Genotype-dependent N-glycosylation and newly exposed O-glycosylation affect plasmin-induced cleavage of histidine-rich glycoprotein (HRG).

Histidine-rich glycoprotein (HRG) is an abundant plasma protein harboring at least three N-glycosylation sites. HRG integrates many biological processes, such as coagulation, antiangiogenic activity, and pathogen clearance. Importantly, HRG is known to exhibit five genetic variants with minor allele frequencies of more than 10%. Among them, Pro204Ser can induce a fourth N-glycosylation site (Asn202). Considerable efforts have been made to reveal the biological function of HRG, whereas data on HRG glycosylation are scarcer. To close this knowledge gap, we used C18-based LC-MS/MS to study the glycosylation characteristics of six HRG samples from different sources. We used endogenous HRG purified from human plasma and compared its glycosylation to that of the recombinant HRG produced in Chinese hamster ovary cells or human embryonic kidney 293 cells, targeting distinct genotypic isoforms. In endogenous plasma HRG, every N-glycosylation site was occupied predominantly with a sialylated diantennary complex-type glycan. In contrast, in the recombinant HRGs, all glycans showed different antennarities, sialylation, and core fucosylation, as well as the presence of oligomannose glycans, LacdiNAcs, and antennary fucosylation. Furthermore, we observed two previously unreported O-glycosylation sites in HRG on residues Thr273 and Thr274. These sites together showed more than 90% glycan occupancy in all HRG samples studied. To investigate the potential relevance of HRG glycosylation, we assessed the plasmin-induced cleavage of HRG under various conditions. These analyses revealed that the sialylation of the N- and O-glycans as well as the genotype-dependent N-glycosylation significantly influenced the kinetics and specificity of plasmin-induced cleavage of HRG.

Complex N-glycans are important for interspecies transmission of H7 influenza A viruses.

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.

Contributing roles of mitochondrial dysfunction and hepatocyte apoptosis in liver diseases through oxidative stress, post-translational modifications, inflammation, and intestinal barrier dysfunction.

This review provides an update on recent findings from basic, translational, and clinical studies on the molecular mechanisms of mitochondrial dysfunction and apoptosis of hepatocytes in multiple liver diseases, including but not limited to alcohol-associated liver disease (ALD), metabolic dysfunction-associated steatotic liver disease (MASLD), and drug-induced liver injury (DILI). While the ethanol-inducible cytochrome P450-2E1 (CYP2E1) is mainly responsible for oxidizing binge alcohol via the microsomal ethanol oxidizing system, it is also responsible for metabolizing many xenobiotics, including pollutants, chemicals, drugs, and specific diets abundant in n-6 fatty acids, into toxic metabolites in many organs, including the liver, causing pathological insults through organelles such as mitochondria and endoplasmic reticula. Oxidative imbalances (oxidative stress) in mitochondria promote the covalent modifications of lipids, proteins, and nucleic acids through enzymatic and non-enzymatic mechanisms. Excessive changes stimulate various post-translational modifications (PTMs) of mitochondrial proteins, transcription factors, and histones. Increased PTMs of mitochondrial proteins inactivate many enzymes involved in the reduction of oxidative species, fatty acid metabolism, and mitophagy pathways, leading to mitochondrial dysfunction, energy depletion, and apoptosis. Unique from other organelles, mitochondria control many signaling cascades involved in bioenergetics (fat metabolism), inflammation, and apoptosis/necrosis of hepatocytes. When mitochondrial homeostasis is shifted, these pathways become altered or shut down, likely contributing to the death of hepatocytes with activation of inflammation and hepatic stellate cells, causing liver fibrosis and cirrhosis. This review will encapsulate how mitochondrial dysfunction contributes to hepatocyte apoptosis in several types of liver diseases in order to provide recommendations for targeted therapeutics.

Oxonium Ion-Guided Optimization of Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro.

