Biology subjects

Snijder, J.

Publications and source records attributed to Snijder, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Reference-free protein sequencing by consensus assembly of redundant de novo peptide reads

Reading a proteins sequence from tandem mass spectra without a reference is limited by single-spectrum accuracy, most acutely across the hypervariable complementaritydetermining regions of antibodies. Broadly specific proteases tile a protein with long, overlapping peptides, so every residue is covered by many independent de novo reads. borgonovo assembles their per-step probability profiles into a reference-free per-residue consensus, seeding templates from mass-closure-consistent reads and recruiting the rest by substitution- tolerant alignment and per-column voting. Re-decoding each spectrum with a prior from its consensus position lifts amino acid accuracy on placed spectra from 0.80 to 0.87. On the therapeutic antibody trastuzumab, nine proteases cover its heavy and light chains completely at 0.88 fixed-window identity, and 0.93 on the pruned assembly once local indels are accommodated. Applied unchanged to five secretome proteins and trastuzumab with three proteases, it reaches 0.87 mean fixed-window identity over 82% coverage. borgonovo is open source and works with most de novo sequencers, so redundant digestion turns any of them into a protein sequencer where no reference exists.

bioinformatics↗

bioRxiv2026

Structure of SARS-CoV-2 spike in complex with its co-receptor the neuronal cell adhesion protein contactin 1

The emergence of SARS-CoV-2 has caused millions of deaths and excess morbidity in the worldwide population. In addition to its respiratory symptoms, SARS-CoV-2 has become known for its neurotropism and long-term neurological sequelae, with a post-acute infection syndrome commonly referred to as long-COVID. Next to the host receptor angiotensin-converting enzyme 2 (ACE2) additional interactions of the SARS-CoV-2 spike (S) protein have been described for neuronal co-receptors specific to the nervous system including cell adhesion protein contactin 1 (CNTN1). Details of the spike-CNTN1 interaction have remained elusive. Here, we quantified the spike-CNTN1 interaction by surface plasmon resonance and resolved the structure of the complex by single particle cryo-electron microscopy (cryo-EM). Spike and CNTN1 interact with nanomolar affinity, driven by an avidity effect and mediated by the horseshoe moiety of CNTN1. The cryo-EM structure reveals that the CNTN1 Ig1-4 horseshoe is wedged in between two receptor binding domains (RBDs) and interacts, through Ig3, with a unique receptor interface at the base of the RBD in the up-conformation. This receptor interface is not previously described for other spike receptors but overlaps with the epitopes of several neutralizing monoclonal antibodies. Comparison of our data with available spike structures suggests one spike trimer can bind three CNTN1 molecules, or alternatively, different co-receptors such as ACE2 and CNTN1, simultaneously. These findings shed new light on the molecular determinants of SARS-CoV-2 neurotropism.

biochemistry↗

bioRxiv2026

Host cell remodeling via cyclin dependent kinases drives Ebola virus replication and transcription

Ebola virus (EBOV) causes re-emerging outbreaks of viral hemorrhagic fever with high case-fatality rates. As a negative-sense single stranded RNA virus, EBOV depends on its RNA-dependent RNA polymerase (L protein) to transcribe and replicate the viral genome. This takes place in cytoplasmic inclusion bodies that form following host cell remodeling involving yet unknown signaling pathways. Here, using mass spectrometry-based (phospho-)proteomics, we profiled global protein abundance and site-specific phosphorylation in HEK293T cells expressing an EBOV minigenome system. Our data reveal EBOV-induced rewiring of the host proteome and phosphorylation signaling landscape, including perturbations in cell cycle control, cytoskeletal organization, innate immune regulation, and DNA damage response. Kinase network analysis revealed that Hippo pathway kinases and especially cyclin dependent kinases like CDK2 are central drivers of EBOV replication and transcription. The functional necessity of these signaling pathways is demonstrated via inhibition of CDK family kinases with small molecule inhibitors, which halted EBOV minigenome replication and transcription when administered in the low micromolar range, demonstrating that these pathways represent putative antiviral targets.

molecular biology↗

bioRxiv2026

The glycan shield of alphaherpesvirus glycoprotein B modulates host co-receptor binding

