Biology subjects

Stadlmann, J.

Publications and source records attributed to Stadlmann, J..

12 recordsLinked to original sources

bioRxiv2024

The wondrous and worrying diversity of the N-glycans of Chlorella food supplements

N-glycans have recently emerged as highly varied elements of Chlorella strains and products. Four years and many samples later, the ever-growing N-glycan diversity shall be revisited in the light of concepts of species definition and product authenticity. N-glycans of commercial products were analyzed by matrix-assisted time-of-flight mass spectrometry (MALDI-TOF MS) supported by chromatography on porous graphitic carbon with mass spectrometric detection. While 36% of 172 products were labeled C. vulgaris, only few had matching N-glycan patterns. 5 and 20 % of the products matched with C. sorokiniana strains SAG 211-8k and SAG 211-34, respectively, which, however, carry entirely different structures. 41 % presented with four frequently occurring glyco-types while 26 % of the samples showed unique or rare N-glycan patterns. The rest presented what could be taken as a C. vulgaris type N-glycan pattern. Identical masses derive from different structures in many cases. By no means do we want to question the presumed health benefits of the products or the honest intentions of manufacturers. We rather wish to raise awareness for the fascinating but also worrying variety of microalgal N-glycans and suggest it as a means for defining product identity and taxonomic assignments.

plant biology↗

bioRxiv2023

Tumor sialylation is a barrier against CD8 T cell-mediated anti-tumor responses and anti-PD-1 resistance in breast cancer

Breast cancer is the most common cancer among women. However, the use of immune checkpoint inhibitors, that have revolutionized treatment of multiple cancers, unfortunately remain largely ineffective in most breast cancer patients. Here, we report the most comprehensive glycoproteome map in breast tumor cells, pointing to a key role of sialic acid modifications in mammary cancer. Genetic and pharmacologic inhibition of sialylation repolarizes the tumor microenvironment, leading to a reduction in myeloid-derived suppressor cells and a significant increase in Tcf7+ memory and CD8+ effector T cells. Mechanistically, sialylation controls cell surface expression of MHC class I and PD-1-ligand on the tumor cells. Functionally, in vivo interference with sialylation on breast cancer cells licenses CD8+ T cells to effectively kill the tumors. In multiple immunotherapy-resistant breast tumor models, we also show that the abrogation of sialylation sensitizes to anti-PD-1 immune checkpoint therapy. We further demonstrate that hyper-sialylation occurs in over half of human breast cancers tested and correlates with poor T cell infiltration. Our results establish sialylation as a central immunoregulator in breast cancer, orchestrating multiple pathways of immune evasion. Targeting tumor sialylation licenses immunologically inert mammary tumors to be efficiently eliminated by anti-cancer immunity and sensitizes to immune checkpoint therapy.

cancer biology↗

bioRxiv2023

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.

neuroscience↗

bioRxiv2022

The Mouse N-Glycome Atlas - High-resolution N-glycan analysis of 23 mouse tissues.

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.

biochemistry↗

bioRxiv2021

Clinical grade ACE2 effectively inhibits SARS-CoV-2 Omicron infections

The recent emergence of the SARS-CoV-2 variant Omicron has caused considerable concern due to reduced vaccine efficacy and escape from neutralizing antibody therapeutics. Omicron is spreading rapidly around the globe and is suspected to account for most new COVID-19 cases in several countries, though the severity of Omicron-mediated disease is still under debate. It is therefore paramount to identify therapeutic strategies that inhibit the Omicron SARS-CoV-2 variant. Here we report using 3D structural modelling that Spike of Omicron can still associate with human ACE2. Sera collected after the second mRNA-vaccination did not exhibit a protective effect against Omicron while strongly neutralizing infection of VeroE6 cells with the reference Wuhan strain, confirming recent data by other groups on limited vaccine and convalescent sera neutralization efficacy against Omicron. Importantly, clinical grade recombinant human soluble ACE2, a drug candidate currently in clinical development, potently neutralized Omicron infection of VeroE6 cells with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. These data show that SARS-CoV-2 variant Omicron can be readily inhibited by soluble ACE2, providing proof of principle of a viable and effective therapeutic approach against Omicron infections.

molecular biology↗

bioRxiv2021

FIBCD1 is a Conserved Receptor for Chondroitin Sulphate Proteoglycans of the Brain Extracellular Matrix and a Candidate Gene for a Complex Neurodevelopmental Disorder