Spatial separation of ions in the gas phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro (Bruker Daltonics) series combines a trapped ion mobility device with a quadrupole, collision cell, and a time-of-flight analyzer to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from nonmodified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared with nonmodified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and nonmodified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. We designed an approach where we (1) focused on a region of interest in the ion mobilogram and (2) applied stepped collision energies to obtain informative glycopeptide tandem mass spectra on the timsTOF Pro:glyco-polygon-stepped collision energy-parallel accumulation serial fragmentation. This method was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical separation in the region of interest allows for improved extraction of information from the samples, even at shorter liquid chromatography gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known makeup, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.

The pleiotropic phenotype of FlbA of Aspergillus niger is explained in part by the activity of seven of its downstream-regulated transcription factors.

Inactivation of flbA in Aspergillus niger results in thinner cell walls, increased cell lysis, abolished sporulation, and an increased secretome complexity. A total of 36 transcription factor (TF) genes are differentially expressed in ΔflbA. Here, seven of these genes (abaA, aslA, aslB, azf1, htfA, nosA, and srbA) were inactivated. Inactivation of each of these genes affected sporulation and, with the exception of abaA, cell wall integrity and protein secretion. The impact on secretion was strongest in the case of ΔaslA and ΔaslB that showed increased pepsin, cellulase, and amylase activity. Biomass was reduced of agar cultures of ΔabaA, ΔaslA, ΔnosA, and ΔsrbA, while biomass was higher in liquid shaken cultures of ΔaslA and ΔaslB. The ΔaslA and ΔhtfA strains showed increased resistance to H2O2, while ΔaslB was more sensitive to this reactive oxygen species. Together, inactivation of the seven TF genes impacted biomass formation, sporulation, protein secretion, and stress resistance, and thereby these genes explain at least part of the pleiotropic phenotype of ΔflbA of A. niger.

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. 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.

Key changes in bovine milk immunoglobulin G during lactation: NeuAc sialylation is a hallmark of colostrum immunoglobulin G N-glycosylation.

We monitored longitudinal changes in bovine milk IgG in samples from four cows at 9 time points in between 0.5 and 28 days following calving. We used peptide-centric LC-MS/MS on proteolytic digests of whole bovine milk, resulting in the combined identification of 212 individual bovine milk protein sequences, with IgG making up >50 percent of the protein content of every 0.5 d colostrum sample, which reduced to ≤3 percent in mature milk. In parallel, we analyzed IgG captured from the bovine milk samples to characterize its N-glycosylation, using dedicated methods for bottom-up glycoproteomics employing product ion-triggered hybrid fragmentation; data are available via ProteomeXchange with identifier PXD037755. The bovine milk IgG N-glycosylation profile was revealed to be very heterogeneous, consisting of >40 glycoforms. Furthermore, these N-glycosylation profiles changed substantially over the period of lactation, but consistently across the four individual cows. We identified NeuAc sialylation as the key abundant characteristic of bovine colostrum IgG, significantly decreasing in the first days of lactation, and barely detectable in mature bovine milk IgG. We also report, for the first time to our knowledge, the identification of subtype IgG3 in bovine milk, alongside the better-documented IgG1 and IgG2. The detailed molecular characteristics we describe of the bovine milk IgG, and their dynamic changes during lactation, are important not only for the fundamental understanding of the calf's immune development, but also for understanding bovine milk and its bioactive components in the context of human nutrition.

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection.

Recent human H3N2 influenza A viruses have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells, which are commonly employed to propagate influenza A virus, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases ß-1,3-N-acetylglucosaminyltransferase and ß-1,4-galactosyltransferase 1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAcs), would result in improved A/H3N2 propagation. Stable overexpression of ß-1,3-N-acetylglucosaminyltransferase and ß-1,4-galactosyltransferase 1 in Madin-Darby Canine Kidney and "humanized" Madin-Darby Canine Kidney cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the ß-1,3-N-acetylglucosaminyltransferase and/or ß-1,4-galactosyltransferase 1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on "humanized" Madin-Darby Canine Kidney-ß-1,3-N-acetylglucosaminyltransferase cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 influenza A viruses require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.

