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1.
J Proteome Res ; 22(10): 3213-3224, 2023 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-37641533

RESUMO

Inflammatory bowel diseases (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC), are chronic and relapsing inflammations of the digestive tract with increasing prevalence, yet they have unknown origins or cure. CD and UC have similar symptoms but respond differently to surgery and medication. Current diagnostic tools often involve invasive procedures, while laboratory markers for patient stratification are lacking. Large glycomic studies of immunoglobulin G and total plasma glycosylation have shown biomarker potential in IBD and could help determine disease mechanisms and therapeutic treatment choice. Hitherto, the glycosylation signatures of plasma immunoglobulin A, an important immunoglobulin secreted into the intestinal mucin, have remained undetermined in the context of IBD. Our study investigated the associations of immunoglobulin A1 and A2 glycosylation with IBD in 442 IBD cases (188 CD and 254 UC) and 120 healthy controls by reversed-phase liquid chromatography electrospray-ionization mass spectrometry of tryptic glycopeptides. Differences of IgA O- and N-glycosylation (including galactosylation, bisection, sialylation, and antennarity) between patient groups were associated with the diseases, and these findings led to the construction of a statistical model to predict the disease group of the patients without the need of invasive procedures. This study expands the current knowledge about CD and UC and could help in the development of noninvasive biomarkers and better patient care.


Assuntos
Colite Ulcerativa , Doença de Crohn , Doenças Inflamatórias Intestinais , Humanos , Doença de Crohn/diagnóstico , Doença de Crohn/epidemiologia , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/epidemiologia , Glicosilação , Imunoglobulina A , Biomarcadores
2.
Diabetes Metab Res Rev ; 39(7): e3685, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37422864

RESUMO

AIMS/HYPOTHESIS: Inflammation is important in the development of type 2 diabetes complications. The N-glycosylation of IgG influences its role in inflammation. To date, the association of plasma IgG N-glycosylation with type 2 diabetes complications has not been extensively investigated. We hypothesised that N-glycosylation of IgG may be related to the development of complications of type 2 diabetes. METHODS: In three independent type 2 diabetes cohorts, plasma IgG N-glycosylation was measured using ultra performance liquid chromatography (DiaGene n = 1815, GenodiabMar n = 640) and mass spectrometry (Hoorn Diabetes Care Study n = 1266). We investigated the associations of IgG N-glycosylation (fucosylation, galactosylation, sialylation and bisection) with incident and prevalent nephropathy, retinopathy and macrovascular disease using Cox- and logistic regression, followed by meta-analyses. The models were adjusted for age and sex and additionally for clinical risk factors. RESULTS: IgG galactosylation was negatively associated with prevalent and incident nephropathy and macrovascular disease after adjustment for clinical risk factors. Sialylation was negatively associated with incident diabetic nephropathy after adjustment for clinical risk factors. For incident retinopathy, similar associations were found for galactosylation, adjusted for age and sex. CONCLUSIONS: We showed that IgG N-glycosylation, particularly galactosylation and to a lesser extent sialylation, is associated with a higher prevalence and future development of macro- and microvascular complications of diabetes. These findings indicate the predictive potential of IgG N-glycosylation in diabetes complications and should be analysed further in additional large cohorts to obtain the power to solidify these conclusions.

