Biology subjects

Sutherland, E.

Publications and source records attributed to Sutherland, E..

8 recordsLinked to original sources

bioRxiv2026

Defining glycoproteoform landscapes through an integrated glycoproteomics approach enabled by high-resolving power proton transfer charge reduction tandem mass spectrometry

Glycan heterogeneity is a fundamental property of glycoproteins. A holistic understanding of glycan modification states is critical to translating glycoproteome regulation to biological function, but the high degree of glycosite-level heterogeneity leads to technical challenges in measuring glycoproteoforms. Common bottom-up glycoproteomics provide some insights but cannot recapitulate the full ensemble of glycoproteoforms from glycopeptide measurements alone. Promising efforts to profile masses of intact glycoproteins have recently explored data-independent acquisition (DIA) coupled with proton-transfer charge reduction (PTCR) or electron-capture-induced charge reduction mass spectrometry (MS). While valuable for generating broad glycoproteoform mass distributions, these approaches have remained limited in their ability to generate discrete glycoproteoform mass measurements, largely because they rely on low-resolving power measurements and deconvolution that does not account for isotopic information. Here, we develop a DIA-PTCR workflow that couples high-resolving power (Rp ~240,000 at m/z 200) tandem mass spectra with an open-source processing suite to define glycoproteoform populations within 20 ppm mass accuracy thresholds. We demonstrate the glycoproteoform characterization capabilities of this platform using a collection of glycoproteins with well-described translational interests (EpCAM, TIGIT, CD40, PDL1, and CD24). With a focus on EpCAM, we showcase how intact glycoproteoform masses acquired using our high-resolving power DIA-PTCR (hRp-DIA-PTCR) approach can be integrated with bottom-up intact glycoproteomics and Direct-Mass Technology (i.e., Orbitrap-based charge-detection MS) acquisitions to inform structural and biological insights. Altogether, our hRp-DIA-PTCR method extends the current capabilities of intact glycoprotein analyses by enabling robust characterization of isotopically resolved proteoforms and facilitating deep biological interpretation of glycosylation heterogeneity. Our open-source informatics platform includes a GUI-based tool called PTsliCR to clean PTCR spectra directly from DIA-PTCR raw files and a deconvolution R package called IsoTrac, both of which are freely available on GitHub at https://github.com/riley-research.

systems biology↗

bioRxiv2025

Sialoglycans on human T cells attenuate death programs executed through the Fas pathway

T cells are critical executors of adaptive immune responses and their persistence is tightly regulated. Part of this regulation relies on programmed cell death driven by the Tumor Necrosis Factor (TNF) receptor superfamily. The addition of glycans that terminate in the monosaccharide sialic acid (sialoglycans) to these cell death receptors has been shown to attenuate their apoptotic functions. While this is now understood to be a pro-survival mechanism in settings of cancer pathophysiology, the specific roles of sialoglycans in regulating cell death receptor activity on human T cells remains unexplored. This is of particular importance given the rising interest in T cell glycan editing for therapeutic benefit. Here, we address this gap using both immortalized (Jurkat) and primary human T cells deficient in sialoglycans. We found that T cell sialoglycans suppressed apoptosis induced by the Fas receptor (FasR) but not other TNF receptor superfamily members such as TNFR1 and TRAIL-R1. Dynamic reorganization of FasR was increased on sialoglycan-deficient Jurkat cells, suggesting that these glycans limit receptor clustering. This model was further supported by phosphoproteomics results, which confirmed that loss of sialoglycans negatively regulated the pro-survival MAPK/ERK signalling pathway. Finally, we used a recombinant sialic acid cleaving enzyme (sialidase) to confirm that sialoglycans on primary human T cells are bona fide immunophysiological regulators of FasR-driven programmed cell death. Combined, our results demonstrate that sialoglycan remodelling on T cells influences cell fate driven by the Fas pathway and provide motivation to further characterize the immunoregulatory roles of the glycocalyx in health and disease.

immunology↗

bioRxiv2025

Extracting informative glycan-specific ions from glycopeptide MS/MS spectra with GlyCounter