Glycoproteomics-Compatible MS/MS-Based Quantification of Glycopeptide Isomers.

Glycosylation is an essential protein modification occurring on the majority of extracellular human proteins, with mass spectrometry (MS) being an indispensable tool for its analysis, that not only determines glycan compositions, but also the position of the glycan at specific sites via glycoproteomics. However, glycans are complex branching structures with monosaccharides interconnected in a variety of biologically relevant linkages, isomeric properties that are invisible when the readout is mass alone. Here, we developed an LC-MS/MS-based workflow for determining glycopeptide isomer ratios. Making use of isomerically defined glyco(peptide) standards, we observed marked differences in fragmentation behavior between isomer pairs when subjected to collision energy gradients, specifically in terms of the galactosylation/sialylation branching and linkage. These behaviors were developed into component variables that allowed for relative quantification of isomerism within mixtures. Importantly, at least for small peptides, the isomer quantification appeared to be largely independent from the peptide portion of the conjugate, allowing a broad application of the method.

Human monoclonal antibodies inhibit invasion of transgenic Plasmodium knowlesi expressing Plasmodium vivax Duffy binding protein.

BACKGROUND: Plasmodium vivax has been more resistant to various control measures than Plasmodium falciparum malaria because of its greater transmissibility and ability to produce latent parasite forms. Therefore, developing P. vivax vaccines and therapeutic monoclonal antibodies (humAbs) remains a high priority. The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to P. vivax invasion of reticulocytes. P. vivax expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and the DARC: PvDBP interaction is critical for P. vivax blood stage malaria. Therefore, PvDBP is a leading vaccine candidate for P. vivax and a target for therapeutic human monoclonal antibodies (humAbs). METHODS: Here, the functional activity of humAbs derived from naturally exposed and vaccinated individuals are compared for the first time using easily cultured Plasmodium knowlesi (P. knowlesi) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. This model was used to evaluate the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs). RESULTS: The PvDBP-specific humAbs demonstrated 70-100% inhibition of PkPvDBPOR invasion with the IC50 values ranging from 51 to 338 µg/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 µg/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10 and 100 µg/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 µg/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. Invasion inhibition efficacy by some mAbs shown with PkPvDBPOR was closely replicated using P. vivax clinical isolates. CONCLUSION: The PkPvDBPOR transgenic model is a robust surrogate of P. vivax to assess invasion and growth inhibition of human monoclonal Abs recognizing PvDBP individually and in combination. There was no synergistic interaction for growth inhibition with the humAbs tested here that target different epitopes or subdomains of PvDBP, suggesting little benefit in clinical trials using combinations of these humAbs.

Meta-heterogeneity: Evaluating and Describing the Diversity in Glycosylation Between Sites on the Same Glycoprotein.

Mass spectrometry-based glycoproteomics has gone through some incredible developments over the last few years. Technological advances in glycopeptide enrichment, fragmentation methods, and data analysis workflows have enabled the transition of glycoproteomics from a niche application, mainly focused on the characterization of isolated glycoproteins, to a mature technology capable of profiling thousands of intact glycopeptides at once. In addition to numerous biological discoveries catalyzed by the technology, we are also observing an increase in studies focusing on global protein glycosylation and the relationship between multiple glycosylation sites on the same protein. It has become apparent that just describing protein glycosylation in terms of micro- and macro-heterogeneity, respectively, the variation and occupancy of glycans at a given site, is not sufficient to describe the observed interactions between sites. In this perspective we propose a new term, meta-heterogeneity, to describe a higher level of glycan regulation: the variation in glycosylation across multiple sites of a given protein. We provide literature examples of extensive meta-heterogeneity on relevant proteins such as antibodies, erythropoietin, myeloperoxidase, and a number of serum and plasma proteins. Furthermore, we postulate on the possible biological reasons and causes behind the intriguing meta-heterogeneity observed in glycoproteins.

Predicting accidental drug overdose as the cause of fatality in near real-time using the Suspected Potential Overdose Tracker (SPOT): public health implications.