Alphaherpesviruses can infect a wide range of cell types and are notorious for their neurotropism. They enter cells through the fusion activity of the essential viral glycoprotein gB. Interactions of gB with co-receptors Myelin Associated Glycoprotein (MAG), paired immunoglobulin-like type 2 receptor alpha (PILR) and Non-muscle Myosin Heavy Chain IIA (NMHC-IIA) influence cell tropism and infectivity. Both MAG and PILR are known to bind to sialic acids, highlighting the potential role for gB glycosylation in mediating the co-receptor interactions. Here, using glycoproteomics, cryo electron microscopy and surface plasmon resonance, we study the specificity of the three co-receptor interactions with gB from herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), and varicella zoster virus (VZV). We show that all gB variants are heterogeneously N-glycosylated at six or seven predicted sites with moderate degrees of sialylation, complemented with extensive O-linked glycosylation at the disordered N-terminus and a flexible loop within domain II. All three gB variants bind to MAG with similar affinity, mediated by sialic acids on gB O-glycans. In contrast, PILR binds only to HSV-1 and HSV-2, but not VZV gB. PILR binding is also mediated by sialic acids on gB O-glycans, of which we identify multiple candidate sites in HSV-1 and HSV-2, and none in VZV gB. NMHC-IIA binds to HSV-1 gB and HSV-2 gB with high affinity through a glycan-independent interaction, and not to VZV gB. These data reveal divergent co-receptor specificity and interaction strength and provide an improved biophysical basis to understand human neurotropic herpesvirus infection.

biochemistry↗

bioRxiv2025

Stabilized Full-Length Measles Fusion Protein Elicits Potent Immunity and Protection In Vivo

Measles virus (MeV) is a highly contagious pathogen that causes significant morbidity and mortality in populations with low vaccination coverage. Infection typically leads to immune amnesia and, in rare cases, fatal neurological disease. While current live-attenuated vaccines are highly effective, they primarily elicit neutralizing antibodies against the hemagglutinin (H) glycoprotein, with a less robust response to the fusion (F) protein, a key protein for viral entry. To improve the immunogenicity of the F protein, we designed and characterized stabilized, prefusion MeV F protein antigens. We engineered both soluble ectodomains (FECTO) and full-length, membrane-embedded proteins (FFL) with mutations that confer thermal stability. Cryo-electron microscopy confirmed that these engineered antigens faithfully maintain the native prefusion conformation. When evaluated in a cotton rat model, immunization with either FECTO or FFL constructs induced neutralizing antibodies and elicited protection against viral challenge. The most stable full-length construct (FFL 3M) elicited a more potent neutralizing antibody response than its ectodomain counterpart. Importantly, no evidence of vaccine-enhanced respiratory disease was observed. These findings establish that a thermostable, full-length F protein is a superior immunogen to its soluble ectodomain. This work presents a promising candidate for next-generation, non-replicating measles vaccines intended to complement current vaccination strategies and provide a safe option for immunocompromised individuals and others who cannot receive live-virus vaccines. One-Sentence SummaryA prefusion-stabilized, full-length measles Fusion glycoprotein immunogen induces strong neutralizing responses and offers protection against challenge with wild-type virus.

immunology↗

bioRxiv2025

Integrative Structure of Norovirus NS3 Suggests a Role in RNA Transport

Human noroviruses (HuNoVs) are the leading global cause of non-bacterial gastroenteritis, yet no vaccines or antiviral therapies are currently approved1. The viral non-structural protein NS3 is a membrane-bound AAA+ ATPase of superfamily 3 (SF3) with multiple proposed roles in the norovirus replication cycle. However, the structure of NS3, and the mechanisms by which it contributes to genome replication and membrane remodeling, have remained unknown. We engineered a soluble, hexameric, and catalytically active form of NS3 and determined its cryo-EM structure in the presence of a nucleotide analogue at 2.9 [A] resolution. The structure adopts a split lock-washer architecture characteristic of AAA+ motors that operate via a hand-over-hand translocation mechanism2. Modeling of the nucleotide-binding site reveals conserved features governing ATP binding and hydrolysis. Using integrative modeling with AlphaFold3, we generated a full-length, membrane-associated model of NS3, in which an N-terminal transmembrane domain, central helical bundle, and AAA+ motor form a continuous conduit. This model supports a role for NS3 as a candidate membrane-spanning RNA translocase that couples ATP hydrolysis to genome movement. This structural framework helps address long-standing gaps in our understanding of norovirus replication and establishes a basis for mechanistic studies and structure-guided antiviral design.

microbiology↗

bioRxiv2025

Deep Coverage and Extended Sequence Reads Obtained with a Single Archaeal Protease Expedite de novo Protein Sequencing by Mass Spectrometry

