Comprehensive characterization of bacterial glycoconjugate vaccines by liquid chromatography - mass spectrometry.

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.

Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level.

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.

Targeting aberrant sialylation and fucosylation in prostate cancer cells using potent metabolic inhibitors.

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.

Immunoglobulin A Glycosylation Differs between Crohn's Disease and Ulcerative Colitis.

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.

-Glycosylation of LRP6 by B3GnT2 Promotes Wnt/β-Catenin Signalling.

Reception of Wnt signals by cells is predominantly mediated by Frizzled receptors in conjunction with a co-receptor, the latter being LRP6 or LRP5 for the Wnt/β-catenin signalling pathway. It is important that cells maintain precise control of receptor activation events in order to properly regulate Wnt/β-catenin signalling as aberrant signalling can result in disease in humans. Phosphorylation of the intracellular domain (ICD) of LRP6 is well known to regulate Wntβ-catenin signalling; however, less is known for regulatory post-translational modification events within the extracellular domain (ECD).

- and -Glycosylation of Serum Immunoglobulin A is Associated with IgA Nephropathy and Glomerular Function.

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 -glycans as post-translational modification, only IgA1 features extensive hinge-region -glycosylation.

Dissecting Total Plasma and Protein-Specific Glycosylation Profiles in Congenital Disorders of Glycosylation.

Protein -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 -glycan biosynthesis. Here, we present insights into the disrupted -glycosylation of various CDG patients exhibiting defects in the transport of nucleotide sugars, Golgi glycosylation or Golgi trafficking.

Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3.

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.

Natural killer cell activation by respiratory syncytial virus-specific antibodies is decreased in infants with severe respiratory infections and correlates with Fc-glycosylation.

Objectives: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, and there is no vaccine available. In early life, the most important contributors to protection against infectious diseases are the innate immune response and maternal antibodies. However, antibody-mediated protection against RSV disease is incompletely understood, as both antibody levels and neutralisation capacity correlate poorly with protection.

Simultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications.

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.

The Glycosylation Site of Myelin Oligodendrocyte Glycoprotein Affects Autoantibody Recognition in a Large Proportion of Patients.

Autoantibodies to myelin oligodendrocytes glycoprotein (MOG) are found in a fraction of patients with inflammatory demyelination and are detected with MOG-transfected cells. While the prototype anti-MOG mAb 8-18C5 and polyclonal anti-MOG responses from different mouse strains largely recognize the FG loop of MOG, the human anti-MOG response is more heterogeneous and human MOG-Abs recognizing different epitopes were found to be pathogenic. The aim of this study was to get further insight into details of antigen-recognition by human MOG-Abs focusing on the impact of glycosylation.

α1-Antichymotrypsin Present in Therapeutic C1-Inhibitor Products Competes with Selectin-Sialyl LewisX Interaction.

Background:  C1-inhibitor (C1-inh) therapeutics can reduce neutrophil activity in various inflammatory conditions. This 'novel' anti-inflammatory effect of C1-inh is attributed to the tetrasaccharide sialyl Lewis (SLe) present on its glycans. Via SLe, C1-inh is suggested to interact with selectins on inflamed endothelium and prevent neutrophil rolling.

Plasma protein N-glycan signatures of type 2 diabetes.

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).

Biophysical analysis of sialic acid recognition by the complement regulator Factor H.

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.

Cutis laxa, exocrine pancreatic insufficiency and altered cellular metabolomics as additional symptoms in a new patient with ATP6AP1-CDG.

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.

The Glycosylation of Mouse Immunoglobulin G (IgG)-Fragment Crystallizable Differs Between IgG Subclasses and Strains.

-linked glycosylation of the fragment crystallizable (Fc)-region of immunoglobulin G (IgG) is known to have a large influence on the activity of the antibody, an effect reported to be IgG subclass specific. This situation applies both to humans and mice. The mouse is often used as experimental animal model to study the effects of Fc-glycosylation on IgG effector functions, and results are not uncommonly translated back to the human situation.

Antigen specificity determines anti-red blood cell IgG-Fc alloantibody glycosylation and thereby severity of haemolytic disease of the fetus and newborn.

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.

Murine Plasma N-Glycosylation Traits Associated with Sex and Strain.

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.

Structural snapshots of the catalytic cycle of the phosphodiesterase Autotaxin.

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.

LaCyTools: A Targeted Liquid Chromatography-Mass Spectrometry Data Processing Package for Relative Quantitation of Glycopeptides.

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.

Acute phase inflammation is characterized by rapid changes in plasma/peritoneal fluid N-glycosylation in mice.

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.

Ethyl Esterification for MALDI-MS Analysis of Protein Glycosylation.

Ethyl esterification is a technique for the chemical modification of sialylated glycans, leading to enhanced stability when performing matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS), as well as allowing the efficient detection of both sialylated and non-sialylated glycans in positive ion mode. In addition, the method shows specific reaction products for α2,3- and α2,6-linked sialic acids, leading to an MS distinguishable mass difference. Here, we describe the ethyl esterification protocol for 96 glycan samples, including enzymatic N-glycan release, the aforementioned ethyl esterification, glycan enrichment, MALDI target preparation, and the MS(/MS) measurement.