Glycoproteomic data are often very complex, reflecting the high structural diversity of peptide and glycan portions. The use of glycopeptide-centered glycoproteomics by mass spectrometry is rapidly... Show moreGlycoproteomic data are often very complex, reflecting the high structural diversity of peptide and glycan portions. The use of glycopeptide-centered glycoproteomics by mass spectrometry is rapidly evolving in many research areas, leading to a demand in reliable data analysis tools. In recent years, several bioinformatic tools were developed to facilitate and improve both the identification and quantification of glycopeptides. Here, a selection of these tools was combined and evaluated with the aim of establishing a robust glycopeptide detection and quantification workflow targeting enriched glycoproteins. For this purpose, a tryptic digest from affinity-purified immunoglobulins G and A was analyzed on a nano-reversed-phase liquid chromatography-tandem mass spectrometry platform with a high-resolution mass analyzer and higher-energy collisional dissociation fragmentation. Initial glycopeptide identification based on MS/MS data was aided by the Byonic software. Additional MS1-based glycopeptide identification relying on accurate mass and retention time differences using Glycopeptide-GraphMS considerably expanded the set of confidently annotated glycopeptides. For glycopeptide quantification, the performance of LaCyTools was compared to Skyline, and GlycopeptideGraphMS. All quantification packages resulted in comparable glycosylation profiles but featured differences in terms of robustness and data quality control. Partial cysteine oxidation was identified as an unexpectedly abundant peptide modification and impaired the automated processing of several IgA glycopeptides. Finally, this study presents a semiautomated workflow for reliable glyco-proteomic data analysis by the combination of software packages for MS/MS- and MS1-based glycopeptide identification as well as the integration of analyte quality control and quantification. Show less
Westhrin, M.; Kovcic, V.; Zhang, Z.J.; Moen, S.H.; Nedal, T.M.V.; Bondt, A.; ... ; Standal, T. 2020
Most patients with multiple myeloma develop a severe osteolytic bone disease. The myeloma cells secrete immunoglobulins, and the presence of monoclonal immunoglobulins in the patient's sera is an... Show moreMost patients with multiple myeloma develop a severe osteolytic bone disease. The myeloma cells secrete immunoglobulins, and the presence of monoclonal immunoglobulins in the patient's sera is an important diagnostic criterion. Here, we show that immunoglobulins isolated from myeloma patients with bone disease promote osteoclast differentiation when added to human preosteoclasts in vitro, whereas immunoglobulins from patients without bone disease do not. This effect was primarily mediated by immune complexes or aggregates. The function and aggregation behavior of immunoglobulins are partly determined by differential glycosylation of the immunoglobulin-Fc part. Glycosylation analyses revealed that patients with bone disease had significantly less galactose on immunoglobulin G (IgG) compared with patients without bone disease and also less sialic acid on IgG compared with healthy persons. Importantly, we also observed a significant reduction of IgG sialylation in serum of patients upon onset of bone disease. In the 5TGM1 mouse myeloma model, we found decreased numbers of lesions and decreased CTX-1 levels, a marker for osteoclast activity, in mice treated with a sialic acid precursor, N-acetylmannosamine (ManNAc). ManNAc treatment increased IgG-Fc sialylation in the mice. Our data support that deglycosylated immunoglobulins promote bone loss in multiple myeloma and that altering IgG glycosylation may be a therapeutic strategy to reduce bone loss. Show less
HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options... Show moreHLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity. Show less
Houvast, R.D.; Vankemmelbeke, M.; Durrant, L.G.; Wuhrer, M.; Baart, V.M.; Kuppen, P.J.K.; ... ; Sier, C.E.M. 2020