The ability to sequence proteins without reliance on a genomic template defines a critical frontier in modern proteomics. This approach, known as de novo protein sequencing, is essential for applications such as antibody sequencing, microbiome proteomics, and antigen discovery, which require accurate reconstruction of peptide and protein sequences. While trypsin remains the gold-standard protease in proteomics, its restricted cleavage specificity limits peptide diversity. This constraint is especially problematic in antibody sequencing, where the functionally critical regions often lack canonical tryptic sites. As a result, exclusively trypsin-based approaches yield sparse reads, leading to sequence gaps. Multi-protease and hybrid-fragmentation strategies can improve the sequence coverage, but they add complexity, compromise scalability and reproducibility. Here, we explore two HyperThermoacidic Archaeal (HTA)-proteases as single-enzyme solutions for de novo antibody sequencing. Each HTA-protease generated about five times more unique peptide reads than trypsin or chymotrypsin, providing high redundancy across all CDRs. Combined with EAciD fragmentation on a ZenoTOF 7600 system, this approach enabled complete, unambiguous antibody sequencing. De novo analysis using PEAKS/DeepNovo and Stitch showed up to fourfold higher alignment scores and reduced the sequence errors within the HTA-generated data. Additionally, the HTA-EAciD approach offers short digestion times, eliminates extensive cleanup, and enables analysis in a single LC-MS/MS run. This streamlined, single-protease approach delivers therefore performance comparable to multi-enzyme workflows, offering a scalable and efficient strategy for de novo protein sequencing across diverse applications.

biochemistry↗

bioRxiv2025

Cetacean coronavirus spikes highlight S glycoprotein structural plasticity

Coronaviruses (CoVs) exhibit a remarkable ability for spill-over infections into naive host populations. While much research has focused on the spike (S) glycoproteins of zoonotic alpha- and betacoronaviruses, the S proteins of gamma- and deltacoronaviruses, which predominantly infect avian hosts, remain poorly understood. Here, we present high-resolution cryo-EM structures of S proteins from two distinct gammacoronaviruses (75.7% sequence identity) that atypically infect marine mammals and belong to the Gammacoronavirus delphinapteri species. The cryo-EM reconstructions reveal that the spikes exhibit a unique quaternary architecture that distinguishes them from other coronaviruses. The S protein features a previously unidentified, tripodal quaternary assembly of the S1 subunit, in which S1B domains are presented in an upright position while their putative receptor binding sites are shielded by extended loops from the S1A domain of the same protomers. Additionally, the CeCoV spike proteins have evolved an additional and unique [~]200 residue N-terminal domain (S10). S10 lacks homology to known protein sequences but displays structural similarity to members of the cupin protein superfamily. This represents a remarkable case of coronaviral exaptation of a host protein integrated into the S glycoprotein. Moreover, glycoproteomic analyses reveal that CeCoV S proteins are extensively N-glycosylated (>100 N-glycans per trimer), with a notable abundance of high-mannose glycans on S10 and O-glycosylation sites within a mucin-like loop at the trimer apex, all contributing to a dense glycan shield and potentially masking immunogenic epitopes. These findings demonstrate the structural diversity and adaptability of CoV S proteins, including alternative quaternary assemblies, additional domains, and diverse glycosylation strategies, offering new insights into the evolutionary mechanisms that enable coronaviruses to expand their host range and establish infections in novel species.

microbiology↗

bioRxiv2025

A universal spectrum annotator for complex peptidoforms in mass spectrometry-based proteomics

Accurate and comprehensive peptide spectrum annotation is a crucial step to interpret mass spectrometry-based proteomics data. While peak assignment in peptide fragmentation spectra is central to a broad range of proteomics applications, current tools tend to be specialized to a specific task. Here we present a more comprehensive tool, the Annotator, which unifies spectrum annotation for bottom-up, middle-down, top-down, cross-linked, and glycopeptide fragmentation mass spectra, from all fragmentation methods, including all ion types: a/b/c, x/y/z, d/v/w, and immonium ions. The Annotator integrates all known post-translational modifications from common databases and additionally allows for the definition of custom fragmentation models and modifications. Modifications allow for diagnostic fragment ions, site specific neutral losses, and multiple breakage sites for cross-linkers. The underlying library used for the theoretical fragmentation and matching is based on the unified peptidoform notation ProForma 2.0 and made available as a Rust library (rustyms) with Python bindings. This enables spectrum annotation of diverse and complex peptidoforms across the broad range of mass spectrometry-based proteomics applications.

bioinformatics↗

bioRxiv2025

Structural basis for postfusion-specific binding to Respiratory Syncytial Virus F protein by the canonical antigenic site I antibody 131-2a