Glycopeptide tandem mass spectra typically contain numerous glycan-specific fragments that can inform several features of glycan modifications, including glycan class, composition, and structure. While these fragment ions are often straightforward to observe by eye, few tools exist to systemically explore these common glycopeptide spectral features or explore their relationships to each other. Instead, most studies rely on manual inspection to understand glycan-informative ion content in their data, or they are restricted to evaluating the presence of these ions only in the small fraction of spectra that are identified by glycopeptide search algorithms. Here we introduce GlyCounter as a freely available, open-source tool to rapidly extract oxonium, Y-type, and custom ion information from raw data files. We highlight GlyCounters utility by evaluating glycan-specific fragments in a diverse selection of publicly available datasets to demonstrate how others in the field can make immediate use of this software. In several cases, we show how conclusions drawn in these publications are evident simply through GlyCounters extracted ion information without requiring database searches or experiment-specific programs. Although one of our goals is to decouple spectral evaluation from glycopeptide identification, we also show that evaluating oxonium ion content with GlyCounter can supplement a database search as valuable spectral evidence to validate conclusions. In all, we present GlyCounter as a user-friendly platform that can be easily incorporated into most glycoproteomic workflows to refine sample preparation, data acquisition, and post-acquisition identification methods through straightforward evaluation of the glycan content of glycoproteomic data. Software and instructions are available at https://github.com/riley-research/GlyCounter.

bioinformatics↗

bioRxiv2024

Overcoming the challenges of cascade reactions and complex substrates in natural product biocatalysis: Immobilization of a cyclodipeptide synthase.

Cyclodipeptide synthases (CDPSs) use aminoacylated tRNAs to produce cyclic dipeptide natural products which can have anticancer and neuroprotective activity. Despite their potential, applications involving CDPSs are hindered by enzyme instability and challenges in producing aminoacylated tRNAs. Immobilizing enzymes can enhance stability and recyclability, yet studies on immobilized enzymes using aminoacylated tRNAs are lacking. Here, we immobilized the CDPS enzyme from Parcubacteria bacterium RAAC4_OD1_1 (PbCDPS) using three sustainable supports: biochar from waste materials, calcium-alginate beads, and chitosan beads. Active PbCDPS immobilization led to production of the cyclodipeptide cyclo (His-Glu) (cHE). Notably, following activation with glutaraldehyde, a five-fold increase in cHE production was observed, while the immobilized enzyme remained active for seven consecutive cycles. Furthermore, we co-immobilized three enzymes required for the cascade reaction yielding cHE, all of which require aminoacyl-tRNA substrates (PbCDPS, histidyl-tRNA synthetase, and glutamyl-tRNA synthetase). This enzymatic cascade successfully generated the cyclic dipeptide of interest, showcasing the potential of immobilizing complex enzymes operating in cascade on a single support. We demonstrated that tRNAs remained free in solution without adsorption onto beads. This work paves the way for the immobilization of enzymes utilizing tRNAs and potentially other complex substrates, expanding the spectrum of reactions exploitable with this technology. Graphical AbstractO_LIFor Table of Contents Only C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/604810v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@12794baorg.highwire.dtl.DTLVardef@1956c09org.highwire.dtl.DTLVardef@e7f800org.highwire.dtl.DTLVardef@14ac97d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Broad substrate scope C-C oxidation in cyclodipeptides catalysed by a flavin-dependent filament

Cyclic dipeptides are produced by organisms in all domains of life. Many possess biological activities as anticancer and antimicrobial compounds. Oxidations are frequently present in these biologically active peptides. C-C bond oxidation is catalysed by tailoring enzymes, including cyclodipeptide oxidases. These flavin-dependent enzymes are underexplored due to their complex three-dimensional arrangement involving multiple copies of two distinct small subunits and unclear mechanistic features underlying substrate selection and catalysis. Here, we determined the structure and mechanism of the cyclodipeptide oxidase from the halophile Nocardiopsis dassonvillei (NdasCDO), part of the biosynthetic pathway for nocazine natural products. We show NdasCDO forms filaments in solution with a covalently bound FMN cofactor in the interface between three distinct subunits. The enzyme is promiscuous, using many cyclic dipeptides as substrates in a distributive manner. The reaction is optimal at high pH, involving the formation of a radical intermediate. Pre-steady state kinetics, a sizeable solvent kinetic isotope effect and lack of viscosity effects support that a step coupled to FMN regeneration determines the rate of the reaction. Our work dissects the complex mechanistic and structural features of this dehydrogenation reaction. This sets the stage to utilizing NdasCDO as a biocatalyst and expands the FMN-dependent oxidase family to include enzyme filaments.