BACKGROUND: Effective responses to the worsening drug overdose epidemic require accurate and timely drug overdose surveillance data. The objectives of this paper are to describe the development, functionality, and accuracy of the Suspected Potential Overdose Tracker (SPOT) for predicting accidental drug overdose as the cause and manner of death in near real-time, and public health implications of adopting the tool. METHODS: SPOT was developed to rapidly identify overdose deaths through a simple and duplicable process using data collected by death investigators. The tool assigns each death a ranking of 1 through 3 based on the likelihood of it being an unintentional drug overdose, with 1 representing the highest likelihood that the death will be confirmed as an unintentional drug overdose and 3 representing the lowest. We measured the accuracy of the tool for predicting overdose deaths by comparing potential overdose deaths in New York City from 2018-2020 that were identified using SPOT to finalized death certificates. We also calculated the proportion of death certificate-confirmed overdoses that were missed by the SPOT tool and the proportion of type 1 errors. RESULTS: SPOT captured up to 77% of unintentional drug overdose deaths using data collected within 72 h of fatality. The tool predicted unintentional drug overdose from 2018 to 2020 with 93-97% accuracy for cases assigned a ranking of 1, 87-91% accuracy for cases assigned a ranking of 2, and 62-73% accuracy for cases assigned a ranking of 3. Among all unintentional overdose deaths in 2018, 2019, and 2020, 21%, 28%, and 33% were missed by the SPOT tool, respectively. During this timeframe, the proportion of type 1 errors ranged from 15%-23%. CONCLUSIONS: SPOT may be used by health departments, epidemiologists, public health programs, and others to monitor overdose fatalities before death certificate data becomes available. Improved monitoring of overdose fatalities allows for rapid data-driven decision making, identification of gaps in public health and public safety overdose response, and evaluation and response to overdose prevention interventions, programs, and policies.

Detection of Bacterial α-l-Fucosidases with an Ortho-Quinone Methide-Based Probe and Mapping of the Probe-Protein Adducts.

Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria.

Plugging the Leak: Empowering Women in Organic Chemistry.

Held June 24-25, 2021, the third annual Empowering Women in Organic Chemistry (EWOC) conference gathered organic chemists at all stages of the career pipeline for rich professional development opportunities and a showcase of recent scientific achievements. This Meeting Review outlines the program.

Hepatitis C Virus Transmission Clusters in Public Health and Correctional Settings, Wisconsin, USA, 2016-20171.

Ending the hepatitis C virus (HCV) epidemic requires stopping transmission among networks of persons who inject drugs. Identifying transmission networks by using genomic epidemiology may inform community responses that can quickly interrupt transmission. We retrospectively identified HCV RNA-positive specimens corresponding to 459 persons in settings that use the state laboratory, including correctional facilities and syringe services programs, in Wisconsin, USA, during 2016-2017. We conducted next-generation sequencing of HCV and analyzed it for phylogenetic linkage by using the Centers for Disease Control and Prevention Global Hepatitis Outbreak Surveillance Technology platform. Analysis showed that 126 persons were linked across 42 clusters. Phylogenetic clustering was higher in rural communities and associated with female sex and younger age among rural residents. These data highlight that HCV transmission could be reduced by expanding molecular-based surveillance strategies to rural communities affected by the opioid crisis.

Potential Influences of the COVID-19 Pandemic on Drug Use and HIV Care Among People Living with HIV and Substance Use Disorders: Experience from a Pilot mHealth Intervention.

People living with HIV (PLWH) and substance use disorder (SUD) are particularly vulnerable to harmful health consequences of the global COVID-19 pandemic. The health and social consequences of the pandemic may exacerbate substance misuse and poor management of HIV among this population. This study compares substance use and HIV care before and during the pandemic using data collected weekly through an opioid relapse prevention and HIV management mobile-health intervention. We found that during the pandemic, PLWH and SUD have increased illicit substance use and contact with other substance-using individuals and decreased their confidence to stay sober and attend recovery meetings. The proportion of people missing their HIV medications also increased, and confidence to attend HIV follow-up appointments decreased. Optimal support for PLWH and SUD is critical during pandemics like COVID-19, as drug-related and HIV antiretroviral therapy (ART) non-adherence risks such as overdose, unsafe sexual behaviors, and transmission of infectious diseases may unfold.