3.
Anal Chem ; 94(12): 4979-4987, 2022 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-35293727

RESUMO

Bacterial glycoconjugate vaccines have a major role in preventing microbial infections. Immunogenic bacterial glycans, such as O-antigen polysaccharides, can be recombinantly expressed and combined with specific carrier proteins to produce effective vaccines. O-Antigen polysaccharides are typically polydisperse, and carrier proteins can have multiple glycosylation sites. Consequently, recombinant glycoconjugate vaccines have a high structural heterogeneity, making their characterization challenging. Since development and quality control processes rely on such characterization, novel strategies are needed for faster and informative analysis. Here, we present a novel approach employing minimal sample preparation and ultrahigh-resolution mass spectrometry analysis for protein terminal sequencing and characterization of the oligosaccharide repeat units of bacterial glycoconjugate vaccines. Three glycoconjugate vaccine candidates, obtained from the bioconjugation of the O-antigen polysaccharides from E. coli serotypes O2, O6A, and O25B with the genetically detoxified exotoxin A from Pseudomonas aeruginosa, were analyzed by MALDI-in-source decay (ISD) FT-ICR MS. Protein and glycan ISD fragment ions were selectively detected using 1,5-diaminonaphtalene and a 2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxybenzoic acid mixture (super-DHB) as a MALDI matrix, respectively. The analysis of protein fragments required the absence of salts in the samples, while the presence of salt was key for the detection of sodiated glycan fragments. MS/MS analysis of O-antigen ISD fragments allowed for the detection of specific repeat unit signatures. The developed strategy requires minute sample amounts, avoids the use of chemical derivatizations, and comes with minimal hands-on time allowing for fast corroboration of key structural features of bacterial glycoconjugate vaccines during early- and late-stage development.


Assuntos
Vacinas contra Escherichia coli , Escherichia coli/metabolismo , Vacinas contra Escherichia coli/metabolismo , Antígenos O , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem
4.
J Am Soc Nephrol ; 32(10): 2455-2465, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34127537

RESUMO

BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerular disease worldwide and is a leading cause of renal failure. The disease mechanisms are not completely understood, but a higher abundance of galactose-deficient IgA is recognized to play a crucial role in IgAN pathogenesis. Although both types of human IgA (IgA1 and IgA2) have several N-glycans as post-translational modification, only IgA1 features extensive hinge-region O-glycosylation. IgA1 galactose deficiency on the O-glycans is commonly detected by a lectin-based method. To date, limited detail is known about IgA O- and N-glycosylation in IgAN. METHODS: To gain insights into the complex O- and N-glycosylation of serum IgA1 and IgA2 in IgAN, we used liquid chromatography-mass spectrometry (LC-MS) for the analysis of tryptic glycopeptides of serum IgA from 83 patients with IgAN and 244 age- and sex-matched healthy controls. RESULTS: Multiple structural features of N-glycosylation of IgA1 and IgA2 were associated with IgAN and glomerular function in our cross-sectional study. These features included differences in galactosylation, sialylation, bisection, fucosylation, and N-glycan complexity. Moreover, IgA1 O-glycan sialylation was associated with both the disease and glomerular function. Finally, glycopeptides were a better predictor of IgAN and glomerular function than galactose-deficient IgA1 levels measured by lectin-based ELISA. CONCLUSIONS: Our high-resolution data suggest that IgA O- and N-glycopeptides are promising targets for future investigations on the pathophysiology of IgAN and as potential noninvasive biomarkers for disease prediction and deteriorating kidney function.


Assuntos
Galactose/metabolismo , Glomerulonefrite por IGA/sangue , Imunoglobulina A/metabolismo , Adulto , Estudos de Casos e Controles , Cromatografia Líquida , Estudos Transversais , Feminino , Galactose/química , Taxa de Filtração Glomerular , Glomerulonefrite por IGA/fisiopatologia , Glicopeptídeos/análise , Glicosilação , Humanos , Imunoglobulina A/química , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/química
5.
Anal Chem ; 92(6): 4518-4526, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32091889

RESUMO

Immunoglobulin (Ig) glycosylation is recognized for its influence on Ig turnover and effector functions. However, the large-scale profiling of Ig glycosylation in a biomedical setting is challenged by the existence of different Ig isotypes and subclasses, their varying serum concentrations, and the presence of multiple glycosylation sites per Ig. Here, a high-throughput nanoliquid chromatography (LC)- mass spectrometry (MS)-based method for simultaneous analysis of IgG and IgA glycopeptides was developed and applied on a serum sample set from 185 healthy donors. Sample preparation from minute amounts of serum was performed in 96-well plate format. Prior to trypsin digestion, IgG and IgA were enriched simultaneously, followed by a one-step denaturation, reduction, and alkylation. The obtained nanoLC-MS data were subjected to semiautomated, targeted feature integration and quality control. The combined and simplified protocol displayed high overall method repeatability, as assessed using pooled plasma and serum standards. Taking all samples together, 143 individual N- and O-glycopeptides were reliably quantified. These glycopeptides were attributable to 11 different peptide backbones, derived from IgG1, IgG2/3, IgG4, IgA1, IgA2, and the joining chain from dimeric IgA. Using this method, novel associations were found between IgA N- and O-glycosylation and age. Furthermore, previously reported associations of IgG Fc glycosylation with age in healthy individuals were confirmed. In conclusion, the new method paves the way for high-throughput multiprotein plasma glycoproteomics.