Simple SummaryDistinguishing malignancy from healthy tissue is essential for oncologic surgery. Targeted imaging during an operation aids the surgeon to operate better. The present tracers for... Show moreSimple SummaryDistinguishing malignancy from healthy tissue is essential for oncologic surgery. Targeted imaging during an operation aids the surgeon to operate better. The present tracers for detecting cancer are directed against proteins that are overexpressed on the membrane of tumor cells. This review evaluates the use of tumor-associated sugar molecules as an alternative for proteins to image cancer tissue. These sugar molecules are present as glycans on glycosylated membrane proteins and glycolipids. Due to their location and large numbers per cell, these sugar molecules might be better targets for tumor imaging than proteins.Real-time tumor imaging techniques are increasingly used in oncological surgery, but still need to be supplemented with novel targeted tracers, providing specific tumor tissue detection based on intra-tumoral processes or protein expression. To maximize tumor/non-tumor contrast, targets should be highly and homogenously expressed on tumor tissue only, preferably from the earliest developmental stage onward. Unfortunately, most evaluated tumor-associated proteins appear not to meet all of these criteria. Thus, the quest for ideal targets continues. Aberrant glycosylation of proteins and lipids is a fundamental hallmark of almost all cancer types and contributes to tumor progression. Additionally, overexpression of glycoproteins that carry aberrant glycans, such as mucins and proteoglycans, is observed. Selected tumor-associated glyco-antigens are abundantly expressed and could, thus, be ideal candidates for targeted tumor imaging. Nevertheless, glycan-based tumor imaging is still in its infancy. In this review, we highlight the potential of glycans, and heavily glycosylated proteoglycans and mucins as targets for multimodal tumor imaging by discussing the preclinical and clinical accomplishments within this field. Additionally, we describe the major advantages and limitations of targeting glycans compared to cancer-associated proteins. Lastly, by providing a brief overview of the most attractive tumor-associated glycans and glycosylated proteins in association with their respective tumor types, we set out the way for implementing glycan-based imaging in a clinical practice. Show less
Over the past decades, the genome and proteome have been widely explored for biomarker discovery and personalized medicine. However, there is still a large need for improved diagnostics and... Show moreOver the past decades, the genome and proteome have been widely explored for biomarker discovery and personalized medicine. However, there is still a large need for improved diagnostics and stratification strategies for a wide range of diseases. Post-translational modification of proteins by glycosylation affects protein structure and function, and glycosylation has been implicated in many prevalent human diseases. Numerous proteins for which the plasma levels are nowadays evaluated in clinical practice are glycoproteins. While the glycosylation of these proteins often changes with disease, their glycosylation status is largely ignored in the clinical setting. Hence, the implementation of glycomic markers in the clinic is still in its infancy. This is for a large part caused by the high complexity of protein glycosylation itself and of the analytical techniques required for their robust quantification. Mass spectrometry-based workflows are particularly suitable for the quantification of glycans and glycoproteins, but still require advances for their transformation from a biomedical research setting to a clinical laboratory. In this review, we describe why and how glycomics is expected to find its role in clinical tests and the status of current mass spectrometry-based methods for clinical glycomics. (C) 2020 The Authors. Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL). Show less
Gstottner, C.; Nicolardi, S.; Haberger, M.; Reusch, D.; Wuhrer, M.; Dominguez Vega, E. 2020
Bispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of... Show moreBispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of the primary sequence of BsAbs guides the proper pairing of the different chains, several side products can often be observed contributing to the macroheterogeneity of these products. Furthermore, changes in the amino acid sequence can result in different protein modifications which can affect the properties of the antibody and further increase the structural complexity. A multi-methods approach can be used for the characterization of their heterogeneity but new analytical strategies are needed for a more accurate and in-depth analysis.Here, we present a combination of intact antibody and subunit-specific mass measurements using sheathless capillary electrophoresis-mass spectrometry for assessing the macro- and microheterogeneity of BsAbs. Two homologous BsAbs with the same bispecificity but slightly different amino acid sequences were analyzed. Intact measurements were performed using a positively coated capillary and a background electrolyte (BGE) consisting of 3% acetic acid. For intact BsAbs, the separation permitted the characterization of free light chains, homo- and