RESUMEN: Personas con VIH y con trastornos por abuso de sustancias son más vulnerable a las consecuencias de la pandemia: COVID-19. Dentro estas poblaciones, las consecuencias sociales y de la salud, causadas por la pandemia, pueden exacerbar el mal uso de las sustancias, y la adherencia a los antiretrovirales. Este estudio compara el abuso de sustancias y el cuidado del VIH, antes y durante la pandemia, usando datos colectados semanal de otro programa que también investigo la prevención entre personas que han recaído con el uso de opioides y que tienen VIH. Nuestro análisis encuentra, que durante la pandemia, incrementaron el uso de sustancias ilícitas, y contacto con otras personas que usan sustancias, y perdieron la capacidad de mantenerse sobrios, y tambien dejaron de asistir reuniones de recuperación/apoyo. También, el porcentaje de personas con VIH no siguiendo con sus planes de tratamiento de VIH, incrementó; perdieron su motivacion en mantener sus citas médicos. Es muy crítico, durante una pandemia como COVID-19, tener recursos para personas que pertenecen a estas poblaciones, si no, casos de sobredosis, sexo sin protección y la transmisión de enfermedades infecciosas van a prevaler.

High-throughput Serum N-Glycomics: Method Comparison and Application to Study Rheumatoid Arthritis and Pregnancy-associated Changes.

N-Glycosylation is a fundamentally important protein modification with a major impact on glycoprotein characteristics such as serum half-life and receptor interaction. More than half of the proteins in human serum are glycosylated, and the relative abundances of protein glycoforms often reflect alterations in health and disease. Several analytical methods are currently capable of analyzing the total serum N-glycosylation in a high-throughput manner.Here we evaluate and compare the performance of three high-throughput released N-glycome analysis methods. Included were hydrophilic-interaction ultra-high-performance liquid chromatography with fluorescence detection (HILIC-UHPLC-FLD) with 2-aminobenzamide labeling of the glycans, multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) with 8-aminopyrene-1,3,6-trisulfonic acid labeling, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with linkage-specific sialic acid esterification. All methods assessed the same panel of serum samples, which were obtained at multiple time points during the pregnancies and postpartum periods of healthy women and patients with rheumatoid arthritis (RA). We compared the analytical methods on their technical performance as well as on their ability to describe serum protein N-glycosylation changes throughout pregnancy, with RA, and with RA disease activity.Overall, the methods proved to be similar in their detection and relative quantification of serum protein N-glycosylation. However, the non-MS methods showed superior repeatability over MALDI-TOF-MS and allowed the best structural separation of low-complexity N-glycans. MALDI-TOF-MS achieved the highest throughput and provided compositional information on higher-complexity N-glycans. Consequentially, MALDI-TOF-MS could establish the linkage-specific sialylation differences within pregnancy and RA, whereas HILIC-UHPLC-FLD and xCGE-LIF demonstrated differences in α1,3- and α1,6-branch galactosylation. While the combination of methods proved to be the most beneficial for the analysis of total serum protein N-glycosylation, informed method choices can be made for the glycosylation analysis of single proteins or samples of varying complexity.

Monitoring Human Milk ß-Casein Phosphorylation and O-Glycosylation Over Lactation Reveals Distinct Differences between the Proteome and Endogenous Peptidome.

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant's best start at a healthy life. One key component of human milk is ß-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk's endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of ß-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of ß-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in ß-casein's known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of ß-casein may be important as it resides on known ß-casein-derived antimicrobial peptide sequences.

Neutrophil myeloperoxidase harbors distinct site-specific peculiarities in its glycosylation.

