Non-targeted isomer-sensitive N-glycome analysis reveals new layers of organ-specific diversity in mice.

N-glycosylation is one of the most common protein modifications in eukaryotes, with immense importance at the molecular, cellular, and organismal level. Accurate and reliable N-glycan analysis is essential to obtain a systems-wide understanding of fundamental biological processes. Due to the structural complexity of glycans, their analysis is still highly challenging. Here we make publicly available a consistent N-glycome dataset of 20 different mouse tissues and demonstrate a multimodal data analysis workflow that allows for unprecedented depth and coverage of N-glycome features. This highly scalable, LC-MS/MS data-driven method integrates the automated identification of N-glycan spectra, the application of non-targeted N-glycome profiling strategies and the isomer-sensitive analysis of glycan structures. Our delineation of critical sub-structural determinants and glycan isomers across the mouse N-glycome uncovered tissue-specific glycosylation patterns, the expression of non-canonical N-glycan structures and highlights multiple layers of N-glycome complexity that derive from organ-specific regulations of glycobiological pathways.

Introduction of a human- and keyboard-friendly N-glycan nomenclature.

In the beginning was the word. But there were no words for N-glycans, at least, no simple words. Next to chemical formulas, the IUPAC code can be regarded as the best, most reliable and yet immediately comprehensible annotation of oligosaccharide structures of any type from any source. When it comes to N-glycans, the venerable IUPAC code has, however, been widely supplanted by highly simplified terms for N-glycans that count the number of antennae or certain components such as galactoses, sialic acids and fucoses and give only limited room for exact structure description. The highly illustrative - and fortunately now standardized - cartoon depictions gained much ground during the last years. By their very nature, cartoons can neither be written nor spoken. The underlying machine codes (e.g., GlycoCT, WURCS) are definitely not intended for direct use in human communication. So, one might feel the need for a simple, yet intelligible and precise system for alphanumeric descriptions of the hundreds and thousands of N-glycan structures. Here, we present a system that describes N-glycans by defining their terminal elements. To minimize redundancy and length of terms, the common elements of N-glycans are taken as granted. The preset reading order facilitates definition of positional isomers. The combination with elements of the condensed IUPAC code allows to describe even rather complex structural elements. Thus, this "proglycan" coding could be the missing link between drawn structures and software-oriented representations of N-glycan structures. On top, it may greatly facilitate keyboard-based mining for glycan substructures in glycan repositories.

Pseudorabies virus hijacks DDX3X, initiating an addictive "mad itch" and immune suppression, to facilitate viral spread.

Infections with defined Herpesviruses, such as Pseudorabies virus (PRV) and Varicella zoster virus (VZV) can cause neuropathic itch, referred to as "mad itch" in multiple species. The underlying mechanisms involved in neuropathic "mad itch" are poorly understood. Here, we show that PRV infections hijack the RNA helicase DDX3X in sensory neurons to facilitate anterograde transport of the virus along axons. PRV induces re-localization of DDX3X from the cell body to the axons which ultimately leads to death of the infected sensory neurons. Inducible genetic ablation of Ddx3x in sensory neurons results in neuronal death and "mad itch" in mice. This neuropathic "mad itch" is propagated through activation of the opioid system making the animals "addicted to itch". Moreover, we show that PRV co-opts and diverts T cell development in the thymus via a sensory neuron-IL-6-hypothalamus-corticosterone stress pathway. Our data reveal how PRV, through regulation of DDX3X in sensory neurons, travels along axons and triggers neuropathic itch and immune deviations to initiate pathophysiological programs which facilitate its spread to enhance infectivity.

A molecularly engineered, broad-spectrum anti-coronavirus lectin inhibits SARS-CoV-2 and MERS-CoV infection in vivo.

