RESUMO
The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies.
Assuntos
Fragmentos Fc das Imunoglobulinas , Imunoglobulina G , Humanos , Glicosilação , Fragmentos Fc das Imunoglobulinas/genética , Linfócitos B/metabolismo , Células Clonais/metabolismoRESUMO
Seizure protein 6 (SEZ6) is required for the development and maintenance of the nervous system, is a major substrate of the protease BACE1 and is linked to Alzheimer's disease (AD) and psychiatric disorders, but its molecular functions are not well understood. Here, we demonstrate that SEZ6 controls glycosylation and cell surface localization of kainate receptors composed of GluK2/3 subunits. Loss of SEZ6 reduced surface levels of GluK2/3 in primary neurons and reduced kainate-evoked currents in CA1 pyramidal neurons in acute hippocampal slices. Mechanistically, loss of SEZ6 in vitro and in vivo prevented modification of GluK2/3 with the human natural killer-1 (HNK-1) glycan, a modulator of GluK2/3 function. SEZ6 interacted with GluK2 through its ectodomain and promoted post-endoplasmic reticulum transport of GluK2 in the secretory pathway in heterologous cells and primary neurons. Taken together, SEZ6 acts as a new trafficking factor for GluK2/3. This novel function may help to better understand the role of SEZ6 in neurologic and psychiatric diseases.
Assuntos
Região CA1 Hipocampal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Piramidais/metabolismo , Receptores de Ácido Caínico/metabolismo , Animais , Glicosilação , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Transporte Proteico , Receptores de Ácido Caínico/genética , Receptor de GluK2 Cainato , Receptor de GluK3 CainatoRESUMO
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 , BiomarcadoresRESUMO
Aberrant glycosylation is a hallmark of cancer and is not just a consequence, but also a driver of a malignant phenotype. In prostate cancer, changes in fucosylated and sialylated glycans are common and this has important implications for tumor progression, metastasis, and immune evasion. Glycans hold huge translational potential and new therapies targeting tumor-associated glycans are currently being tested in clinical trials for several tumor types. Inhibitors targeting fucosylation and sialylation have been developed and show promise for cancer treatment, but translational development is hampered by safety issues related to systemic adverse effects. Recently, potent metabolic inhibitors of sialylation and fucosylation were designed that reach higher effective concentrations within the cell, thereby rendering them useful tools to study sialylation and fucosylation as potential candidates for therapeutic testing. Here, we investigated the effects of global metabolic inhibitors of fucosylation and sialylation in the context of prostate cancer progression. We find that these inhibitors effectively shut down the synthesis of sialylated and fucosylated glycans to remodel the prostate cancer glycome with only minor apparent side effects on other glycan types. Our results demonstrate that treatment with inhibitors targeting fucosylation or sialylation decreases prostate cancer cell growth and downregulates the expression of genes and proteins important in the trajectory of disease progression. We anticipate our findings will lead to the broader use of metabolic inhibitors to explore the role of fucosylated and sialylated glycans in prostate tumor pathology and may pave the way for the development of new therapies for prostate cancer.
Assuntos
Neoplasias da Próstata , Masculino , Humanos , Glicosilação , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Processamento de Proteína Pós-Traducional , Polissacarídeos/metabolismoRESUMO
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/químicaRESUMO
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 , MasculinoRESUMO
Protein N-glycosylation is a multifactorial process involved in many biological processes. A broad range of congenital disorders of glycosylation (CDGs) have been described that feature defects in protein N-glycan biosynthesis. Here, we present insights into the disrupted N-glycosylation of various CDG patients exhibiting defects in the transport of nucleotide sugars, Golgi glycosylation or Golgi trafficking. We studied enzymatically released N-glycans of total plasma proteins and affinity purified immunoglobulin G (IgG) from patients and healthy controls using mass spectrometry (MS). The applied method allowed the differentiation of sialic acid linkage isomers via their derivatization. Furthermore, protein-specific glycan profiles were quantified for transferrin and IgG Fc using electrospray ionization MS of intact proteins and glycopeptides, respectively. Next to the previously described glycomic effects, we report unprecedented sialic linkage-specific effects. Defects in proteins involved in Golgi trafficking (COG5-CDG) and CMP-sialic acid transport (SLC35A1-CDG) resulted in lower levels of sialylated structures on plasma proteins as compared to healthy controls. Findings for these specific CDGs include a more pronounced effect for α2,3-sialylation than for α2,6-sialylation. The diverse abnormalities in glycomic features described in this study reflect the broad range of biological mechanisms that influence protein glycosylation.