Assuntos
Glicopeptídeos/sangue , Ensaios de Triagem em Larga Escala , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Feminino , Voluntários Saudáveis , Humanos , Masculino
6.
Gastroenterology ; 155(3): 829-843, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29792883

RESUMO

BACKGROUND & AIMS: Biomarkers are needed for early detection of Crohn's disease (CD) and ulcerative colitis (UC) or to predict patient outcomes. Glycosylation is a common and complex posttranslational modification of proteins that affects their structure and activity. We compared plasma N-glycosylation profiles between patients with CD or UC and healthy individuals (controls). METHODS: We analyzed the total plasma N-glycomes of 2635 patients with inflammatory bowel diseases and 996 controls by mass spectrometry with a linkage-specific sialic acid derivatization technique. Plasma samples were acquired from 2 hospitals in Italy (discovery cohort, 1989 patients with inflammatory bowel disease [IBD] and 570 controls) and 1 medical center in the United States (validation cohort, 646 cases of IBD and 426 controls). Sixty-three glycoforms met our criteria for relative quantification and were extracted from the raw data with the software MassyTools. Common features shared by the glycan compositions were combined in 78 derived traits, including the number of antennae of complex-type glycans and levels of fucosylation, bisection, galactosylation, and sialylation. Associations of plasma N-glycomes with age, sex, CD, UC, and IBD-related parameters such as disease location, surgery and medication, level of C-reactive protein, and sedimentation rate were tested by linear and logistic regression. RESULTS: Plasma samples from patients with IBD had a higher abundance of large-size glycans compared with controls, a decreased relative abundance of hybrid and high-mannose structures, lower fucosylation, lower galactosylation, and higher sialylation (α2,3- and α2,6-linked). We could discriminate plasma from patients with CD from that of patients with UC based on higher bisection, lower galactosylation, and higher sialylation (α2,3-linked). Glycosylation patterns were associated with disease location and progression, the need for a more potent medication, and surgery. These results were replicated in a large independent cohort. CONCLUSIONS: We performed high-throughput analysis to compare total plasma N-glycomes of individuals with vs without IBD and to identify patterns associated with disease features and the need for treatment. These profiles might be used in diagnosis and for predicting patients' responses to treatment.


Assuntos
Colite Ulcerativa/sangue , Doença de Crohn/sangue , Polissacarídeos/sangue , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Colite Ulcerativa/diagnóstico , Doença de Crohn/diagnóstico , Progressão da Doença , Feminino , Glicosilação , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Processamento de Proteína Pós-Traducional
7.
Biochim Biophys Acta ; 1860(8): 1596-607, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26748235

RESUMO

BACKGROUND: A subset of histo-blood group antigens including ABO and Lewis are oligosaccharide structures which may be conjugated to lipids or proteins. They are known to be important recognition motifs not only in the context of blood transfusions, but also in infection and cancer development. SCOPE OF REVIEW: Current knowledge on the molecular background and the implication of histo-blood group glycans in the prevention and therapy of infectious and non-communicable diseases, such as cancer and cardiovascular disease, is presented. MAJOR CONCLUSIONS: Glycan-based histo-blood groups are associated with intestinal microbiota composition, the risk of various diseases as well as therapeutic success of, e.g., vaccination. Their potential as prebiotic or anti-microbial agents, as disease biomarkers and vaccine targets should be further investigated in future studies. For this, recent and future technological advancements will be of particular importance, especially with regard to the unambiguous structural characterization of the glycan portion in combination with information on the protein and lipid carriers of histo-blood group-active glycans in large cohorts. GENERAL SIGNIFICANCE: Histo-blood group glycans have a unique linking position in the complex network of genes, oncodevelopmental biological processes, and disease mechanisms. Thus, they are highly promising targets for novel approaches in the field of personalized medicine. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.


