O-GalNAc (N-acetylgalactosaminyl) glycosylation is an abundant posttranslational modification in mammalian cells. Dysregulation of O-GalNAc glycosylation is implicated in cancer metastasis and immune evasion; however, our mechanistic understanding remains limited due to the lack of small-molecule tools. O-GalNAc biosynthesis depends heavily on the availability of UDP-GalNAc that is biosynthesised by the cytosolic enzyme UDP-galactose-4-epimerase (GalE). Knockout studies have demonstrated that loss of GalE severely impairs O-GalNAc glycosylation, positioning GalE as a promising enzymatic therapeutic target in oncology. Here, we present an efficient workflow that combines both covalent and high-throughput crystallographic non-covalent fragment screening with structure-based design to identify GalE inhibitors. Using these strategies, we discovered a ligandable pocket adjacent to a reactive tyrosine, enabling the development of a potent, "beyond cysteine" sulfonyl fluoride covalent inhibitor as well as a derived covalent alkyne probe. Structurally-enabled fragment screening methodologies yielded nanomolar non-covalent as well as covalent binders within no more than 22 elaborated compounds. Our work demonstrates synergism in next-generation delivery of chemical matter for GalE inhibition, with the broader potential for targeting non-cysteine residues in chemical biology and therapeutic applications.

ZUSAMMENFASSUNG O‐GalNAc ( N ‐Acetylgalactosaminyl) Glykosylierung ist eine weit verbreitete posttranslationale Modifikation in Säugetierzellen. Die Dysregulation der O‐GalNAc Glykosylierung trägt zur Krebsmetastasierung und der Umgehung des Immunsystems bei; jedoch ist das mechanistische Verständnis durch eine begrenzte Anzahl an kleinmolekularen chemischen Werkzeugen eingeschränkt. Die Biosynthese von O‐GalNAc ist stark abhängig von der Verfügbarkeit von UDP‐GalNAc, welches von dem cytosolischen Enzym UDP‐galactose‐4‐epimerase (GalE) biosynthetisiert wird. Knockout‐Studien zeigten, dass ein Funktionsverlust in GalE die O‐GalNAc Glykosylierung stark einschränkt. Dies deutet auf GalE als ein vielversprechendes enzymatisches Target in der Onkologie hin. In dieser Arbeit stellen wir einen effizienten Workflow vor, der kovalente und High‐Throughput kristallographische nicht‐kovalente Fragmentscreens mit strukturbasiertem Design zur Identifizierung von GalE‐Inhibitoren kombiniert. Unter Verwendung dieser Strategien zeigte sich eine für Liganden zugängliche Bindungstasche angrenzend an ein reaktives Tyrosin. Dadurch wurde die Entwicklung eines potenten kovalenten Inhibitors mit Sulfonylfluoridgruppe und weiterhin die Derivatisierung mit einer kovalenten Alkin‐Sonde ermöglicht. Die strukturbasierten Fragmentscreen‐Methoden ergaben nanomolar nicht‐kovalent und kovalent bindende Strukturen aus nur insgesamt 22 Verbindungen. Unsere Arbeit zeigt Synergismus in der GalE‐Inhibierung durch verbesserte Anwendung chemischer Substanzen mit vielversprechendem Potential für nicht‐Cystein‐gerichtete Strategien in der chemischen Biologie und für therapeutische Zwecke.