heterodimers as well as incomplete assemblies. For subunit-specific measurements, BsAbs were hinge region cleaved using two different enzymes (SpeB and IdeS) followed by disulfide-bond reduction. The six different subunits (Lc1, Lc2, Fd'1, Fd'2, (Fc/2)1 and (Fc/2)2) were separated using the same positively-coated capillary and a BGE consisting of 20% acetic acid and 10% methanol. Mass measurements of hinge region cleaved antibodies were performed at isotopic resolution (resolving power 140000 at m/z 1100) for a more confident analysis of low abundance proteoforms. For both BsAbs several proteoforms with e.g. pyroglutamic acid (Pyro-Glu) or glycation which could not be properly assigned at the intact level, were accurately determined in the subunits showing the complementarity of both approaches. (C) 2020 Elsevier B.V. All rights reserved. Show less
Fc gamma receptors (Fc gamma Rs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel... Show moreFc gamma receptors (Fc gamma Rs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and Fc gamma R impacts their interaction dramatically. Regarding Fc gamma R glycosylation profiling, major analytical challenges are associated with the presence of multiple glycosylation sites in close proximity and large structural heterogeneity. To address these challenges, we developed a straightforward and comprehensive analytical methodology to map Fc gamma RIIIb glycosylation in primary human cells. After neutrophil isolation and immunoprecipitation, glycopeptides containing a single site each were generated by a dual-protease in-gel digestion. The complex mixture was resolved by liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing information on the level of individual donors. In contrast to recently published alternatives for Fc gamma RIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying Fc gamma RIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous Fc gamma RIIIa on natural killer cells. This method will allow assessment of differences in Fc gamma RIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions. Show less
Immunoglobulin G (IgG) glycosylation is a key post-translational modification in regulating IgG function. It is therefore a prominent target for biomarker discovery and a critical quality attribute... Show moreImmunoglobulin G (IgG) glycosylation is a key post-translational modification in regulating IgG function. It is therefore a prominent target for biomarker discovery and a critical quality attribute of antibody-based biopharmaceuticals. A common approach for IgG glycosylation analysis is the measurement of tryptic glycopeptides. Glycosylation stability during sample processing is a key prerequisite for an accurate and robust analysis yet has hitherto hardly been studied. Especially, acid hydrolysis of sialic acids may be a source for instability. Therefore, we investigated acid denaturation, centrifugal vacuum concentration, and glycopeptide storage regarding changes in the IgG glycosylation profile. Intravenous IgG was analyzed employing imaginable deviations from a reference method and stress conditions. All glycosylation features-sialylation, galactosylation, bisection, and fucosylation-remained unchanged for most conditions. Only with prolonged exposure to acidic conditions at 37 degrees C, sialylation decreased significantly and subtle changes occurred for galactosylation. Consequently, provided that long or intense heating in acidic solutions is avoided, sample preparation for bottom-up glycoproteomics does not introduce conceivable biases. Show less
Approximately 20% of patients receiving multiple platelet transfusions develop platelet alloantibodies, which can be directed against human leukocyte antigens (HLA) and, to a lesser extent, against... Show moreApproximately 20% of patients receiving multiple platelet transfusions develop platelet alloantibodies, which can be directed against human leukocyte antigens (HLA) and, to a lesser extent, against human platelet antigens (HPA). These anti-bodies can lead to the rapid clearance of donor platelets, presumably through IgG-Fc receptor (Fc gamma R)-mediated phagocytosis or via complement activation, resulting in platelet refractoriness. Strikingly, not all patients with anti-HLA or-HPA antibodies develop platelet refractoriness upon unmatched platelet transfusions. Previously, we found that IgG Fc glycosylation of anti-HLA antibodies was highly variable between patients with platelet refractoriness, especially with respect to galactosylation and sialylation of the Fc-bound sugar moiety. Here, we produced recombinant glycoengineered anti-HLA and anti-HPA-1a monoclonal antibodies with varying Fc galactosylation and sialylation levels and studied