"Pan-coronavirus" antivirals targeting conserved viral components can be designed. Here, we show that the rationally engineered H84T-banana lectin (H84T-BanLec), which specifically recognizes high mannose found on viral proteins but seldom on healthy human cells, potently inhibits Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (including Omicron), and other human-pathogenic coronaviruses at nanomolar concentrations. H84T-BanLec protects against MERS-CoV and SARS-CoV-2 infection in vivo. Importantly, intranasally and intraperitoneally administered H84T-BanLec are comparably effective. Mechanistic assays show that H84T-BanLec targets virus entry. High-speed atomic force microscopy depicts real-time multimolecular associations of H84T-BanLec dimers with the SARS-CoV-2 spike trimer. Single-molecule force spectroscopy demonstrates binding of H84T-BanLec to multiple SARS-CoV-2 spike mannose sites with high affinity and that H84T-BanLec competes with SARS-CoV-2 spike for binding to cellular ACE2. Modeling experiments identify distinct high-mannose glycans in spike recognized by H84T-BanLec. The multiple H84T-BanLec binding sites on spike likely account for the drug compound's broad-spectrum antiviral activity and the lack of resistant mutants.

FIBCD1 is an endocytic GAG receptor associated with a novel neurodevelopmental disorder.

Whole-exome sequencing of two patients with idiopathic complex neurodevelopmental disorder (NDD) identified biallelic variants of unknown significance within FIBCD1, encoding an endocytic acetyl group-binding transmembrane receptor with no known function in the central nervous system. We found that FIBCD1 preferentially binds and endocytoses glycosaminoglycan (GAG) chondroitin sulphate-4S (CS-4S) and regulates GAG content of the brain extracellular matrix (ECM). In silico molecular simulation studies and GAG binding analyses of patient variants determined that such variants are loss-of-function by disrupting FIBCD1-CS-4S association. Gene knockdown in flies resulted in morphological disruption of the neuromuscular junction and motor-related behavioural deficits. In humans and mice, FIBCD1 is expressed in discrete brain regions, including the hippocampus. Fibcd1 KO mice exhibited normal hippocampal neuronal morphology but impaired hippocampal-dependent learning. Further, hippocampal synaptic remodelling in acute slices from Fibcd1 KO mice was deficient but restored upon enzymatically modulating the ECM. Together, we identified FIBCD1 as an endocytic receptor for GAGs in the brain ECM and a novel gene associated with an NDD, revealing a critical role in nervous system structure, function and plasticity.

Clinical grade ACE2 as a universal agent to block SARS-CoV-2 variants.

The recent emergence of multiple SARS-CoV-2 variants has caused considerable concern due to both reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is, therefore, paramount to develop therapeutic strategies that inhibit all known and future SARS-CoV-2 variants. Here, we report that all SARS-CoV-2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, Delta, and Omicron, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by all current VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. Effective inhibition of infections with SARS-CoV-2 variants was validated and confirmed in two independent laboratories. These data show that SARS-CoV-2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan-SARS-CoV-2 therapeutic.

Targeting glycans for CAR therapy: The advent of sweet CARs.

Chimeric antigen receptor (CAR) T cell therapy has created a paradigm shift in the treatment of hematologic malignancies but has not been as effective toward solid tumors. For such tumors, the primary obstacles facing CAR T cells are scarcity of tumor-specific antigens and the hostile and complex tumor microenvironment. Glycosylation, the process by which sugars are post-translationally added to proteins or lipids, is profoundly dysregulated in cancer. Abnormally glycosylated glycoproteins expressed on cancer cells offer unique targets for CAR T therapy as they are specific to tumor cells. Tumor stromal cells also express abnormal glycoproteins and thus also have the potential to be targeted by glycan-binding CAR T cells. This review will discuss the state of CAR T cells in the therapy of solid tumors, the cancer glycoproteome and its potential for use as a therapeutic target, and the landscape and future of glycan-binding CAR T cell therapy.

A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing.

RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.

Comparative Proteome Signatures of Trace Samples by Multiplexed Data-Independent Acquisition.

Single-cell transcriptomics has revolutionized our understanding of basic biology and disease. Since transcript levels often do not correlate with protein expression, it is crucial to complement transcriptomics approaches with proteome analyses at single-cell resolution. Despite continuous technological improvements in sensitivity, mass-spectrometry-based single-cell proteomics ultimately faces the challenge of reproducibly comparing the protein expression profiles of thousands of individual cells. Here, we combine two hitherto opposing analytical strategies, DIA and Tandem-Mass-Tag (TMT)-multiplexing, to generate highly reproducible, quantitative proteome signatures from ultralow input samples. We developed a novel, identification-independent proteomics data-analysis pipeline that allows to quantitatively compare DIA-TMT proteome signatures across hundreds of samples independent of their biological origin to identify cell types and single protein knockouts. These proteome signatures overcome the need to impute quantitative data due to accumulating detrimental amounts of missing data in standard multibatch TMT experiments. We validate our approach using integrative data analysis of different human cell lines and standard database searches for knockouts of defined proteins. Our data establish a novel and reproducible approach to markedly expand the numbers of proteins one detects from ultralow input samples.