BACKGROUND:Influenza remains a significant threat to human and animal health. Assessing serological protection against influenza has relied upon haemagglutinin inhibition (HAI) assays, which are used to gauge existing immune landscapes, seasonal vaccine decisions and in systems vaccinology studies. HAI assays were first described in the 1940s. Here, we adapt our high-throughput live virus microneutralisation (LV-N) assay for SARS-CoV-2, benchmark against HAI assays, and report serological vaccine responsiveness in a cohort of older (> 65 yo) community dwelling adults. METHODS:Influenza-specific antibody responses were assessed in 73 individuals, before and after receipt of the adjuvanted 2021-22 Northern Hemisphere quadrivalent vaccine. We performed both HAI and LV-N assays against all four viruses represented in the vaccine [A/Cambodia/e0826360/2020 (H3N2), IVR-215 (A/Victoria/2570/2019-like) (H1N1)pdm09, B/Phuket/3073/2013 (B/Yamagata lineage), B/Washington/02/2019 (B/Victoria lineage)], using sera drawn before vaccination [range: d-82 to d-5], and days 7 [d6-10] and 181 [d156-200] after vaccination. We compared serological responses within each assay and between assays. RESULTS:Both the traditional HAI assay and our high-throughput live virus microneutralisation identified vaccine-induced boosts in antibody titres. We found population-level concordance between the two assays (Spearman's correlation coefficient range 0.49-0.88; all p ≤ 1.4 × 10-5). The improved granularity of microneutralisation was better able to estimate fold changes of responses and quantify the inhibitory effect of pre-existing antibody. CONCLUSIONS:Our high-throughput method offers an alternative approach to assess influenza-specific serological responses with improved resolution, with the potential to improve the annual assessment of existing antibody landscapes, to improve new vaccine strain evaluation, and to offer a step-change in systems vaccinology, and a facet of laboratory-based pandemic preparedness.

Proteins with multiple domains are intrinsically prone to misfold, yet fold efficiently during their synthesis on the ribosome. This is especially important in eukaryotes, where multidomain proteins predominate. Here we sought to understand how multidomain protein folding is modulated by the eukaryotic ribosome. We used hydrogen-deuterium exchange mass spectrometry and cryo-electron microscopy to characterize the structure and dynamics of partially synthesized intermediates of a model multidomain protein. We find that nascent subdomains fold progressively during synthesis on the human ribosome, templated by interactions across domain interfaces. The conformational ensemble of the nascent chain is tuned by its unstructured C-terminal segments, which keep interfaces between folded domains in dynamic equilibrium until translation termination. This contrasts with the bacterial ribosome, on which domain interfaces form early and remain stable during synthesis. Delayed domain docking may avoid interdomain misfolding to promote the maturation of multidomain proteins in eukaryotes.

Immunological determinants favouring emergence of broadly neutralising antibodies are crucial to the development of HIV-1 vaccination strategies. Here, we combined RNAseq and B cell cloning approaches to isolate a broadly neutralising antibody (bnAb) ELC07 from an individual living with untreated HIV-1. Using single particle cryogenic electron microscopy (cryo-EM), we show that the antibody recognises a conformational epitope at the gp120-gp41 interface. ELC07 binds the closed state of the viral glycoprotein causing considerable perturbations to the gp41 trimer core structure. Phenotypic analysis of memory B cell subsets from the ELC07 bnAb donor revealed a lack of expected HIV-1-associated dysfunction, specifically no increase in CD21 - /CD27 - cells was observed whilst the resting memory (CD21 + /CD27 + ) population appeared preserved despite uncontrolled HIV-1 viraemia. Moreover, single cell transcriptomes of memory B cells from this bnAb donor showed a resting memory phenotype irrespective of the epitope they targeted or their ability to neutralise diverse strains of HIV-1. Strikingly, single memory B cells from the ELC07 bnAb donor were transcriptionally similar to memory B cells from HIV-negative individuals. Our results demonstrate that potent bnAbs can arise without the HIV-1-induced dysregulation of the memory B cell compartment and suggest that sufficient levels of antigenic stimulation with a strategically designed immunogen could be effective in HIV-negative vaccine recipients.