their ability to activate the classical complement pathway. We observed that anti-HLA monoclonal antibodies with different specificities, binding simultaneously to the same HLA-molecules, or anti-HLA in combination with anti-HPA-1a monoclonal antibodies interacted synergistically with C1q, the first component of the classical pathway. Elevated Fc galactosylation and, to a lesser extent, sialylation significantly increased the complement-activating properties of anti-HLA and anti-HPA-1a monoclonal antibodies. We propose that both the breadth of the polyclonal immune response, with recognition of different HLA epitopes and in some cases HPA antigens, and the type of Fc glycosylation can provide an optimal stoichiometry for C1q binding and sub-sequent complement activation. These factors can shift the effect of a platelet alloimmune response to a clinically relevant response, leading to complement-mediated clearance of donor platelets, as observed in platelet refractoriness. Show less
Ederveen, A.L.H.; Haan, N. de; Baerenfaenger, M.; Lefeber, D.J.; Wuhrer, M. 2020
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... Show moreProtein 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 alpha 2,3-sialylation than for alpha 2,6-sialylation. The diverse abnormalities in glycomic features described in this study reflect the broad range of biological mechanisms that influence protein glycosylation. Show less
Gstottner, C.; Vergoossen, D.L.E.; Wuhrer, M.; Huijbers, M.G.M.; Dominguez Vega, E. 2020
Bispecific monoclonal antibodies (BsAbs) are receiving great attention due to their extensive benefits as biopharmaceuticals and their involvement in IgG4 mediated autoimmune diseases. While the... Show moreBispecific monoclonal antibodies (BsAbs) are receiving great attention due to their extensive benefits as biopharmaceuticals and their involvement in IgG4 mediated autoimmune diseases. While the production of BsAbs is getting more accessible, their analytical characterization remains challenging. We explored the potential of sheathless CE-MS for monitoring exchange efficiency and stability of in-house produced bispecific antibodies. Two IgG4 bispecific antibodies with different molecular characteristics were prepared using controlled Fragment antigen binding (Fab)-arm exchange. Separation of BsAbs from their parent monospecific antibodies was achieved using a polyethyleniimine (PEI)-coated capillary and acidic background electrolytes permitting reliable assessment of the exchange efficiency. This was especially valuable for a Fab-glycosylated BsAb where the high glycan heterogeneity resulted in an overlap of masses with the monospecific parent antibody, hindering their discrimination by MS only. The method showed also good capabilities to monitor the stability of the generated BsAbs under different storage conditions. The levels of degradation products were different for the studied antibodies indicating pronounced differences in stability. Overall, the proposed method represents a useful analytical tool for exchange efficiency and stability studies of bispecific antibodies. Show less
Nicolardi, S.; Kilgour, D.P.A.; Burgt, Y.E.M. van der; Wuhrer, M. 2020
The development of various ionization and fragmentation techniques has been of key importance for establishing mass spectrometry (MS) as a powerful tool for protein characterization. One example of... Show moreThe development of various ionization and fragmentation techniques has been of key importance for establishing mass spectrometry (MS) as a powerful tool for protein characterization. One example of this is matrix-assisted laser desorption/ionization (MALDI) combined with in-source decay (ISD) fragmentation that allows mapping of N- and C-terminal regions of large proteins without the need for proteolysis. Positive ion mode ISD fragments are commonly assigned in the mass region above m/z 1000, while MALDI matrix ions generally hamper the detection of smaller singly charged fragments. The ultrahigh resolving power provided by Fourier transform ion cyclotron resonance (FT-ICR) MS partially overcomes this limitation, but to further increase the detection of smaller fragments we have revisited the application of negative ion mode MALDI-ISD and found good coverage of the peptide chain termini starting from c'2 and z'2 fragment ions. For the first time, we demonstrate that the combination of negative and positive ion MALDI FT-ICR MS is a useful tool to improve the characterization of mAbs. The different specificities of the two ion modes allowed us to selectively cover the sequence of the light and heavy chains of mAbs at increased sensitivity. A comprehensive evaluation of positive and negative ion mode MALDI-ISD FT-ICR MS in the m/z range 46-13 500 showed an increased sequence coverage for three standard proteins, namely, myoglobin, SiLuLite mAb, and NIST mAb. The data obtained in the two ion modes were, in part, complementary. Show less
