Biology subjects

Bineva-Todd, G.

Publications and source records attributed to Bineva-Todd, G..

7 recordsLinked to original sources

bioRxiv2026

An in vivo chemical genetic approach for targeted glycoproteome analysis in Drosophila melanogaster

Glycans impact every aspect of physiology. Despite the rapid increase in relevance and translational potential, there remains a lack of tools for studying glycosylation in vivo. Here, we present FlyMOE, a chemical-genetic platform for targeted glycoprotein profiling in Drosophila melanogaster. Transgenic flies are equipped with biosynthetic capabilities to install bioorthogonally-tagged monosaccharides into the cellular glycoproteome. The genetic tractability of Drosophila allows for versatile application based on developmental stages, genetic drivers and glycosyltransferase enzymes. We employ FlyMOE to create an overview of the glycoproteome of Drosophila tissues and embryos based on mass spectrometry glycoproteomics, and investigate the substrates of individual members of the PGANT glycosyltransferase family in vivo. FlyMOE will allow the detailed investigation of the glycoproteome in Drosophila and similarly genetically modifiable in vivo model systems.

bioengineering↗

bioRxiv2026

Iterative Bump-and-hole engineering creates a bioorthogonal reporter for N-acetylglucosaminyltransferase I

Asparagine-linked protein glycosylation is among the most frequent modifications of proteins trafficking through the secretory pathway. These glycans are manufactured in an assembly line process to a common precursor that is then subject to individual modifications with different levels of complexity. An important biosynthetic modulator is the incorporation of N-acetylglucosamine (GlcNAc) at distinct positions in N-linked glycan biosynthesis, commencing with the activity of the glycosyltransferase MGAT1. While mapping of N-glycans to their corresponding protein attachment sites is generally possible, not much is known about the glycoprotein substrate choice for MGAT1 and related transferases. Analogs of GlcNAc with small bioorthogonal tags can be incorporated into N-glycans. However, due to the promiscuity of some GlcNAc transferases, incorporation is of little specificity towards individual positions. Here, we report an iterative bump-and-hole approach in the design of a bioorthogonal precision tool for the activity of MGAT1 in mammalian cells. Structure-informed protein engineering abrogated the activity of MGAT1 towards the nucleotide-sugar UDP-GlcNAc while retaining activity towards bumped, azide-modified analogs. Kinetic and computational analyses using a neural network approach informed the synthesis of a tailored UDP-GlcNAc analog with preferential acceptance by the engineered enzyme. Following substrate biosynthesis, the strategy allowed selective incorporation of a chemical tag on MGAT1 substrate proteins in living mammalian cells with little background incorporation by other GlcNAc transferases. Our work expands the toolbox for glycan-based reporter compounds.

biochemistry↗

bioRxiv2022

O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Contribute to the Mutational Trajectory in Variants of Concern

The emergence of a polybasic cleavage motif for the protease furin in the SARS-CoV-2 spike protein has been established as a major factor for enhanced viral transmission in humans. The peptide region N-terminal to that motif is extensively mutated in major variants of concern including Alpha, Delta and Omicron. Besides furin, spike proteins from these variants appear to rely on other proteases for maturation, including TMPRSS2 that may share the same cleavage motif. Glycans found near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, with a suite of chemical tools, we establish O-linked glycosylation as a major determinant of SARS-CoV-2 spike cleavage by the host proteases furin and TMPRSS2, and as a likely driving force for the emergence of common mutations in variants of concern. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, and this glycosylation event is suppressed by many, but not all variant mutations. A novel strategy for rapid bioorthogonal modification of Thr678-containing glycopeptides revealed that introduction of a negative charge completely abrogates furin activity. In a panel of synthetic glycopeptides containing elaborated O-glycans, we found that the sole incorporation of N-acetylgalactosamine did not substantially impact furin activity, but the presence of sialic acid in elaborated O-glycans reduced furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on spike cleavage by TMPRSS2. With a chemistry-centered approach, we firmly establish O-glycosylation as a major determinant of spike maturation and propose that a disruption of O-GalNAc glycosylation is a substantial driving force for the evolution of variants of concern. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/508093v3_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@71b54eorg.highwire.dtl.DTLVardef@1361b4aorg.highwire.dtl.DTLVardef@139bdd9org.highwire.dtl.DTLVardef@1df192b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Cell-specific Bioorthogonal Tagging of Glycoproteins in Co-culture

Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.

synthetic biology↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp3 Papain-like Protease

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom chemical library from which we identified Dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

biochemistry↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of nsp5 Main Protease

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5,000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

biochemistry↗

bioRxiv2020

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe GalNAzMe that is specific for cancer-relevant Ser/Thr-N-acetylgalactosamine (O-GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn (N)-linked glycans. We equip cells with the capacity to biosynthesize the nucleotide-sugar donor UDP-GalNAzMe from a caged precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR knock-out (KO) screen, and imaging of intestinal organoids. GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation. Significance statementA large portion of all secreted and cell surface proteins in humans are modified by Ser/Thr(O)-linked glycosylation with N-acetylgalactosamine (GalNAc). While of fundamental importance in health and disease, O-GalNAc glycosylation is technically challenging to study because of a lack of specific tools to be used in biological assays. Here, we design an O-GalNAc specific reporter molecule termed GalNAzMe to selectively label O-GalNAc glycoproteins in living human cells. GalNAzMe is compatible with a range of experiments in quantitative biology to broaden our understanding of glycosylation. We further demonstrate that labeling is genetically programmable by expression of a mutant glycosyltransferase, allowing application even to experiments with low inherent sensitivity.

synthetic biology↗