Correct elaboration of N-linked glycans in the secretory pathway of human cells is essential in physiology. Early N-glycan biosynthesis follows an assembly line principle before undergoing crucial elaboration points that feature the sequential incorporation of the sugar N-acetylglucosamine (GlcNAc). The activity of GlcNAc transferase V (MGAT5) primes the biosynthesis of an N-glycan antenna that is heavily upregulated in cancer. Still, the functional relevance and substrate choice of MGAT5 are ill-defined. Here, we employ protein engineering to develop a bioorthogonal substrate analog for the activity of MGAT5. Chemoenzymatic synthesis is used to produce a collection of nucleotide-sugar analogs with bulky, bioorthogonal acylamide side chains. We find that WT-MGAT5 displays considerable activity toward such substrate analogues. Protein engineering yields an MGAT5 variant that loses activity against the native nucleotide sugar and increases activity toward a 4-azidobutyramide-containing substrate analogue. By such restriction of substrate specificity, we show that the orthogonal enzyme-substrate pair is suitable to bioorthogonally tag glycoproteins. Through X-ray crystallography and molecular dynamics simulations, we establish the structural basis of MGAT5 engineering, informing the design rules for bioorthogonal precision chemical tools.

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformations, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron variant emergence.

Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O -glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O -glycosylation as an important driver of prostate cancer progression.

Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineered transferases and enzymes for biosynthesis of bioorthogonal substrates, the tactic complements methods in molecular biology. For complete details on the use and execution of this protocol, please refer to Schumann et al. (2020)1 , Cioce et al. (2021)2 , and Cioce et al. (2022)3

SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.

The emergence of a polybasic cleavage motif for the protease furin in SARS-CoV-2 spike has been established as a major factor for human viral transmission. The region N-terminal to that motif is extensively mutated in variants of concern (VOCs). Besides furin, spikes from these variants appear to rely on other proteases for maturation, including TMPRSS2. Glycans near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, we identify that sialic acid-containing O-linked glycans on Thr678 of SARS-CoV-2 spike influence furin and TMPRSS2 cleavage and posit O-linked glycosylation as a likely driving force for the emergence of VOC mutations. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, an event that is suppressed by mutations in the VOCs Alpha, Delta, and Omicron. We found that the sole incorporation of N-acetylgalactosamine did not impact furin activity in synthetic O-glycopeptides, but the presence of sialic acid reduced the furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on TMPRSS2 cleavage. With a chemistry-centered approach, we substantiate O-glycosylation as a major determinant of spike maturation and propose disruption of O-glycosylation as a substantial driving force for VOC evolution.

Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.

Bio-orthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain a bio-orthogonal functionality, such as azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bio-orthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g., N-linked vs O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization, we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat unexpected, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.

ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy.

At-home sampling is key to large scale seroprevalence studies. Dried blood spot (DBS) self-sampling removes the need for medical personnel for specimen collection but facilitates specimen referral to an appropriately accredited laboratory for accurate sample analysis. To establish a highly sensitive and specific antibody assay that would facilitate self-sampling for prevalence and vaccine-response studies. Paired sera and DBS eluates collected from 439 sero-positive, 382 sero-negative individuals and DBS from 34 vaccine recipients were assayed by capture ELISAs for IgG and IgM antibody to SARS-CoV-2. IgG and IgM combined on DBS eluates achieved a diagnostic sensitivity of 97.9% (95%CI 96.6 to 99.3) and a specificity of 99.2% (95% CI 98.4 to 100) compared to serum, displaying limits of detection equivalent to 23 and 10 WHO IU/ml, respectively. A strong correlation (r = 0.81) was observed between serum and DBS reactivities. Reactivity remained stable with samples deliberately rendered inadequate, (p = 0.234) and when samples were accidentally damaged or 'invalid'. All vaccine recipients were sero-positive. This assay provides a secure method for self-sampling by DBS with a sensitivity comparable to serum. The feasibility of DBS testing in sero-prevalence studies and in monitoring post-vaccine responses was confirmed, offering a robust and reliable tool for serological monitoring at a population level.