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... Show moreBackground &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 proteinN-glycan profiles from PDAC patients with regard to their applicability to serve as a disease biomarker panel. Methods Total serumN-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. ProteinN-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 x 10(-11)) and (antenna)fucosylation (OR 13.27,P-value 2.31 x 10(-9)) ofN-glycans was found. Furthermore, the ratio of alpha 2,6- vs alpha 2,3-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 SerumN-glycome analysis revealed glycosylation differences that allow classification of PDAC patients from healthy controls. It was demonstrated that glycosylation traits rather than singleN-glycan structures obtained in this clinical glycomics study can serve as a basis for further development of a blood-based diagnostic test. Show less
Background: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of... Show moreBackground: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of inflammatory (auto)immune disorders and have also been linked to protection against viral infection and discussed in the context of vaccine-induced protection. In contrast, sialylated IgG Abs have shown immunosuppressive effects.Objective: We sought to investigate IgG glycosylation programming during the germinal center (GC) reaction following immunization of mice with a foreign protein antigen and different adjuvants.Methods: Mice were analyzed for GC T-cell, B-cell, and plasma cell responses, as well as for antigen-specific serum IgG subclass titers and Fc glycosylation patterns.Results: Different adjuvants induce distinct IgG(+) GC B-cell responses with specific transcriptomes and expression levels of the alpha 2,6-sialyltransferase responsible for IgG sialylation that correspond to distinct serum IgG Fc glycosylation patterns. Low IgG Fc sialylation programming in GC B cells was overall highly dependent on the Foxp3(-) follicular helper T (TFH) cell-inducing cytokine IL-6, here in particular induced by water-inoil adjuvants and Mycobacterium tuberculosis. Furthermore, low IgG Fc sialylation programming was dependent on adjuvants that induced IL-27 receptor-dependent IFN-gamma(+) TFH1 cells, IL-6/IL-23-dependent IL-17A(+) T-FH17 cells, and high ratios of TFH cells to Foxp31 follicular regulatory T cells. Here, the 2 latter were dependent on M tuberculosis and its cord factor.Conclusion: This study's findings regarding adjuvant-dependent GC responses and IgG glycosylation programming may aid in the development of novel vaccination strategies to induce IgG Abs with both high affinity and defined Fc glycosylation patterns in the GC. Show less
Laan, E.A.N.Z. van der; Velden, S. van der; Bentlage, A.E.H.; Larsen, M.D.; Osch, T.L.J. van; Mok, J.Y.; ... ; Kapur, R. 2020
Transfusion-related acute lung injury (TRALI) remains a leading cause of transfusion-related deaths. In most cases, anti-leukocyte antibodies in the transfusion product trigger TRALI, but not all... Show moreTransfusion-related acute lung injury (TRALI) remains a leading cause of transfusion-related deaths. In most cases, anti-leukocyte antibodies in the transfusion product trigger TRALI, but not all anti-leukocyte antibodies cause TRALI. It has been shown that the anti-major histocompatibility complex (MHC) class I antibody 34-1-2S (anti-H-2K(d)) causes TRALI in BALB/c mice (MHC class I haplotype H-2K(d)), whereas SF1.1.10 (anti-H-2K(d)) does not. In C57BL/6 mice (MHC class I haplotype H-2K(b)), TRALI only occurs when anti-MHC class I antibody AF6-88.5.5.3 (anti-H-2K(b)) is administered together with a high dose of 34-1-2S. It remains unknown which specific antibody characteristics are responsible for eliciting TRALI. We therefore investigated several biological and structural features of 34-1-2S compared with other anti-MHC class I antibodies, which on their own do not cause TRALI: SF1.1.10 and AF6-88.5.5.3. No substantial differences were observed between the TRALI-causing 34-1-2S and the TRALI-resistant SF1.1.10 regarding binding affinity to H-2K(d). Regarding binding affinity to H-2K(b), only AF6-88.5.5.3 potently bound to H-2K(b), whereas 34-1-2S exhibited weak but significant cross-reactivity. Furthermore, the binding affinity to Fc gamma Rs as well as the Fc glycan composition seemed to be similar for all antibodies. Similar Fc glycosylation profiles were also observed for human TRALI-causing donor anti-HLA antibodies compared with human anti-HLA antibodies from control donors. 34-1-2S, however, displayed superior complement activation capacity, which was fully Fc dependent and not significantly dependent on Fc glycosylation. We conclude that TRALI induction is not correlated with Fab- and Fc-binding affinities for antigen and Fc gamma Rs, respectively, nor with the composition of Fc glycans; but increased Fc-mediated complement activation is correlated with TRALI induction. Show less