Assuntos
Sistema ABO de Grupos Sanguíneos , Doenças Cardiovasculares , Antígenos do Grupo Sanguíneo de Lewis , Neoplasias , Oligossacarídeos , Medicina de Precisão/métodos , Sistema ABO de Grupos Sanguíneos/genética , Sistema ABO de Grupos Sanguíneos/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/terapia , Humanos , Antígenos do Grupo Sanguíneo de Lewis/genética , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Oligossacarídeos/genética , Oligossacarídeos/metabolismo
8.
Glycobiology ; 26(12): 1308-1316, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27613801

RESUMO

Beneficial effects have been proposed for human milk oligosaccharides (HMO), as deduced from in vitro and animal studies. To date, in vivo evidence of the link between certain oligosaccharide structures in milk and their consumption by infant gut microbiota is still missing, although likely. Whereas many studies have described HMO patterns in human milk from larger cohorts, data on the excretion of HMO and possible metabolites produced in the infant gut are still very limited. From smaller-scale studies, an age-dependency according to infant gut maturation and microbiota adaptation has previously been hypothesized. To further investigate this, we profiled neutral fecal oligosaccharides from term-born infants who were exclusively breastfed, formula-fed or mixed-fed at the age of 2 months, and from a follow-up of a subgroup at 7 months of age (INFABIO study). Data on maternal antibiotic exposure was also included. Automated matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analyses revealed the presence of HMO and metabolites in the feces of most, but not all breastfed infants at 2 months, with highly varying patterns that appeared not to differ with maternal antibiotics exposure. Formula-fed infants at 2 months and most of the breastfed infants at 7 months did not excrete HMO-like structures in their feces, the latter corresponding to the hypothesis of age-dependency. Together with our previous results that were partly contradictory to what has been proposed by others, here, we suggest alternative explanations for the described association of oligosaccharide excretion with age and feeding type in infants below 7 months of age.


Assuntos
Aleitamento Materno , Fezes/química , Fórmulas Infantis/química , Leite Humano/química , Oligossacarídeos/química , Humanos , Lactente
9.
Glycobiology ; 24(2): 185-94, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24253766

RESUMO

Human milk oligosaccharides (HMOs) have been paid much attention due to their beneficial effects observed in vitro, e.g., prebiotic, anti-infective and anti-inflammatory properties. However, in vivo investigations with regard to HMO metabolism and functions are rare. The few data available indicate that HMOs are absorbed to a low extent and excreted via urine without noteworthy modifications, whereas the major proportion reaches infant's colon undigested. Via intrinsic (13)C-labeling of HMOs during their biosynthesis in the mammary gland of 10 lactating women, we were able to follow the fate of (13)C-labeled oligosaccharides (OSs) from their secretion in milk to the excretion in the urine of their breastfed infants. To a certain extent, we could therefore discriminate between original HMOs and non-labeled OSs derived from degradation of HMOs or endogenous glycoconjugates. By means of our novel, rapid, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based approach, we found a homogeneous time pattern of isotopomer enrichment in milk among all subjects and between single OS species. In contrast, the time curves from infants' urine varied strongly between individuals and OS species, though the overall MALDI-TOF MS profile resembled those of the mothers' milk. Our data suggest that neutral HMOs might be processed and/or utilized differentially after or upon absorption from the gut, as deduced from their structure-dependent variation in the extent of tracer enrichment and in the retention times in infant's organism. This sheds new light on the role of HMOs within infant's body, beyond the intestine and its microbiota alone.