The Respiratory Syncytial Virus (RSV) Fusion (F) protein is a major target of antiviral antibodies following natural infection or vaccination and responsible for mediating fusion between the viral envelope and the host membrane. The fusion process is driven by a large-scale conformational change in F, switching irreversibly from the metastable prefusion state to the stable postfusion conformation. Previous research has identified six distinct antigenic sites in RSV-F, termed sites O, I, II, III, IV, and V. Of these, only antigenic site I is fully specific to the postfusion conformation of F. A monoclonal antibody 131-2a that specifically targets postfusion F has been widely used as a research tool to probe for postfusion F and to define antigenic site I in serological studies, yet its sequence and precise epitope have remained unknown. Here we use mass spectrometry-based de novo sequencing of 131-2a to reverse engineer a recombinant product and study the epitope to define antigenic site I with molecular detail, revealing the structural basis for the antibodys specificity towards postfusion RSV-F.

immunology↗

bioRxiv2024

Structural and immunological characterization of the H3 influenza hemagglutinin during antigenic drift

The quest for a universal influenza vaccine holds great promise for mitigating the global burden of influenza-related morbidity and mortality. However, challenges persist in identifying conserved epitopes capable of inducing protection. In this study, we explore the influence of glycan evolution on H3 hemagglutinin from 1968 to present day and its impacts on antigenicity and immunogenicity. We observe that the appearance of potential N-linked glycosylation sites in Sing/16 hemagglutinin head domain reduces the binding of broadly neutralizing antibodies and shifts the polyclonal immune response upon vaccination to target the stem. Furthermore, structural characterization of HK/68 and Sing/16 by cryo-electron microscopy shows that while HK/68 is resistant to enzymatic deglycosylation, removal of glycans destabilizes the hyperglycosylated head and membrane-proximal region in Sing/16. These insights expand our understanding of glycans beyond their role in protein folding and highlight the interplay among glycan integration and immune recognition to design a universal influenza vaccine.

immunology↗

bioRxiv2024

Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry - a perspective.

Antibodies are a major component of adaptive immunity against invading pathogens. Here we explore possibilities for an analytical approach to characterize the antigen-specific antibody repertoire directly from the secreted proteins in convalescent serum. This approach aims to perform simultaneous antibody sequencing and epitope mapping using a combination of single particle cryo-electron microscopy (cryoEM) and bottom-up proteomics techniques based on mass spectrometry (LC-MS/MS). We evaluate the performance of the deep-learning tool ModelAngelo in determining de novo antibody sequences directly from reconstructed 3D volumes of antibody-antigen complexes. We demonstrate that while map quality is a critical bottleneck, it is possible to sequence antibody variable domains from cryoEM reconstructions with accuracies of up to 80-90%. While the rate of errors exceeds the typical levels of somatic hypermutation, we show that the ModelAngelo-derived sequences can be used to assign the used V-genes. This provides a functional guide to assemble de novo peptides from LC-MS/MS data more accurately and improves the tolerance to a background of polyclonal antibody sequences. Following this proof-of-principle, we discuss the feasibility and future directions of this approach to characterize antigen-specific antibody repertoires.

biochemistry↗

bioRxiv2024

Structural basis for recognition of the FLAG-1 tag by anti-FLAG M2

The FLAG-tag/anti-FLAG system is a widely used biochemical tool for protein detection and purification. Anti-FLAG M2 is the most popular antibody against the FLAG-tag, due to its ease of use, versatility, and availability in pure form or as bead conjugate. M2 binds N-terminal, C-terminal and internal FLAG-tags and binding is calcium-independent, but the molecular basis for the FLAG-tag specificity and recognition remains unresolved. Here we present an atomic resolution (1.17 [A]) structure of the FLAG peptide in complex with the Fab of anti-FLAG M2, revealing key binding determinants. Five of the eight FLAG peptide residues form direct interactions with paratope residues. The FLAG peptide adopts a 310 helix conformation in complex with the Fab. These structural insights allowed us to rationally introduce point mutations on both the peptide and antibody side. We tested these by surface plasmon resonance, leading us to propose a shorter yet equally binding version of the FLAG-tag for the M2 antibody.

biochemistry↗

bioRxiv2024

A handle on mass coincidence errors in de novo sequencing of antibodies by bottom-up proteomics