Assuntos
Defeitos Congênitos da Glicosilação/sangue , Glicopeptídeos/sangue , Adolescente , Adulto , Proteínas Sanguíneas/metabolismo , Criança , Pré-Escolar , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Feminino , Glicômica/métodos , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Lactente , Masculino , Espectrometria de Massas/métodos , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteômica/métodos , Ácidos Siálicos/metabolismoRESUMO
Complement factor H (FH), an elongated and substantially glycosylated 20-domain protein, is a soluble regulator of the complement alternative pathway (AP). It contains several glycan binding sites which mediate recognition of α2-3-linked sialic acid (FH domain 20) and glycosaminoglycans (domains 6-8 and 19-20). FH also binds the complement C3-activation product C3b, a powerful opsonin and focal point for the formation of C3-convertases of the AP feedback loop. In freely circulating FH the C3b binding site in domains 19-20 is occluded, a phenomenon that is not fully understood and could be mediated by an intramolecular interaction between FH's intrinsic sialylated glycosylation and its own sialic acid binding site. In order to assess this possibility, we characterized FH's sialylation with respect to glycosidic linkage type and searched for further potential, not yet characterized sialic acid binding sites in FH and its seven-domain spanning splice variant and fellow complement regulator FH like-1 (FHL-1). We also probed FH binding to the sialic acid variant Neu5Gc which is not expressed in humans but on heterologous erythrocytes that restrict the human AP and in FH transgenic mice. We find that FH contains mostly α2-6-linked sialic acid, making an intramolecular interaction with its α2-3-sialic acid specific binding site and an associated self-lock mechanism unlikely, substantiate that there is only a single sialic acid binding site in FH and none in FHL-1, and demonstrate direct binding of FH to the nonhuman sialic acid Neu5Gc, supporting the use of FH transgenic mouse models for studies of complement-related diseases.
Assuntos
Ácido N-Acetilneuramínico/análise , Animais , Sítios de Ligação , Configuração de Carboidratos , Fator H do Complemento/química , Fator H do Complemento/isolamento & purificação , Fator H do Complemento/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismoRESUMO
Congenital disorders of glycosylation (CDG) are genetic defects in the glycoconjugate biosynthesis. >100 types of CDG are known, most of them cause multi-organ diseases. Here we describe a boy whose leading symptoms comprise cutis laxa, pancreatic insufficiency and hepatosplenomegaly. Whole exome sequencing identified the novel hemizygous mutation c.542T>G (p.L181R) in the X-linked ATP6AP1, an accessory protein of the mammalian vacuolar H+-ATPase, which led to a general N-glycosylation deficiency. Studies of serum N-glycans revealed reduction of complex sialylated and appearance of truncated diantennary structures. Proliferation of the patient's fibroblasts was significantly reduced and doubling time prolonged. Additionally, there were alterations in the fibroblasts' amino acid levels and the acylcarnitine composition. Especially, short-chain species were reduced, whereas several medium- to long-chain acylcarnitines (C14-OH to C18) were elevated. Investigation of the main lipid classes revealed that total cholesterol was significantly enriched in the patient's fibroblasts at the expense of phophatidylcholine and phosphatidylethanolamine. Within the minor lipid species, hexosylceramide was reduced, while its immediate precursor ceramide was increased. Since catalase activity and ACOX3 expression in peroxisomes were reduced, we assume an ATP6AP1-dependent impact on the ß-oxidation of fatty acids. These results help to understand the complex clinical characteristics of this new patient.