The Xbp1-regulated transcription factor Mist1 restricts antibody secretion by restraining Blimp1 expression in plasma cells.

Antibody secretion by plasma cells provides acute and long-term protection against pathogens. The high secretion potential of plasma cells depends on the unfolded protein response, which is controlled by the transcription factor Xbp1. Here, we analyzed the Xbp1-dependent gene expression program of plasma cells and identified Bhlha15 (Mist1) as the most strongly activated Xbp1 target gene. As Mist1 plays an important role in other secretory cell types, we analyzed in detail the phenotype of Mist1-deficient plasma cells in Cd23-Cre Bhlha15 fl/fl mice under steady-state condition or upon NP-KLH immunization. Under both conditions, Mist1-deficient plasma cells were 1.4-fold reduced in number and exhibited increased IgM production and antibody secretion compared to control plasma cells. At the molecular level, Mist1 regulated a largely different set of target genes compared with Xbp1. Notably, expression of the Blimp1 protein, which is known to activate immunoglobulin gene expression and to contribute to antibody secretion, was 1.3-fold upregulated in Mist1-deficient plasma cells, which led to a moderate downregulation of most Blimp1-repressed target genes in the absence of Mist1. Importantly, a 2-fold reduction of Blimp1 (Prdm1) expression was sufficient to restore the cell number and antibody expression of plasma cells in Prdm1 Gfp/+ Cd23-Cre Bhlha15 fl/fl mice to the same level seen in control mice. Together, these data indicate that Mist1 restricts antibody secretion by restraining Blimp1 expression, which likely contributes to the viability of plasma cells.

Structure-guided glyco-engineering of ACE2 for improved potency as soluble SARS-CoV-2 decoy receptor.

Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.

Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

Identification of lectin receptors for conserved SARS-CoV-2 glycosylation sites.

New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N-glycan sites of Spike remain highly conserved among SARS-CoV-2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single-molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.

The Structural Difference of Isobaric N-Glycans of Two Microalgae Samples Reveals Taxonomic Distance.

Microalgae of the Chlorella clade are extensively investigated as an environmentally friendly source of renewable biofuels and high-value nutrients. In addition, essentially unprocessed Chlorella serves as wholesome food additive. A recent study on 80 commercial Chlorella preparations revealed an unexpected variety of protein-linked N-glycan patterns with unprecedented structural features, such as the occurrence of arabinose. Two groups of products exhibited a characteristic major N-glycan isobaric to the Man2GlcNAc2XylFuc N-glycan known from pineapple stem bromelain, but tandem mass spectrometry (MS/MS) analysis pointed at two types of N-glycan different from the bromelain structure, as well as from each other. Here we report the exact structures of these two novel N-glycan structures, elucidated by nuclear magnetic resonance spectroscopy and MS/MS, as well as on their phylogenetic context. Despite their humble size, these two N-glycans exhibited a very different design with structural features unrelated to those recently described for other Chlorella-clade strains. The major glycans of this study presented several novel structural features such as substitution by arabinose or xylose of the internal N-acetylglucosamine, as well as methylated sugars. ITS1-5.8S-ITS2 rDNA barcode analyses revealed that the xylose-containing structure derived from a product primarily comprising Scenedesmus species, and the arabinose-containing glycan type related to Chlorella species (SAG211-34 and FACHB-31) and to Auxenochlorella. This is another example where characteristic N-glycan structures distinguish phylogenetically different groups of microalgae.

Naphthylphthalamic acid associates with and inhibits PIN auxin transporters.