Antibody sequences can be determined at 99% accuracy directly from the polypeptide product using bottom-up proteomics techniques. This circumvents the need to isolate the antibody-producing B-cell clone and enables reverse engineering of monoclonal antibodies from lost hybridoma cell lines, as well as the secreted protein in bodily fluid. Sequencing accuracy at the peptide level is limited by common mass coincidences of isobaric residues like leucine/isoleucine, but also by incomplete fragmentation spectra in which the order of two or more residues remains ambiguous due to lacking fragment ions for the intermediate positions. Likewise, different combinations of amino acids, of potentially different length, can also coincide to the same mass (e.g. GG=N, GA=Q etc.). Here we present several updates to Stitch (v1.5), which performs template-based assembly of de novo peptide reads to reconstruct antibody sequences. This version introduces a mass-based alignment algorithm that explicitly accounts for mass coincidence errors. In addition, it incorporates a postprocessing procedure to assign I/L residues based on secondary fragments (satellite ions, i.e. w-ions). Moreover, evidence for sequence assignments can now be directly evaluated with the addition of an integrated spectrum viewer. This version of Stitch also allows input data from a wider selection of de novo peptide sequencing algorithms, now including Casanovo, PEAKS, Novor.Cloud, pNovo, and MaxNovo, in addition to flat text and FASTA. Combined, these changes make Stitch compatible with a larger range of data processing pipelines and improve its tolerance to peptide-level sequencing errors.

bioinformatics↗

bioRxiv2023

Reverse engineering the anti-MUC1 hybridoma antibody 139H2 by mass spectrometry-based de novo sequencing

Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at different mucosal surfaces including breast and intestine. In the gastrointestinal tract, MUC1 has a barrier function against bacterial invasion, but can also serve as an entry receptor for pathogenic Salmonella bacteria. Moreover, MUC1 is well known for its aberrant expression and glycosylation in adenocarcinomas The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily O-linked glycosylated.. Monoclonal antibodies against the MUC1 VNTR can be powerful tools because of their multiplicity of binding and possible applications in the diagnosis and treatment of MUC1-expressing cancers. One such antibody is the hybridoma mouse monoclonal 139H2, which is also widely used as a research tool to study non-cancer MUC1. Here we report direct mass spectrometry-based sequencing of hybridoma-derived 139H2 IgG, which enabled reverse engineering of a recombinant 139H2. The performance of the reverse engineered 139H2 IgG and its Fab fragment were validated by comparison to the hybridoma-derived product in Western blot and immunofluorescence microscopy. The reverse engineering of 139H2 allowed us to characterize binding to the VNTR peptide epitope by surface plasmon resonance (SPR) and solve the crystal structure of the 139H2 Fab fragment in complex with the MUC1 VNTR peptide. These analyses reveal the molecular basis for 139H2 binding specificity to MUC1 and its tolerance to O-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostics, targeting and treatment strategies.

biochemistry↗

bioRxiv2023

Direct mass spectrometry-based detection and antibody sequencing of Monoclonal Gammopathy of Undetermined Significance from patient serum - a case study.

Monoclonal gammopathy of undetermined significance (MGUS) is a plasma cell disorder, characterized by the presence of a predominant monoclonal antibody (i.e., M-protein) in serum, without clinical symptoms. Here we present a case study in which we detect MGUS by liquid-chromatography coupled with mass spectrometry (LC-MS) profiling of IgG1 in human serum. We detected a Fab-glycosylated M-protein and determined the full heavy and light chain sequences by bottom-up proteomics techniques using multiple proteases, further validated by top-down LC-MS. Moreover, the composition and location of the Fab-glycan could be determined in CDR1 of the heavy chain. The outlined approach adds to an expanding mass spectrometry-based toolkit to characterize monoclonal gammopathies such as MGUS and multiple myeloma, with fine molecular detail. The ability to detect monoclonal gammopathies and determine M-protein sequences straight from blood samples by mass spectrometry provides new opportunities to understand the molecular mechanisms of such diseases.

immunology↗

bioRxiv2023

Sialoglycan binding triggers spike opening in a human coronavirus

Coronavirus (CoV) spikes mediate receptor binding and membrane fusion, making them prime targets for neutralising antibodies. In the cases of SARS-CoV, SARS-CoV-2, and MERS-CoV, spikes transition freely between open and closed conformations to balance host cell attachment and immune evasion. The open conformation exposes domain S1B, allowing it to bind to proteinaceous cell surface receptors. It also facilitates protein refolding during spike-mediated membrane fusion. However, with a single exception, the pre-fusion spikes of all other CoVs studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, where spikes more commonly transition to open states in response to specific cues, rather than spontaneously. In our study, using cryo-EM and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes upon binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1B domains to the open state via allosteric inter-domain cross-talk. Our findings paint a more elaborate picture of CoV attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

microbiology↗

bioRxiv2022

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

Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in 2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by cells commonly employed to propagate these viruses (MDCK and hCK), resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK 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 B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 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 IAVs 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.

microbiology↗