Anti-neutrophil cytoplasmic autoantibodies (ANCAs) are directed against lysosomal components of neutrophils. ANCAs directed to proteinase 3 and myeloperoxidase (MPO) in particular are associated with distinct forms of small vessel vasculitides. MPO is an abundant neutrophil-derived heme protein that is part of the antimicrobial defense system. The protein is typically present in the azurophilic granules of neutrophils, but a large portion may also enter the extracellular space. It remains unclear why MPO is frequently the target of antibody-mediated autoimmune responses. MPO is a homodimeric glycoprotein, posttranslationally modified with complex sugars at specific sites. Glycosylation can strongly influence protein function, affecting its folding, receptor interaction, and backbone accessibility. MPO potentially can be heavily modified as it harbors 5 putative N-glycosylation sites (10 in the mature dimer). Although considered important for MPO structure and function, the full scope and relative abundance of the glycans attached to MPO is unknown. Here, combining bottom-up glycoproteomics and native MS approaches, we structurally characterized MPO from neutrophils of healthy human donors. We quantified the relative occupancy levels of the glycans at each of the five sites and observed complex heterogeneity and site-specific glycosylation. In particular, we detected glycosylation phenotypes uncommon for glycoproteins in the extracellular space, such as a high abundance of phosphorylated high-mannose species and severely truncated small glycans having the size of paucimannose or smaller. We hypothesize that the atypical glycosylation pattern found on MPO might contribute to its specific processing and presentation as a self-antigen by antigen-presenting cells.

Promoting Safe Injection Practices, Substance Use Reduction, Hepatitis C Testing, and Overdose Prevention Among Syringe Service Program Clients Using a Computer-Tailored Intervention: Pilot Randomized Controlled Trial.

BACKGROUND: Syringe service programs (SSPs) are safe, highly effective programs for promoting health among people who inject drugs. However, resource limitations prevent the delivery of a full package of prevention services to many clients in need. Computer-tailored interventions may represent a promising approach for providing prevention information to people who inject drugs in resource-constrained settings. OBJECTIVE: The aim of this paper is to assess the effect of a computer-tailored behavioral intervention, called Hep-Net, on safe injection practices, substance use reduction, overdose prevention, and hepatitis C virus (HCV) testing among SSP clients. METHODS: Using a social network-based recruitment strategy, we recruited clients of an established SSP in Wisconsin and peers from their social networks. Participants completed a computerized baseline survey and were then randomly assigned to receive the Hep-Net intervention. Components of the intervention included an overall risk synthesis, participants' selection of a behavioral goal, and an individualized risk reduction exercise. Individuals were followed up 3 months later to assess their behavior change. The effect of Hep-Net on receiving an HCV screening test, undergoing Narcan training, reducing the frequency of drug use, and sharing drug equipment was assessed. The individual's readiness to change each behavior was also examined. RESULTS: From 2014 to 2015, a total of 235 people who injected drugs enrolled into the Hep-Net study. Of these, 64.3% (151/235) completed the follow-up survey 3-6 months postenrollment. Compared with the control group, individuals who received the Hep-Net intervention were more likely to undergo HCV testing (odds ratio [OR] 2.23, 95% CI 1.05-4.74; P=.04) and receive Narcan training (OR 2.25, 95% CI 0.83-6.06; P=.11), and they shared drug equipment less frequently (OR 0.06, 95% CI 0.55-0.65; P<.001). Similarly, individuals who received the intervention were more likely to advance in their stage of readiness to change these 3 behaviors. However, intervention participants did not appear to reduce the frequency of drug use or increase their readiness to reduce drug use more than control participants, despite the fact that the majority of the intervention participants selected this as the primary goal to focus on after participation in the baseline survey. CONCLUSIONS: Implementing computer-based risk reduction interventions in SSPs may reduce harms associated with the sharing of injection equipment and prevent overdose deaths; however, brief computerized interventions may not be robust enough to overcome the challenges associated with reducing and ceasing drug use when implemented in settings centered on the delivery of prevention services. TRIAL REGISTRATION: ClinicalTrials.gov NCT02474043; https://clinicaltrials.gov/ct2/show/NCT02474043. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR1-10.2196/resprot.4830.