Current approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they... Show moreCurrent approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they also result in a loss of valuable information regarding certain subclasses, allotypes and co-occuring post-translational modifications (PTMs). Unfortunately, the high variability of polyclonal IgGs makes their intact mass spectrometry (MS) analysis extremely challenging. We propose here a middle-up strategy for the analysis of the intact fragment crystallizable (Fc) region of human plasma IgGs, with the aim of acquiring integrated information of theN-glycosylation and other PTMs of subclasses and allotypes. Human plasma IgG was isolated using Fc-specific beads followed by an on-bead C(H)2 domain digestion with the enzyme IdeS. The obtained mixture of Fc subunits was analyzed by capillary electrophoresis (CE) and hydrophilic interaction liquid chromatography (HILIC) hyphenated with MS. CE-MS provided separation of different IgG-subclasses and allotypes, while HILIC-MS allowed resolution of the different glycoforms and their oxidized variants. The orthogonality of these techniques was key to reliably assign Fc allotypes. Five individual donors were analyzed using this approach. Heterozygosis was observed in all the analyzed donors resulting in a total of 12 allotypes identified. The assignments were further confirmed using recombinant monoclonal IgG allotypes as standards. While the glycosylation patterns were similar within allotypes of the same subclass, clear differences were observed between IgG subclasses and donors, highlighting the relevance of the proposed approach. In a single analysis, glycosylation levels specific for each allotype, relative abundances of subclasses and information on co-occurring modifications are obtained. This middle-up method represents an important step toward a comprehensive analysis of immunoglobulin G-Fc variants. Show less
The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin... Show moreThe thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states. Epithelial cells that line the gut secrete complex glycoproteins that form a mucus layer to protect the gut wall from enteric pathogens. Here, the authors provide a comprehensive characterisation of endo-acting glycoside hydrolases expressed by mucin-degrading members of the microbiome that are able to cleave the O-glycan chains of a range of different animal and human mucins. Show less
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... Show moreSeizure 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 SEZ6in vitroandin vivoprevented 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. Show less
Colorectal cancer (CRC) is the second-leading cause of cancer death worldwide due in part to a high proportion of patients diagnosed at advanced stages of the disease. For this reason, many efforts... Show moreColorectal cancer (CRC) is the second-leading cause of cancer death worldwide due in part to a high proportion of patients diagnosed at advanced stages of the disease. For this reason, many efforts have been made towards new approaches for early detection and prognosis. Cancer-associated aberrant glycosylation, especially the Tn and STn antigens, can be detected using the macrophage galactose-type C-type lectin (MGL/CLEC10A/CD301), which has been shown to be a promising tool for CRC prognosis. We had recently identified the major MGL-binding glycoproteins in two high-MGL-binding CRC cells lines, HCT116 and HT29. However, we failed to detect the presence ofO-linked Tn and STn glycans on most CRC glycoproteins recognized by MGL. We therefore investigated here the impact ofN-linked andO-linked glycans carried by these proteins for the binding to MGL. In addition, we performed quantitative proteomics to study the major differences in proteins involved in glycosylation in these cells. Our results showed thatN-glycans have a significant, previously underestimated, importance in MGL binding to CRC cell lines. Finally, we highlighted both common and cell-specific processes associated with a high-MGL-binding phenotype, such as differential levels of enzymes involved in protein glycosylation, and a transcriptional factor (CDX-2) involved in their regulation. Show less