The brain extracellular matrix (ECM) is enriched in chondroitin sulphate proteoglycans (CSPGs) with variable sulphate modifications that intimately participate in brain maturation and function. Very little is known about how the changing biophysical properties of the CSPGs are signalled to neurons. Here, we report Fibrinogen C Domain Containing 1 (FIBCD1), a known chitin-binding receptor of the innate immune system, to be highly expressed in the hippocampus and to specifically bind CSPGs containing 4-O sulphate modification (CS-4S). Cultured Fibcd1 knockout (KO) neurons lack phenotypic and transcriptomic responses to CSPG stimulation. Further, Fibcd1 KO mice exhibit accumulation of CS-4S, likely resulting in deficits of hippocampal-dependent learning tasks and abrogated synaptic remodelling, a phenotype rescued by enzymatic digestion of CSPGs. Likewise, neuronal specific knockdown of a Fibcd1 orthologue in flies results in neuronal morphological changes at the neuromuscular junctions and behavioural defects. Finally, we report two undiagnosed patients with a complex neurodevelopmental disorder with deleterious variants in FIBCD1, strongly implicating FIBCD1 in the development of the disease. Taken together, our results demonstrate that FIBCD1 is a novel, evolutionarily conserved component of ECM sulphation recognition that is crucial for neuronal development and function.

neuroscience↗

bioRxiv2021

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.

biochemistry↗

bioRxiv2021

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. All 22 N-glycan sites of SARS-CoV-2 Spike remain highly conserved among the variants B.1.1.7, 501Y.V2 and P.1, 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.

molecular biology↗

bioRxiv2021

Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Glycopeptide Data

Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project - Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance for N- and O-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved "high coverage" and "high accuracy" glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.

biochemistry↗

bioRxiv2021

Comparative proteome signatures of ultra-low input samples by TMT 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 ultra-low input samples. While conventional, data-dependent shotgun proteomics (DDA) of ultra-low input samples critically suffers from the accumulation of missing values with increasing sample-cohort size, data-independent acquisition (DIA) strategies do usually not take full advantage of isotope-encoded sample multiplexing. 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, and to identify cell types and single protein knockouts. We validate our approach using integrative data analysis of different human cell lines and standard database searches for knockouts of defined proteins. These data establish a novel and reproducible approach to markedly expand the numbers of proteins one detects from ultra-low input samples, such as single cells.

biochemistry↗

bioRxiv2019

A synthetic peptide library for benchmarking crosslinking mass spectrometry search engines

We have created synthetic peptide libraries to benchmark crosslinking mass spectrometry search engines for different types of crosslinker. The unique benefit of using a library is knowing which identified crosslinks are true and which are false. Here we have used mass spectrometry data generated from measurement of the peptide libraries to evaluate the most frequently applied search algorithms in crosslinking mass-spectrometry. When filtered to an estimated false discovery rate of 5%, false crosslink identification ranged from 5.2% to 11.3% for search engines with inbuilt validation strategies for error estimation. When different external validation strategies were applied to one single search output, false crosslink identification ranged from 2.4% to a surprising 32%, despite being filtered to an estimated 5% false discovery rate. Remarkably, the use of MS-cleavable crosslinkers did not reduce the false discovery rate compared to non-cleavable crosslinkers, results from which have far-reaching implications in structural biology. We anticipate that the datasets acquired during this research will further drive optimisation and development of search engines and novel data-interpretation technologies, thereby advancing our understanding of vital biological interactions.

bioinformatics↗

bioRxiv2019

Improved Sensitivity in Low-Input Proteomics using Micro-Pillar Array-based Chromatography

Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nano-LC-ESI-MS/MS is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples.\n\nHowever, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for nano-flow HPLC, current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology.\n\nWith the recent introduction of the {micro}PAC system, which provides perfectly ordered micro-pillar array based chromatographic support materials, completely new chromatographic concepts for optimization towards the needs of ultra-sensitive proteomics become available.\n\nHere we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micro-pillar array column to a widely used nano-flow HPLC column for the proteomics analysis of 10 ng tryptic HeLa cell digest.\n\nComparative analysis of LC-MS/MS-data corroborated that micro-pillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, increase 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 nano-flow HPLC columns.

biochemistry↗