Biology subjects

Boons, G.-J. P. H.

Publications and source records attributed to Boons, G.-J. P. H..

3 recordsLinked to original sources

bioRxiv2023

Glycoproteomics-compatible MS/MS-based quantification of glycopeptide isomers

Glycosylation is an essential protein modification occurring on the majority of extracellular human proteins, mass spectrometry (MS) being an indispensable tool for its analysis. Not only can MS determine glycan compositions, but also position the glycan at specific sites via glycoproteomics. However, glycans are complex branching structures with monosaccharides interconnected in a variety of biologically relevant linkages - isomeric properties which are invisible when the readout is mass alone. Here, we developed an LC-MS/MS-based workflow for determining glycopeptide isomer ratios. Making use of isomerically-defined glyco(peptide) standards, we observed marked differences in fragmentation behavior between isomer pairs when subjected to collision energy gradients, specifically in terms of galactosylation/sialylation branching and linkage. These behaviors were developed into component variables that allowed relative quantification of isomerism within mixtures. Importantly, at least for small peptides, the isomer quantification appeared largely independent from the peptide portion of the conjugate, allowing broad application of the method.

biochemistry↗

bioRxiv2020

Coronavirus hemagglutinin-esterase and spike proteins co-evolve for functional balance and optimal virion avidity

Human coronaviruses OC43 and HKU1 are respiratory pathogen of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spill-over. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase HE acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity towards clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked-out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor-binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations selected for cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and co-evolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses (IAVs). Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.

microbiology↗

bioRxiv2018

Identification of lead compounds targeting the dTDP-L-rhamnose biosynthesis pathway using Streptococcus pyogenes

Biosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS) and Streptococcus mutans. Those pathogens require dTDP-L-rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirm that GAS RmlB and RmlC are critical for dTDP-L-rhamnose biosynthesis through their action as dTDP-glucose-4,6-dehydratase and dTDP-4-keto-6-deoxyglucose-3,5-epimerase enzymes, respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues in these enzymes. Bio-layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP-rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS as well as several other rhamnose-dependent streptococcal pathogens with an MIC50 of 120-410 M. We therefore conclude that inhibition of dTDP-L-rhamnose biosynthesis such as Ri03 affect streptococcal viability and can serve as a lead compound for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria.

microbiology↗