Assuntos
Defeitos Congênitos da Glicosilação/genética , Cútis Laxa/genética , Insuficiência Pancreática Exócrina/genética , Metaboloma/genética , ATPases Vacuolares Próton-Translocadoras/genética , Acil-CoA Oxidase/metabolismo , Catalase/metabolismo , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/metabolismo , Cútis Laxa/diagnóstico , Cútis Laxa/metabolismo , Insuficiência Pancreática Exócrina/diagnóstico , Insuficiência Pancreática Exócrina/metabolismo , Ácidos Graxos/metabolismo , Genes Ligados ao Cromossomo X/genética , Humanos , Lactente , Masculino , Metabolômica , Oxirredução , ATPases Vacuolares Próton-Translocadoras/deficiência , Sequenciamento do ExomaRESUMO
Haemolytic disease of the fetus and newborn (HDFN) is a severe disease in which fetal red blood cells (RBC) are destroyed by maternal anti-RBC IgG alloantibodies. HDFN is most often caused by anti-D but may also occur due to anti-K, -c- or -E. We recently found N-linked glycosylation of anti-D to be skewed towards low fucosylation, thereby increasing the affinity to IgG-Fc receptor IIIa and IIIb, which correlated with HDFN disease severity. Here, we analysed 230 pregnant women with anti-c, -E or -K alloantibodies from a prospective screening cohort and investigated the type of Fc-tail glycosylation of these antibodies in relation to the trigger of immunisation and pregnancy outcome. Anti-c, -E and -K show - independent of the event that had led to immunisation - a different kind of Fc-glycosylation compared to that of the total IgG fraction, but with less pronounced differences compared to anti-D. High Fc-galactosylation and sialylation of anti-c correlated with HDFN disease severity, while low anti-K Fc-fucosylation correlated with severe fetal anaemia. IgG-Fc glycosylation of anti-RBC antibodies is shaped depending on the antigen. These features influence their clinical potency and may therefore be used to predict severity and identify those needing treatment.
Assuntos
Eritroblastose Fetal/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Isoanticorpos/sangue , Adulto , Antígenos de Grupos Sanguíneos/imunologia , Eritroblastose Fetal/diagnóstico , Eritrócitos/imunologia , Feminino , Glicosilação , Humanos , Recém-Nascido , Masculino , Gravidez , Índice de Gravidade de DoençaRESUMO
Glycosylation is an abundant and important protein modification with large influence on the properties and interactions of glycoconjugates. Human plasma N-glycosylation has been the subject of frequent investigation, revealing strong associations with physiological and pathological conditions. Less well-characterized is the plasma N-glycosylation of the mouse, the most commonly used animal model for studying human diseases, particularly with regard to differences between strains and sexes. For this reason, we used MALDI-TOF(/TOF)-MS(/MS) assisted by linkage-specific derivatization of the sialic acids to comparatively analyze the plasma N-glycosylation of both male and female mice originating from BALB/c, CD57BL/6, CD-1, and Swiss Webster strains. The combined use of this analytical method and the recently developed data processing software named MassyTools allowed the relative quantification of the N-glycan species within plasma, the distinction between α2,3- and α2,6-linked N-glycolylneuraminic acids (due to respective lactonization and ethyl esterification), the detection of sialic acid O-acetylation, as well as the characterization of branching sialylation (Neu5Gcα2,3-Hex-[Neu5Gcα2,6-]HexNAc). When analyzing the glycosylation according to mouse sex, we found that female mice present a considerably higher degree of core fucosylation (2-4-fold depending on the strain), galactosylation, α2,6-linked sialylation, and larger high-mannose type glycan species compared with their male counterparts. Male mice, on the contrary, showed on average higher α2,3-linked sialylation, branching sialylation, and putative bisection. These differences together with sialic acid acetylation proved to be strain-specific as well. Interestingly, the outbred strains CD-1 and Swiss Webster displayed considerably larger interindividual variation than inbred strains BALB/c and CD57BL/6, suggesting a strong hereditable component of the observed plasma N-glycome.
Assuntos
Glicosilação , Polissacarídeos/química , Animais , Animais Endogâmicos/metabolismo , Animais não Endogâmicos/metabolismo , Feminino , Humanos , Masculino , Camundongos , Polissacarídeos/sangue , Fatores SexuaisRESUMO
Bottom-up glycoproteomics by liquid chromatography-mass spectrometry (LC-MS) is an established approach for assessing glycosylation in a protein- and site-specific manner. Consequently, tools are needed to automatically align, calibrate, and integrate LC-MS glycoproteomics data. We developed a modular software package designed to tackle the individual aspects of an LC-MS experiment, called LaCyTools. Targeted alignment is performed using user defined m/z and retention time (tr) combinations. Subsequently, sum spectra are created for each user defined analyte group. Quantitation is performed on the sum spectra, where each user defined analyte can have its own tr, minimum, and maximum charge states. Consequently, LaCyTools deals with multiple charge states, which gives an output per charge state if desired, and offers various analyte and spectra quality criteria. We compared throughput and performance of LaCyTools to combinations of available tools that deal with individual processing steps. LaCyTools yielded relative quantitation of equal precision (relative standard deviation <0.5%) and higher trueness due to the use of MS peak area instead of MS peak intensity. In conclusion, LaCyTools is an accurate automated data processing tool for high-throughput analysis of LC-MS glycoproteomics data. Released under the Apache 2.0 license, it is freely available on GitHub ( https://github.com/Tarskin/LaCyTools ).