Assuntos
Aleitamento Materno , Lactação/metabolismo , Leite Humano/química , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Sequência de Carboidratos , Isótopos de Carbono/farmacocinética , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Individualidade , Recém-Nascido , Intestinos/química , Leite Humano/metabolismo , Dados de Sequência Molecular , Oligossacarídeos/urina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Urinálise
10.
Carbohydr Polym ; 341: 122327, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-38876725

RESUMO

Bacterial pathogens can cause a broad range of infections with detrimental effects on health. Vaccine development is essential as multi-drug resistance in bacterial infections is a rising concern. Recombinantly produced proteins carrying O-antigen glycosylation are promising glycoconjugate vaccine candidates to prevent bacterial infections. However, methods for their comprehensive structural characterization are lacking. Here, we present a bottom-up approach for their site-specific characterization, detecting N-glycopeptides by nano reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS). Glycopeptide analyses revealed information on partial site-occupancy and site-specific glycosylation heterogeneity and helped corroborate the polysaccharide structures and their modifications. Bottom-up analysis was complemented by intact glycoprotein analysis using nano RP-LC-MS allowing the fast visualization of the polysaccharide distribution in the intact glycoconjugate. At the glycopeptide level, the model glycoconjugates analyzed showed different repeat unit (RU) distributions that spanned from 1 to 21 RUs attached to each of the different glycosylation sites. Interestingly, the intact glycoprotein analysis displayed a RU distribution ranging from 1 to 28 RUs, showing the predominant species when the different glycopeptide distributions are combined in the intact glycoconjugate. The complete workflow based on LC-MS measurements allows detailed and comprehensive analysis of the glycosylation state of glycoconjugate vaccines.


Assuntos
Vacinas Bacterianas , Glicoconjugados , Glicopeptídeos , Glicoconjugados/química , Glicoconjugados/imunologia , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/química , Glicosilação , Glicopeptídeos/química , Glicopeptídeos/análise , Espectrometria de Massas/métodos , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologia , Cromatografia Líquida/métodos , Cromatografia de Fase Reversa/métodos
11.
iScience ; 26(7): 107021, 2023 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-37485378

RESUMO

Visceral leishmaniasis (VL) is a clinical form of leishmaniasis with high mortality rates when not treated. Diagnosis suffers from invasive techniques and sub-optimal sensitivities. The current (affordable) treatment with pentavalent antimony as advised by the WHO is possibly harmful to the patient. There is need for an improved diagnosis to prevent possibly unnecessary treatment. N-glycan analysis may aid in diagnosis. We evaluated the N-glycan profiles from active VL, asymptomatic infections (ASYMP) and controls from non-endemic (NC) and endemic (EC) areas. Active VL has a distinct N-glycome profile that associates with disease severity. Our study suggests that the observed glycan signatures could be a valuable additive to diagnosis and assist in identifying possible markers of disease and understanding the pathogenesis of VL. Further studies are warranted to assess a possible future role of blood glycome analysis in active VL diagnosis and should aim at disease specificity.

12.
Anal Bioanal Chem ; 401(8): 2495-510, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21898157

RESUMO

The structural diversity of human milk oligosaccharides (HMOs) strongly depends on the Lewis (Le) blood group status of the donor which allows a classification of these glycans into three different groups. Starting from 50 µL of human milk, a new high-throughput, standardized, and widely automated mass spectrometric approach has been established which can be used for correlation of HMO structures with the respective Lewis blood groups on the basis of mass profiles of the entire mixture of glycans together with selected fragment ion spectra. For this purpose, the relative abundance of diagnostically relevant compositional species, such as Hex(2)Fuc(2) and Hex(3)HexNAc(1)Fuc(2), as well as the relative intensities of characteristic fragment ions obtained thereof are of key importance. For each Lewis blood group, i.e., Le(a-b+), Le(a+b-), and Le(a-b-), specific mass profile and fragment ion patterns could be thus verified. The described statistically proven classification of the derived glycan patterns may be a valuable tool for analysis and comparison of large sets of milk samples in metabolic studies. Furthermore, the outlined protocol may be used for rapid screening in clinical studies and quality control of milk samples donated to milk banks.