N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar) transport of the hormone auxin in plants. For decades, it has been a pivotal tool in elucidating the unique polar auxin transport-based processes underlying plant growth and development. Its exact mode of action has long been sought after and is still being debated, with prevailing mechanistic schemes describing only indirect connections between NPA and the main transporters responsible for directional transport, namely PIN auxin exporters. Here we present data supporting a model in which NPA associates with PINs in a more direct manner than hitherto postulated. We show that NPA inhibits PIN activity in a heterologous oocyte system and that expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to specific saturable NPA binding. We thus propose that PINs are a bona fide NPA target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent auxin transport and a logical parsimonious explanation for the known physiological effects of NPA on plant growth, as well as an alternative hypothesis to interpret past and future results. We also introduce PIN dimerization and describe an effect of NPA on this, suggesting that NPA binding could be exploited to gain insights into structural aspects of PINs related to their transport mechanism.

Generation of enzymatically competent SARS-CoV-2 decoy receptor ACE2-Fc in glycoengineered Nicotiana benthamiana.

Human angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding and cell entry. Administration of high concentrations of soluble ACE2 can be utilized as a decoy to block the interaction of the virus with cellular ACE2 receptors and potentially be used as a strategy for treatment or prevention of coronavirus disease 2019. Human ACE2 is heavily glycosylated and its glycans impact on binding to the SARS-CoV-2 spike protein and virus infectivity. Here, we describe the production of a recombinant soluble ACE2-fragment crystallizable (Fc) variant in glycoengineered Nicotiana benthamiana. Our data reveal that the produced dimeric ACE2-Fc variant is glycosylated with mainly complex human-type N-glycans and functional with regard to enzyme activity, affinity to the SARS-CoV-2 receptor-binding domain, and wild-type virus neutralization.

A crucial role for Jagunal homolog 1 in humoral immunity and antibody glycosylation in mice and humans.

Jagunal homolog 1 (JAGN1) has been identified as a critical regulator of neutrophil biology in mutant mice and rare-disease patients carrying JAGN1 mutations. Here, we report that Jagn1 deficiency results in alterations in the endoplasmic reticulum (ER) of antibody-producing cells as well as decreased antibody production and secretion. Consequently, mice lacking Jagn1 in B cells exhibit reduced serum immunoglobulin (Ig) levels at steady state and fail to mount an efficient humoral immune response upon immunization with specific antigens or when challenged with viral infections. We also demonstrate that Jagn1 deficiency in B cells results in aberrant IgG N-glycosylation leading to enhanced Fc receptor binding. Jagn1 deficiency in particular affects fucosylation of IgG subtypes in mice as well as rare-disease patients with loss-of-function mutations in JAGN1. Moreover, we show that ER stress affects antibody glycosylation. Our data uncover a novel and key role for JAGN1 and ER stress in antibody glycosylation and humoral immunity in mice and humans.

A synthetic peptide library for benchmarking crosslinking-mass spectrometry search engines for proteins and protein complexes.

Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. Numerous search engines for crosslink identification exist, but lack of ground truth samples containing known crosslinks has precluded their systematic validation. Here we report on XL-MS data arising from measuring synthetic peptide libraries that provide the unique benefit of knowing which identified crosslinks are true and which are false. The data are analysed with the most frequently used search engines and the results filtered to an estimated false discovery rate of 5%. We find that the actual false crosslink identification rates range from 2.4 to 32%, depending on the analysis strategy employed. Furthermore, the use of MS-cleavable crosslinkers does not reduce the false discovery rate compared to non-cleavable crosslinkers. We anticipate that the datasets acquired during this research will further drive optimisation and development of XL-MS search engines, thereby advancing our understanding of vital biological interactions.

Improved Sensitivity in Low-Input Proteomics Using Micropillar Array-Based Chromatography.

Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nanoliquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS) is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples. However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for nanoflow high-pressure liquid chromatography (HPLC), current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology. With the recent introduction of the µPAC system, which provides perfectly ordered micropillar array based chromatographic support materials, completely new chromatographic concepts for optimization toward the needs of ultrasensitive proteomics become available. Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micropillar array column to a widely used nanoflow HPLC column for the proteomics analysis of 10 ng of tryptic HeLa cell digest. Comparative analysis of LC-MS/MS-data corroborated that micropillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, and increased sensitivity in the analysis of low-input proteomics samples and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional nanoflow HPLC columns.