Biology subjects

Janssen, B. J. C.

Publications and source records attributed to Janssen, B. J. C..

6 recordsLinked to original sources

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

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↗

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↗

bioRxiv2023

Alternative splicing controls teneurin-3 compact dimer formation for neuronalrecognition

Neuronal network formation is facilitated by recognition between synaptic cell adhesion molecules (CAMs) at the cell surface. Alternative splicing of CAMs provides additional specificity in forming neuronal connections. For the teneurin family of CAMs, alternative splicing of the EGF-repeats and NHL domain controls protein-protein interactions at the synapse. Here we present a 3.2 [A] cryo-EM structure of the compact dimeric ectodomain of teneurin-3 harbouring both splice inserts. This dimer is stabilised by an EGF8-ABD contact between subunits. Cryo-EM reconstructions of all four splice variants, together with SAXS and negative stain EM, reveal compacted dimers for each, with variant-specific dimeric arrangements. This results in specific trans-cellular interactions, as tested in cell clustering and stripe assays. The compact conformations provide a structural basis for teneurin homo- and heterophilic interactions. Altogether, our findings demonstrate how alternative splicing results in rearrangements of the dimeric subunits, influencing neuronal recognition and circuit wiring.

biochemistry↗

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↗

bioRxiv2021

Notch-Jagged signaling complex defined by an interaction mosaic

The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand Jagged1 to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical and structural techniques on the full extracellular complex and targeted sites,we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network.The Notch1 membrane-proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8-13 and Jagged1 C2-EGF3 activation sites, as well as to two additional Jagged1 regions, EGF 8-11 and cysteine-rich domain (CRD). XL-MS and quantitative interaction experiments show that the three Notch1 binding sites on Jagged1 also engage intramolecularly.These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility determined by small-angle X-ray scattering (SAXS), support the formation of backfolded architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal, but engage directly to control Notch1 signaling, thereby redefining the Notch1-Jagged1 activation mechanism and indicating new routes for therapeutic applications.

molecular biology↗