Assuntos
Antígenos do Grupo Sanguíneo de Lewis/análise , Leite Humano/química , Oligossacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Carboidratos , Análise Discriminante , Feminino , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economia
13.
Front Chem ; 9: 678883, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34026735

RESUMO

Apolipoprotein-CIII (apo-CIII) is a glycoprotein involved in lipid metabolism and its levels are associated with cardiovascular disease risk. Apo-CIII sialylation is associated with improved plasma triglyceride levels and its glycosylation may have an effect on the clearance of triglyceride-rich lipoproteins by directing these particles to different metabolic pathways. Large-scale sample cohort studies are required to fully elucidate the role of apo-CIII glycosylation in lipid metabolism and associated cardiovascular disease. In this study, we revisited a high-throughput workflow for the analysis of intact apo-CIII by ultrahigh-resolution MALDI FT-ICR MS. The workflow includes a chemical oxidation step to reduce methionine oxidation heterogeneity and spectrum complexity. Sinapinic acid matrix was used to minimize the loss of sialic acids upon MALDI. MassyTools software was used to standardize and automate MS data processing and quality control. This method was applied on 771 plasma samples from individuals without diabetes allowing for an evaluation of the expression levels of apo-CIII glycoforms against a panel of lipid biomarkers demonstrating the validity of the method. Our study supports the hypothesis that triglyceride clearance may be regulated, or at least strongly influenced by apo-CIII sialylation. Interestingly, the association of apo-CIII glycoforms with triglyceride levels was found to be largely independent of body mass index. Due to its precision and throughput, the new workflow will allow studying the role of apo-CIII in the regulation of lipid metabolism in various disease settings.

14.
Artigo em Inglês | MEDLINE | ID: mdl-34645615

RESUMO

INTRODUCTION: Although associations of total plasma N-glycome (TPNG) with type 2 diabetes have been reported, little is known on the role of TPNG in type 2 diabetes complications, a major cause of type 2 diabetes-related morbidity and mortality. Here, we assessed TPNG in relation to type 2 diabetes complications in subsamples of two Dutch cohorts using mass spectrometry (n=1815 in DiaGene and n=1518 in Hoorn Diabetes Care System). RESEARCH DESIGN AND METHODS: Blood plasma samples and technical replicates were pipetted into 96-well plates in a randomized manner. Peptide:N-glycosidase F (PNGase F) was used to release N-glycans, whereafter sialic acids were derivatized for stabilization and linkage differentiation. After total area normalization, 68 individual glycan compositions were quantified in total and were used to calculate 45 derived traits which reflect structural features of glycosylation. Associations of glycan features with prevalent and incident microvascular or macrovascular complications were tested in logistic and Cox regression in both independent cohorts and the results were meta-analyzed. RESULTS: Our results demonstrated similarities between incident and prevalent complications. The strongest association for prevalent cardiovascular disease was a high level of bisection on a group of diantennary glycans (A2FS0B; OR=1.38, p=1.34×10-11), while for prevalent nephropathy the increase in 2,6-sialylation on triantennary glycans was most pronounced (A3E; OR=1.28, p=9.70×10-6). Several other TPNG features, including fucosylation, galactosylation, and sialylation, firmly demonstrated associations with prevalent and incident complications of type 2 diabetes. CONCLUSIONS: These findings may provide a glance on how TPNG patterns change before complications emerge, paving the way for future studies on prediction biomarkers and potentially disease mechanisms.


Assuntos
Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Doenças Retinianas , Proteínas Sanguíneas , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/etiologia , Diabetes Mellitus Tipo 2/complicações , Glicosilação , Humanos , Plasma
15.
Cancer Med ; 9(22): 8519-8529, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32898301

