP are critical for the adhesion properties and infectivity of S. pneumoniae Here, we characterize the putative S. pneumoniaepsrP locus glycosyltransferases responsible for PsrP glycosylation. We also begin to elucidate their roles in S. pneumoniae virulence. We show that four glycosyltransferases within the psrP locus are indispensable for S. pneumoniae biofilm formation, lung epithelial cell adherence, and establishment of lung infection in a mouse model of pneumococcal pneumonia.IMPORTANCE PsrP has previously been identified as a necessary virulence factor for many serotypes of S. pneumoniae and studied as a surface glycoprotein. Thus, studying the effects on virulence of each glycosyltransferase (GT) that builds the PsrP glycan is of high importance. Our work elucidates the influence of GTs in vivo We have identified at least four GTs that are required for lung infection, an indication that it is worthwhile to consider glycosylated PsrP as a candidate for serotype-independent pneumococcal vaccine design.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/patogenicidade , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Proteínas de Bactérias/genética , Feminino , Glicosiltransferases/genética , Humanos , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Virulência
4.
Pharm Res ; 37(12): 236, 2020 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-33140159

RESUMO

PURPOSE: Streptococcus pneumoniae (Spn) serotype 3 (Spn3) is considered one of the most virulent serotypes with resistance to conventional vaccine and treatment regimens. Pn3Pase is a glycoside hydrolase that we have previously shown to be highly effective in degrading the capsular polysaccharide of type 3 Spn, sensitizing it to host immune clearance. To begin assessing the value and safety of this enzyme for future clinical studies, we investigated the effects of high doses of Pn3Pase on host cells and immune system. METHODS: We assessed the enzyme's catalytic activity following administration in mice, and performed septic infection models to determine if prior administration of the enzyme inhibited repeat treatments of Spn3-challenged mice. We assessed immune populations in mouse tissues following administration of the enzyme, and tested Pn3Pase toxicity on other mammalian cell types in vitro. RESULTS: Repeated administration of the enzyme in vivo does not prevent efficacy of the enzyme in promoting bacterial clearance following bacterial challenge, with insignificant antibody response generated against the enzyme. Immune homeostasis is maintained following high-dose treatment with Pn3Pase, and no cytotoxic effects were observed against mammalian cells. CONCLUSIONS: These data indicate that Pn3Pase has potential as a therapy against Spn3. Further development as a drug product could overcome a great hurdle of pneumococcal infections.


Assuntos
Proteínas de Bactérias/farmacologia , Glicosídeo Hidrolases/farmacologia , Paenibacillus/enzimologia , Infecções Pneumocócicas/tratamento farmacológico , Streptococcus pneumoniae/efeitos dos fármacos , Animais , Cápsulas Bacterianas/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/uso terapêutico , Modelos Animais de Doenças , Feminino , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/uso terapêutico , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Infecções Pneumocócicas/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Streptococcus pneumoniae/isolamento & purificação
5.
Nat Commun ; 11(1): 2550, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32439962

RESUMO

The inherent molecular complexity of human pathogens requires that mammals evolved an adaptive immune system equipped to handle presentation of non-conventional MHC ligands derived from disease-causing agents, such as HIV-1 envelope (Env) glycoprotein. Here, we report that a CD4+ T cell repertoire recognizes a glycopeptide epitope on gp120 presented by MHCII pathway. This glycopeptide is strongly immunogenic in eliciting glycan-dependent cellular and humoral immune responses. The glycopeptide specific CD4+ T cells display a prominent feature of Th2 and Th17 differentiation and exert high efficacy and potency to help Env trimer humoral immune responses. Glycopeptide-induced CD4+ T cell response prior to Env trimer immunization elicits neutralizing antibody development and production of antibodies facilitating uptake of immunogens by antigen-presenting cells. Our identification of gp120 glycopeptide-induced, T cell-specific immune responses offers a foundation for developing future knowledge-based vaccines that elicit strong and long-lasting protective immune responses against HIV-1 infection.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Imunidade Humoral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Citocinas/metabolismo , Epitopos de Linfócito T/química , Glicopeptídeos/química , Glicopeptídeos/imunologia , Anticorpos Anti-HIV/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Celular , Imunização , Camundongos , Polissacarídeos/imunologia , Células Th17/imunologia , Células Th2/imunologia
6.
J Vis Exp ; (146)2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-31009013

RESUMO

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.