Assuntos
Processamento Eletrônico de Dados/métodos , Glicopeptídeos/análise , Proteômica/métodos , Cromatografia Líquida , Espectrometria de Massas , Proteômica/normas , SoftwareRESUMO
Autotaxin (ATX) is a secreted phosphodiesterase that produces the signalling lipid lysophosphatidic acid (LPA). The bimetallic active site of ATX is structurally related to the alkaline phosphatase superfamily. Here, we present a new crystal structure of ATX in complex with orthovanadate (ATX-VO5), which binds the Oγ nucleophile of Thr209 and adopts a trigonal bipyramidal conformation, following the nucleophile attack onto the substrate. We have now a portfolio of ATX structures we discuss as intermediates of the catalytic mechanism: the new ATX-VO5 structure; a unique structure where the nucleophile Thr209 is phosphorylated (ATX-pThr). Comparing these to a complex with the LPA product (ATX-LPA) and with a complex with a phosphate ion (ATX-PO4), that represent the Michaelis complex of the reaction, we observe movements of Thr209, changes in the relative displacement of the zinc ions, and a water molecule that likely fulfils the second nucleophilic attack. We propose that ATX follows the associative two-step in-line displacement mechanism.
Assuntos
Lisofosfolipídeos/química , Diester Fosfórico Hidrolases/química , Conformação Proteica , Vanadatos/química , Fosfatase Alcalina/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Humanos , Lipídeos/química , Fosfatos/química , Fosforilação , Zinco/químicaRESUMO
Murine zymosan-induced peritonitis is a widely used model for studying the molecular and cellular events responsible for the initiation, persistence and/or resolution of inflammation. Among these events, it is becoming increasingly evident that changes in glycosylation of proteins, especially in the plasma and at the site of inflammation, play an important role in the inflammatory response. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based glycosylation profiling, we investigated the qualitative and quantitative effect of zymosan-induced peritonitis on N-glycosylation in mouse plasma and peritoneal fluid. Our results show that both N-glycomes exhibit highly similar glycosylation patterns, consisting mainly of diantennary and triantennary complex type N-glycans with high levels (>95 %) of galactosylation and sialylation (mostly NeuGc) and a medium degree of core fucosylation (30 %). Moreover, MS/MS structural analysis, assisted by linkage-specific derivatization of sialic acids, revealed the presence of O-acetylated sialic acids as well as disialylated antennae ("branching sialylation") characterized by the presence of α2-6-linked NeuGc on the GlcNAc of the NeuGcα2-3-Galß1-3-GlcNAc terminal motif. A significant decrease of (core) fucosylation together with an increase of both α2-3-linked NeuGc and "branching sialylation" were observed in N-glycomes of mice challenged with zymosan, but not in control mice injected with PBS. Importantly, substantial changes in glycosylation were already observed 12 h after induction of peritonitis, thereby demonstrating an unexpected velocity of the biological mechanisms involved.
Assuntos
Reação de Fase Aguda/metabolismo , Líquido Ascítico/metabolismo , Glicoproteínas/metabolismo , Peritonite/metabolismo , Processamento de Proteína Pós-Traducional , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Reação de Fase Aguda/sangue , Animais , Líquido Ascítico/química , Feminino , Glicoproteínas/sangue , Glicoproteínas/química , Glicosilação , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/sangueRESUMO
The study of N-linked glycosylation has long been complicated by a lack of bioinformatics tools. In particular, there is still a lack of fast and robust data processing tools for targeted (relative) quantitation. We have developed modular, high-throughput data processing software, MassyTools, that is capable of calibrating spectra, extracting data, and performing quality control calculations based on a user-defined list of glycan or glycopeptide compositions. Typical examples of output include relative areas after background subtraction, isotopic pattern-based quality scores, spectral quality scores, and signal-to-noise ratios. We demonstrated MassyTools' performance on MALDI-TOF-MS glycan and glycopeptide data from different samples. MassyTools yielded better calibration than the commercial software flexAnalysis, generally showing 2-fold better ppm errors after internal calibration. Relative quantitation using MassyTools and flexAnalysis gave similar results, yielding a relative standard deviation (RSD) of the main glycan of ~6%. However, MassyTools yielded 2- to 5-fold lower RSD values for low-abundant analytes than flexAnalysis. Additionally, feature curation based on the computed quality criteria improved the data quality. In conclusion, we show that MassyTools is a robust automated data processing tool for high-throughput, high-performance glycosylation analysis. The package is released under the Apache 2.0 license and is freely available on GitHub ( https://github.com/Tarskin/MassyTools ).