RESUMO

BACKGROUND &AIMS: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type with loco-regional spread that makes the tumor surgically unresectable. Novel diagnostic tools are needed to improve detection of PDAC and increase patient survival. In this study we explore serum protein N-glycan profiles from PDAC patients with regard to their applicability to serve as a disease biomarker panel. METHODS: Total serum N-glycome analysis was applied to a discovery set (86 PDAC cases/84 controls) followed by independent validation (26 cases/26 controls) using in-house collected serum specimens. Protein N-glycan profiles were obtained using ultrahigh resolution mass spectrometry and included linkage-specific sialic acid information. N-glycans were relatively quantified and case-control classification performance was evaluated based on glycosylation traits such as branching, fucosylation, and sialylation. RESULTS: In PDAC patients a higher level of branching (OR 6.19, P-value 9.21 × 10-11 ) and (antenna)fucosylation (OR 13.27, P-value 2.31 × 10-9 ) of N-glycans was found. Furthermore, the ratio of α2,6- vs α2,3-linked sialylation was higher in patients compared to healthy controls. A classification model built with three glycosylation traits was used for discovery (AUC 0.88) and independent validation (AUC 0.81), with sensitivity and specificity values of 0.85 and 0.71 for the discovery set and 0.75 and 0.72 for the validation set. CONCLUSION: Serum N-glycome analysis revealed glycosylation differences that allow classification of PDAC patients from healthy controls. It was demonstrated that glycosylation traits rather than single N-glycan structures obtained in this clinical glycomics study can serve as a basis for further development of a blood-based diagnostic test.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Ductal Pancreático/sangue , Glicômica , Glicoproteínas/sangue , Neoplasias Pancreáticas/sangue , Idoso , Carcinoma Ductal Pancreático/diagnóstico , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Feminino , Glicosilação , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Neoplasias Pancreáticas/diagnóstico , Valor Preditivo dos Testes , Reprodutibilidade dos Testes
16.
Artigo em Inglês | MEDLINE | ID: mdl-32616483

RESUMO

INTRODUCTION: Recent studies revealed N-glycosylation signatures of type 2 diabetes, inflammation and cardiovascular risk factors. Most people with diabetes use medication to reduce cardiovascular risk. The association of these medications with the plasma N-glycome is largely unknown. We investigated the associations of metformin, statin, ACE inhibitor/angiotensin II receptor blocker (ARB), sulfonylurea (SU) derivatives and insulin use with the total plasma N-glycome in type 2 diabetes. RESEARCH DESIGN AND METHODS: After enzymatic release from glycoproteins, N-glycans were measured by matrix-assisted laser desorption/ionization mass spectrometry in the DiaGene (n=1815) and Hoorn Diabetes Care System (n=1518) cohorts. Multiple linear regression was used to investigate associations with medication, adjusted for clinical characteristics. Results were meta-analyzed and corrected for multiple comparisons. RESULTS: Metformin and statins were associated with decreased fucosylation and increased galactosylation and sialylation in glycans unrelated to immunoglobulin G. Bisection was increased within diantennary fucosylated non-sialylated glycans, but decreased within diantennary fucosylated sialylated glycans. Only few glycans were associated with ACE inhibitor/ARBs, while none associated with insulin and SU derivative use. CONCLUSIONS: We conclude that metformin and statins associate with a total plasma N-glycome signature in type 2 diabetes. Further studies are needed to determine the causality of these relations, and future N-glycomic research should consider medication a potential confounder.


Assuntos
Diabetes Mellitus Tipo 2 , Inibidores de Hidroximetilglutaril-CoA Redutases , Metformina , Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Proteínas Sanguíneas , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glicosilação , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Metformina/uso terapêutico
17.
FEBS Lett ; 593(21): 2966-2976, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31509238

RESUMO

N-glycome analysis in total plasma or serum yields information about the levels and glycosylation patterns of major plasma glycoproteins, including immunoglobulins, acute-phase proteins, and apolipoproteins. Until recently, glycomic studies in disease settings largely suffered from small cohort sizes, poor analytical resolution, and poor comparability of results owing to the diversity of analytical techniques. Here, we report on recent advances in high-throughput mass spectrometry glycomics technology that enabled elucidation of N-glycome signatures in the plasma of patients with type 2 diabetes, inflammatory bowel disease, or colorectal cancer. Use of this technology revealed both commonalities and differences among disease fingerprints. Moreover, we summarize findings on glycomic signatures associated with age, sex, and body mass index. High-throughput, high-resolution glycomics technologies, together with robust data analysis workflows, will advance clinical translation.