Assuntos
Bactérias/imunologia , Bioensaio , Proteínas Opsonizantes/metabolismo , Fagocitose , Sobrevivência Celular/imunologia , Células HL-60 , Humanos
7.
Org Biomol Chem ; 17(10): 2646-2650, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30778481

RESUMO

Chemoselective ligation of carbohydrates and polypeptides was achieved using an adipic acid dihydrazide cross-linker. The reducing end of a carbohydrate is efficiently attached to peptides in two steps, constructing a glycoconjugate in high yield and with high regioselectivity, enabling the production of homogeneous glycoconjugates.


Assuntos
Glicoconjugados/química , Glicoconjugados/síntese química , Adipatos/química , Sequência de Aminoácidos , Técnicas de Química Sintética , Glicopeptídeos/síntese química , Glicopeptídeos/química , Modelos Moleculares , Conformação Molecular , Especificidade por Substrato
8.
J Biol Chem ; 293(39): 15178-15194, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30115684

RESUMO

The glycan shield on the envelope glycoprotein gp120 of HIV-1 has drawn immense attention as a vulnerable site for broadly neutralizing antibodies and for its significant impact on host adaptive immune response to HIV-1. Glycosylation sites and glycan composition/structure at each site on gp120 along with the interactions of gp120 glycan shield with broadly neutralizing antibodies have been extensively studied. However, a method for directly and selectively tracking gp120 glycans has been lacking. Here, we integrate metabolic labeling and click chemistry technology with recombinant gp120 expression to demonstrate that gp120 glycans could be specifically labeled and directly detected. Selective labeling of gp120 by N-azidoacetylmannosamine (ManNAz) and N-azidoacetylgalactosamine (GalNAz) incorporation into the gp120 glycan shield was characterized by MS of tryptic glycopeptides. By using metabolically labeled gp120, we investigated the impact of gp120 glycosylation on its interaction with host cells and demonstrated that oligomannose enrichment and sialic acid deficiency drastically enhanced gp120 uptake by bone marrow-derived dendritic cells. Collectively, our data reveal an effective labeling and detection method for gp120, serving as a tool for functional characterization of the gp120 glycans and potentially other glycosylated proteins.


Assuntos
Anticorpos Neutralizantes/imunologia , Glicopeptídeos/imunologia , Proteína gp120 do Envelope de HIV/isolamento & purificação , HIV-1/isolamento & purificação , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Antígenos/química , Antígenos/imunologia , Azidas/química , Azidas/metabolismo , Células da Medula Óssea/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Glicopeptídeos/química , Glicopeptídeos/genética , Glicosilação , Células HEK293 , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/genética , HIV-1/imunologia , HIV-1/patogenicidade , Hexosaminas/química , Hexosaminas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Metabolismo/imunologia , Polissacarídeos/química , Polissacarídeos/genética , Polissacarídeos/imunologia
9.
Infect Immun ; 86(8)2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29866907