Assuntos
Glicômica/métodos , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Anticorpos Monoclonais/análise , Calibragem , Processamento Eletrônico de Dados/métodos , Glicopeptídeos/análise , Glicosilação , Humanos , Polissacarídeos/análise , Controle de Qualidade , Razão Sinal-RuídoRESUMO
Glycosylation is a post-translational modification of key importance with heterogeneous structural characteristics. Previously, we have developed a robust, high-throughput MALDI-TOF-MS method for the comprehensive profiling of human plasma N-glycans. In this approach, sialic acid residues are derivatized with linkage-specificity, namely the ethylation of α2,6-linked sialic acid residues with parallel lactone formation of α2,3-linked sialic acids. In the current study, this procedure was used as a starting point for the automation of all steps on a liquid-handling robot system. This resulted in a time-efficient and fully standardized procedure with throughput times of 2.5 h for a first set of 96 samples and approximately 1 h extra for each additional sample plate. The mass analysis of the thus-obtained glycans was highly reproducible in terms of relative quantification, with improved interday repeatability as compared to that of manual processing.
Assuntos
Automação/métodos , Glicoproteínas/sangue , Glicoproteínas/química , Ensaios de Triagem em Larga Escala/métodos , Ácido N-Acetilneuramínico/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Esterificação , Glicoproteínas/metabolismo , Glicosilação , Ácido N-Acetilneuramínico/química , Reprodutibilidade dos TestesRESUMO
BACKGROUND: RhIG is obtained from hyperimmunized healthy anti-D donors (HIDs) boosted with D+ red blood cells (RBCs). One hypothesis for its mechanism of action is fast clearance of opsonized D+ RBCs through Fcγ receptor (FcγR)III. Levels of immunoglobulin (Ig)G Fc-fucosylation influence interactions with FcγRIII, with less Fc-fucosylation strengthening the interaction. STUDY DESIGN AND METHODS: Anti-D IgG1 Fc-glycosylation patterns in 93 plasma samples from 28 male and 28 female Dutch HIDs and RhIG were analyzed with mass spectrometry. The Fc-glycosylation profiles of HIDs were evaluated with regard to their immunization history. RESULTS: HID sera demonstrated clearly lowered anti-D Fc-fucosylation compared to normal IgG fucosylation (93%); this was more pronounced for female than for male HIDs (47% vs. 65%, p = 0.001). RhIG preparations from seven manufacturers varied greatly in the level of Fc-fucosylation (56%-91%). The level of fucosylation slightly increased upon repeated immunization, although it remained fairly constant over time. The RhIG from the different manufacturers all demonstrated increased Fc-galactosylation (64%-82%) compared to total IgG (38%-51%). CONCLUSION: RhIG preparations vary in Fc-fucosylation and all demonstrate increased galactosylation. Despite not knowing the exact working mechanism, immunoprophylaxis could perhaps be optimized by selection of donors whose anti-D have low amounts of Fc-fucose, to increase the clearance activity of anti-D preparations, as well as high amounts of galactosylation, for anti-inflammatory effects. Implementing a biologic assay in the standardization of RhIG preparations might be considered.