Assuntos
Neoplasias Colorretais/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glicômica/métodos , Doenças Inflamatórias Intestinais/metabolismo , Plasma/metabolismo , Biomarcadores/sangue , Glicoproteínas/sangue , Ensaios de Triagem em Larga Escala , Humanos , Espectrometria de Massas
19.
Front Immunol ; 9: 2436, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30405629

RESUMO

The N-glycosylation of immunoglobulin (Ig) G, the major antibody in the circulation of human adults, is well known for its influence on antibody effector functions and its alterations with various diseases. In contrast, knowledge on the role of glycans attached to IgA, which is a key immune defense agent in secretions, is very scarce. In this study we aimed to characterize the glycosylation of salivary (secretory) IgA, including the IgA joining chain (JC), and secretory component (SC) and to compare IgA and IgG glycosylation between human plasma and saliva samples to gain a first insight into oral cavity-specific antibody glycosylation. Plasma and whole saliva were collected from 19 healthy volunteers within a 2-h time window. IgG and IgA were affinity-purified from the two biofluids, followed by tryptic digestion and nanoLC-ESI-QTOF-MS(/MS) analysis. Saliva-derived IgG exhibited a slightly lower galactosylation and sialylation as compared to plasma-derived IgG. Glycosylation of IgA1, IgA2, and the JC showed substantial differences between the biofluids, with salivary proteins exhibiting a higher bisection, and lower galactosylation and sialylation as compared to plasma-derived IgA and JC. Additionally, all seven N-glycosylation sites, characterized on the SC of secretory IgA in saliva, carried highly fucosylated and fully galactosylated diantennary N-glycans. This study lays the basis for future research into the functional role of salivary Ig glycosylation as well as its biomarker potential.


Assuntos
Proteínas Sanguíneas/metabolismo , Glicômica , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Testes Imunológicos/métodos , Saliva/metabolismo , Adulto , Biomarcadores/metabolismo , Proteínas Sanguíneas/química , Feminino , Glicosilação , Voluntários Saudáveis , Humanos , Imunoglobulina A/química , Imunoglobulina G/química , Masculino , Adulto Jovem
20.
Biochim Biophys Acta Gen Subj ; 1862(12): 2613-2622, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30251656

RESUMO

BACKGROUND: Little is known about enzymatic N-glycosylation in type 2 diabetes, a common posttranslational modification of proteins influencing their function and integrating genetic and environmental influences. We sought to gain insights into N-glycosylation to uncover yet unexplored pathophysiological mechanisms in type 2 diabetes. METHODS: Using a high-throughput MALDI-TOF mass spectrometry method, we measured N-glycans in plasma samples of the DiaGene case-control study (1583 cases and 728 controls). Associations were investigated with logistic regression and adjusted for age, sex, body mass index, high-density lipoprotein-cholesterol, non-high-density lipoprotein-cholesterol, and smoking. Findings were replicated in a nested replication cohort of 232 cases and 108 controls. RESULTS: Eighteen glycosylation features were significantly associated with type 2 diabetes. Fucosylation and bisection of diantennary glycans were decreased in diabetes (odds ratio (OR) = 0.81, p = 1.26E-03, and OR = 0.87, p = 2.84E-02, respectively), whereas total and, specifically, alpha2,6-linked sialylation were increased (OR = 1.38, p = 9.92E-07, and OR = 1.40, p = 5.48E-07). Alpha2,3-linked sialylation of triantennary glycans was decreased (OR = 0.60, p = 6.38E-11). CONCLUSIONS: While some glycosylation changes were reflective of inflammation, such as increased alpha2,6-linked sialylation, our finding of decreased alpha2,3-linked sialylation in type 2 diabetes patients is contradictory to reports on acute and chronic inflammation. Thus, it might have previously unreported immunological implications in type 2 diabetes. GENERAL SIGNIFICANCE: This study provides new insights into N-glycosylation patterns in type 2 diabetes, which can fuel studies on causal mechanisms and consequences of this complex disease.


Assuntos
Proteínas Sanguíneas/metabolismo , Diabetes Mellitus Tipo 2/sangue , Polissacarídeos/metabolismo , Idoso , Feminino , Glicosilação , Humanos , Masculino , Pessoa de Meia-Idade , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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