RESUMO

Despite a century of investigation, Streptococcus pneumoniae remains a major human pathogen, causing a number of diseases, such as pneumonia, meningitis, and otitis media. Like many encapsulated pathogens, the capsular polysaccharide (CPS) of S. pneumoniae is a critical component for colonization and virulence in mammalian hosts. This study aimed to evaluate the protective role of a glycoside hydrolase, Pn3Pase, targeting the CPS of type 3 S. pneumoniae, which is one of the most virulent serotypes. We have assessed the ability of Pn3Pase to degrade the capsule on a live type 3 strain. Through in vitro assays, we observed that Pn3Pase treatment increases the bacterium's susceptibility to phagocytosis by macrophages and complement-mediated killing by neutrophils. We have demonstrated that in vivo Pn3Pase treatment reduces nasopharyngeal colonization and protects mice from sepsis caused by type 3 S. pneumoniae Due to the increasing shifts in serotype distribution, the rise in drug-resistant strains, and poor immune responses to vaccine-included serotypes, it is necessary to investigate approaches to combat pneumococcal infections. This study evaluates the interaction of pneumococcal CPS with the host at molecular, cellular, and systemic levels and offers an alternative therapeutic approach for diseases caused by S. pneumoniae through enzymatic hydrolysis of the CPS.


Assuntos
Cápsulas Bacterianas/metabolismo , Glicosídeo Hidrolases/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Fagocitose/fisiologia , Infecções Pneumocócicas/fisiopatologia , Polissacarídeos Bacterianos/metabolismo , Streptococcus pneumoniae/metabolismo , Animais , Humanos , Hidrólise , Camundongos
10.
Glycobiology ; 28(2): 90-99, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29190349

RESUMO

Bacillus circulans Jordan 32352 was isolated from decaying organic matter in the New Jersey soil in the early 1930s. This soil-dwelling bacterium produced an enzyme capable of degrading the type 3 capsular polysaccharide (Pn3P) of Streptococcus pneumoniae (Spn). Early reports of this enzyme, Pn3Pase, demonstrated its inducibility by, and specificity for Pn3P. We set out to identify and clone this enzyme for its recombinant expression and characterization. We first sequenced the genome of this bacterial species, and reclassified the Pn3Pase producing bacterium as Paenibacillus species 32352. We identified the putative protein of Pn3Pase through mass spectrometry-based proteomics and cloned the gene for recombinant expression. We then characterized the oligosaccharide products generated upon the enzymatic depolymerization of Pn3P. Sequence analysis suggests that this glycoside hydrolase belongs to a new carbohydrate-active enzyme GH family. To our knowledge, this is the only enzyme to demonstrate Pn3P depolymerization activity.


Assuntos
Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Paenibacillus/enzimologia , Polissacarídeos Bacterianos/metabolismo , Streptococcus pneumoniae/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética
11.
J Immunol ; 199(2): 598-603, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28566369

RESUMO

Most pathogenic bacteria express surface carbohydrates called capsular polysaccharides (CPSs). CPSs are important vaccine targets because they are easily accessible and recognizable by the immune system. However, CPS-specific adaptive humoral immune responses can only be achieved by the covalent conjugation of CPSs with carrier proteins to produce glycoconjugate vaccines. We previously described a mechanism by which a model glycoconjugate vaccine can activate the adaptive immune system and demonstrated that the mammalian CD4+ T cell repertoire contains a population of carbohydrate-specific T cells. In this study, we use glycoconjugates of type 3 Streptococcus pneumoniae CPS (Pn3P) to assess whether the carbohydrate-specific adaptive immune response exemplified in our previous study can be applied to the conjugates of this lethal pathogen. In this article, we provide evidence for the functional roles of Pn3P-specific CD4+ T cells utilizing mouse immunization schemes that induce Pn3P-specific IgG responses in a carbohydrate-specific T cell-dependent manner.


Assuntos
Cápsulas Bacterianas/imunologia , Linfócitos T CD4-Positivos/imunologia , Glicoconjugados/imunologia , Imunidade Humoral , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Imunidade Adaptativa , Animais , Cápsulas Bacterianas/química , Linfócitos T CD4-Positivos/metabolismo , Carboidratos/imunologia , Feminino , Glicoconjugados/administração & dosagem , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Streptococcus pneumoniae/patogenicidade , Vacinação , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologia
12.
Genome Announc ; 5(19)2017 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-28495770

RESUMO

Here, we report the complete genome sequence for the Bacillus circulans Jordan strain 32352. This species is a soil dwelling bacterium that expresses glycosyl hydrolase enzymes degrading pneumococcal capsular polysaccharides.