Assuntos
Fucose/sangue , Imunoglobulina G/química , Isoanticorpos/química , Adulto , Doadores de Sangue , Feminino , Galactose/sangue , Glicosilação , Humanos , Imunização , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/isolamento & purificação , Isoanticorpos/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Gravidez , Processamento de Proteína Pós-Traducional , Receptores de IgG/metabolismo , Isoimunização Rh/terapia , Imunoglobulina rho(D) , Caracteres Sexuais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Congenital disorders of glycosylation comprise a group of genetic defects with a high frequency of intellectual disability, caused by deficient glycosylation of proteins and lipids. The molecular basis of the majority of the congenital disorders of glycosylation type I subtypes, localized in the cytosol and endoplasmic reticulum, has been solved. However, elucidation of causative genes for defective Golgi glycosylation (congenital disorders of glycosylation type II) remains challenging because of a lack of sufficiently specific diagnostic serum methods. In a single patient with intellectual disability, whole-exome sequencing revealed MAN1B1 as congenital disorder of glycosylation type II candidate gene. A novel mass spectrometry method was applied for high-resolution glycoprofiling of intact plasma transferrin. A highly characteristic glycosylation signature was observed with hybrid type N-glycans, in agreement with deficient mannosidase activity. The speed and robustness of the method allowed subsequent screening in a cohort of 100 patients with congenital disorder of glycosylation type II, which revealed the characteristic glycosylation profile of MAN1B1-congenital disorder of glycosylation in 11 additional patients. Abnormal hybrid type N-glycans were also observed in the glycoprofiles of total serum proteins, of enriched immunoglobulins and of alpha1-antitrypsin in variable amounts. Sanger sequencing revealed MAN1B1 mutations in all patients, including severe truncating mutations and amino acid substitutions in the alpha-mannosidase catalytic site. Clinically, this group of patients was characterized by intellectual disability and delayed motor and speech development. In addition, variable dysmorphic features were noted, with truncal obesity and macrocephaly in â¼65% of patients. In summary, MAN1B1 deficiency appeared to be a frequent cause in our cohort of patients with unsolved congenital disorder of glycosylation type II. Our method for analysis of intact transferrin provides a rapid test to detect MAN1B1-deficient patients within congenital disorder of glycosylation type II cohorts and can be used as efficient diagnostic method to identify MAN1B1-deficient patients in intellectual disability cohorts. In addition, it provides a functional confirmation of MAN1B1 mutations as identified by next-generation sequencing in individuals with intellectual disability.
Assuntos
Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Adolescente , Adulto , Pré-Escolar , Análise Mutacional de DNA/métodos , Proteínas de Ligação a DNA , Feminino , Glicosilação , Humanos , Lactente , Deficiência Intelectual/sangue , Masculino , Proteínas de Membrana/sangue , Mutação , Proteínas Nucleares/sangue , Adulto JovemRESUMO
Haemolytic disease of the fetus and newborn (HDFN) may occur when maternal IgG antibodies against red blood cells (RBCs), often anti-RhD (anti-D) antibodies, cross the placenta and mediate the destruction of RBCs via phagocytic IgG-Fc-receptors (FcγR). Clinical severity is not strictly related to titre and is more accurately predicted by the diagnostically-applied monocyte-based antibody-dependent cellular cytotoxicity (ADCC), a sensitive test with relatively low specificity. This suggests that other factors are involved in the pathogenesis of HDFN. Binding of IgG to FcγR requires the N-linked glycan at position 297 in the IgG-Fc-region, consisting of several different glycoforms. We therefore systematically analysed IgG-derived glycopeptides by mass spectrometry from 70 anti-D IgG1 antibodies purified from the plasma of alloimmunized pregnant women. This revealed a variable decrease in Fc-fucosylation in the majority of anti-D IgG1 (even down to 12%), whereas the total IgG of these patients remained highly fucosylated, like in healthy individuals (>90%). The degree of anti-D fucosylation correlated significantly with CD16 (FcγRIIIa)-mediated ADCC, in agreement with increased affinity of defucosylated IgG to human FcγRIIIa. Additionally, low anti-D fucosylation correlated significantly with low fetal-neonatal haemoglobin levels, thus with increased haemolysis, suggesting IgG-fucosylation to be an important pathological feature in HDFN with diagnostic potential.
Assuntos
Anticorpos Anti-Idiotípicos/metabolismo , Eritroblastose Fetal/etiologia , Isoanticorpos/metabolismo , Eritroblastose Fetal/imunologia , Feminino , Fucose/metabolismo , Humanos , Recém-Nascido , Espectrometria de Massas , Gravidez , Receptores Fc/imunologia , Receptores Fc/metabolismo , Imunoglobulina rho(D)/metabolismoRESUMO
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.