Recently emerged variants of SARS-CoV-2 contain in their surface spike glycoproteins multiple substitutions associated with increased transmission and resistance to neutralising antibodies. We have examined the structure and receptor binding properties of spike proteins from the B.1.1.7 (Alpha) and B.1.351 (Beta) variants to better understand the evolution of the virus in humans. Spikes of both variants have the same mutation, N501Y, in the receptor-binding domains. This substitution confers tighter ACE2 binding, dependent on the common earlier substitution, D614G. Each variant spike has acquired other key changes in structure that likely impact virus pathogenesis. The spike from the Alpha variant is more stable against disruption upon binding ACE2 receptor than all other spikes studied. This feature is linked to the acquisition of a more basic substitution at the S1-S2 furin site (also observed for the variants of concern Delta, Kappa, and Omicron) which allows for near-complete cleavage. In the Beta variant spike, the presence of a new substitution, K417N (also observed in the Omicron variant), in combination with the D614G, stabilises a more open spike trimer, a conformation required for receptor binding. Our observations suggest ways these viruses have evolved to achieve greater transmissibility in humans.

Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity. The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA), providing the most sensitive format has been exploited in a novel hybrid manner employing a solid-phase S1 preferentially presenting RBD, coupled with a labelled RBD conjugate, used in a two-step sequential assay for detection and measurement of antibody to RBD (anti-RBD). This class and species neutral assay showed a specificity of 100 % on 825 pre COVID-19 samples and a po-tential sensitivity of 99.6 % on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralization and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine and in humans immunised with both AstraZeneca and Pfizer vaccines. This assay detects anti-RBD at presentation with illness, demonstrates its elevation with disease severity, its sequel to asymptomatic infection and its persistence after the loss of antibody to the nucleoprotein (anti-NP). It also provides serological confirmation of prior infection and offers a secure measure for seroprevalence and studies of vaccine immunisation in human and animal populations. The hybrid DABA also displays the attributes necessary for the detection and quantification of anti-RBD to be used in clinical practice. An absence of detectable anti-RBD by this assay predicates the need for passive immune prophylaxis in at-risk patients.

Mucin-type O-glycosylation is among the most complex post-translational modifications. Despite mediating many physiological processes, O-glycosylation remains understudied compared to other modifications, simply because the right analytical tools are lacking. In particular, analysis of intact O-glycopeptides by mass spectrometry is challenging for several reasons; O-glycosylation lacks a consensus motif, glycopeptides have low charge density which impairs ETD fragmentation, and the glycan structures modifying the peptides are unpredictable. Recently, we introduced chemically modified monosaccharide analogues that allowed selective tracking and characterization of mucin-type O-glycans after bioorthogonal derivatization with biotin-based enrichment handles. In doing so, we realized that the chemical modifications used in these studies have additional benefits that allow for improved analysis by tandem mass spectrometry. In this work, we built on this discovery by generating a series of new GalNAc analogue glycopeptides. We characterized the mass spectrometric signatures of these modified glycopeptides and their signature residues left by bioorthogonal reporter reagents. Our data indicate that chemical methods for glycopeptide profiling offer opportunities to optimize attributes such as increased charge state, higher charge density, and predictable fragmentation behavior.

Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines show protective efficacy, which is most likely mediated by neutralizing antibodies recognizing the viral entry protein, spike. Because new SARS-CoV-2 variants are emerging rapidly, as exemplified by the B.1.1.7, B.1.351, and P.1 lineages, it is critical to understand whether antibody responses induced by infection with the original SARS-CoV-2 virus or current vaccines remain effective. In this study, we evaluate neutralization of a series of mutated spike pseudotypes based on divergence from SARS-CoV and then compare neutralization of the B.1.1.7 spike pseudotype and individual mutations. Spike-specific monoclonal antibody neutralization is reduced dramatically; in contrast, polyclonal antibodies from individuals infected in early 2020 remain active against most mutated spike pseudotypes, but potency is reduced in a minority of samples. This work highlights that changes in SARS-CoV-2 spike can alter neutralization sensitivity and underlines the need for effective real-time monitoring of emerging mutations and their effect on vaccine efficacy.