13.
Glycobiology ; 27(3): 206-212, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28177478

RESUMO

Determining the correct enzymatic activity of putative glycosyltransferases (GTs) can be challenging as these enzymes can utilize multiple donor and acceptor substrates. Upon initial determination of the donor-sugar nucleotide(s), a GT utilizes various acceptor molecules that can then be tested. Here, we describe a quick method to screen sugar-nucleotide donor specificities of GTs utilizing a sensitive, nonradioactive, commercially available bioluminescent uridine diphosphate detection kit. This in vitro method allowed us to validate the sugar-nucleotide donor-substrate specificities of recombinantly expressed human, bovine, bacterial and protozoan GTs. Our approach, which is less time consuming than many traditional assays that utilize radiolabeled sugars and chromatographic separations, should facilitate discovery of novel GTs that participate in diverse biological processes.


Assuntos
Glicosiltransferases/isolamento & purificação , Nucleotídeos/química , Açúcares/química , Animais , Bactérias/enzimologia , Bovinos , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Humanos , Especificidade por Substrato
14.
Glycobiology ; 26(10): 1029-1040, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27236197

RESUMO

Glycosylation is arguably the most ubiquitous post-translational modification on proteins in microbial and mammalian cells. During the past few years, there has been intensive research demonstrating that carbohydrates, either in pure forms or in conjunction with proteins or lipids, evoke and modulate adaptive immune responses. We now know that carbohydrates can be directly recognized by T cells or participate in T-cell stimulation as components of T-cell epitopes. T-cell recognition of carbohydrate antigens takes place via their presentation by major histocompatibility complex pathways on antigen-presenting cells. In this review, we summarize studies on carbohydrates as T-cell antigens modulating adaptive immune responses. Through discussion of glycan-containing antigens, such as glycoproteins, glycolipids, zwitterionic polysaccharides and carbohydrate-based glycoconjugate vaccines, we will illustrate the key molecular and cellular interactions between carbohydrate antigens and T cells and the implications of these interactions in health and disease.


Assuntos
Antígenos/imunologia , Carboidratos/imunologia , Doença , Saúde , Linfócitos T/imunologia , Animais , Glicoconjugados/imunologia , Glicolipídeos/imunologia , Glicoproteínas/imunologia , Glicosilação , Humanos , Polissacarídeos/imunologia
15.
J Chromatogr A ; 1397: 43-51, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25913329

RESUMO

Pneumococcal type-3 polysaccharide (Pn3P) is considered a major target for the development of a human vaccine to protect against Streptococcus pneumoniae infection. Thus, it is critical to develop methods for the preparation and analysis of Pn3P-derived oligosaccharides to better understand its immunological properties. In this paper, we profile oligosaccharides, generated by the free radical depolymerization of Pn3P, using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Hydrophilic liquid interaction chromatography (HILIC)-mass spectrometry (MS) revealed a series of oligosaccharides with an even- and odd-number of saccharide residues, ranging from monosaccharide, degree of polymerization (dp1) to large oligosaccharides up to dp 20, generated by free radical depolymerization. Isomers of oligosaccharides with an even number of sugar residues were easily separated on a HILIC column, and their sequences could be distinguished by comparing MS/MS of these oligosaccharides and their reduced alditols. Fluorescent labeling with 2-aminoacridone (AMAC) followed by reversed phase (RP)-LC-MS/MS was applied to analyze and sequence poorly separated product mixtures, as RP-LC affords higher resolution of AMAC-labeled oligosaccharides than does HILIC-based separation. The present methodology can be potentially applied to profiling other capsular polysaccharides.


Assuntos
Técnicas de Química Analítica , Cromatografia Líquida , Oligossacarídeos/química , Streptococcus pneumoniae/química , Espectrometria de Massas em Tandem , Aminoacridinas/química , Interações Hidrofóbicas e Hidrofílicas , Isomerismo
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