biochemistry↗

bioRxiv2021

Structure, dynamics, and inhibition of Staphylococcus aureus m1A22-tRNA methyltransferase

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group from SAM to the N1 of adenine 22 in tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we describe the structural and functional characterisation of S. aureus TrmK. Crystal structures are reported for S. aureus TrmK apoenzyme and in complexes with SAM and SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. Activity assays for S. aureus TrmK-catalysed methylation of WT and position 22 mutants of tRNALeu demonstrate that the enzyme requires an adenine at position 22 of the tRNA. Intriguingly, a small RNA hairpin of 18 nucleotides is methylated by TrmK depending on the position of the adenine. In-silico screening of compounds suggested plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92, in the vicinity of the SAM-binding site, as the sole residue modified. These results these results identify a cryptic binding pocket of S. aureus TrmK and lay the foundation for future structure-based drug discovery.

biochemistry↗

bioRxiv2017

Temporal differentiation of bovine airway epithelial cells grown at an air-liquid interface

The respiratory epithelium is exposed to assault by toxins and pathogens through the process of inhalation, which has numerous implications on both human and animal health. As such, there is a need to develop and characterise an in vitro model of the airway epithelium to study respiratory pathologies during infection or toxicology experiments. This has been achieved by growing airway epithelial cells at an air-liquid interface (ALI). Characterisation of ALI models are not well-defined for airway epithelial cells derived from non-human species. In this study we have fully characterised a bovine airway epithelial cell models (AECM) grown at an ALI in relation to ex vivo tissue. The morphology of the model was monitored at three day intervals, to identify the time-period at which the culture was optimally differentiated. The model was shown to be fully-differentiated by day 21 post-ALI. The culture formed a stereotypical pseudostratified, columnar epithelium containing the major cell types of the bronchial epithelium (ciliated-, goblet- and basal cells). Once fully differentiated the bovine AECM displayed both barrier function, through the formation of tight-junctions, and active mucociliary clearance, important properties of the mucosal barrier. The bovine bronchial epithelial cells remained stable for three weeks, with no evidence of deterioration or dedifferentiation. The window in which the model displayed full differentiation was determined to be between day 21-42 post-ALI. Through comparison with ex vivo tissue derived from donor animals, our bovine AECM was shown to be highly representative of the in vivo bovine bronchial epithelium and can be utilised in the study of respiratory pathologies.

microbiology↗

bioRxiv2017

Interactions of pathogenic and commensal strains of Mannheimia haemolytica with differentiated bovine airway epithelial cells grown at an air-liquid interface

Mannheimia haemolytica serotype A2 is a common commensal species present in the nasopharynx of healthy cattle. However, prior to the onset of bovine pneumonic pasteurellosis, there is sudden increase in M. haemolytica serotype A1 within the upper respiratory tract. The events during this selective proliferation of serotype A1 strains are poorly characterised. In this investigation, a differentiated bovine airway epithelial cell culture was used to study the interactions of A1 and A2 bovine isolates with the respiratory epithelium. This model reproduced the key defences of the airway epithelium, including tight junctions and mucociliary clearance. Although initial adherence of the serotype A1 strains was low, by 12 hours post-infection the bacteria was able to traverse the tight junctions to form foci of infection below the apical surface. The size, density and number of these foci increased with time, as did the cytopathic effects observed in the bovine bronchial epithelial cells. Penetration of M. haemolytica A1 into the sub-apical epithelium was shown to be through transcytosis but not paracytosis. Commensal A2 bovine isolates however were not capable of colonising the model to a high degree, and did not penetrate the epithelium following initial adherence at the apical surface. This difference in their ability to colonise the respiratory epithelium may account for the sudden proliferation of serotype A1 in the onset of pneumonia pasteurellosis. The pathogenesis observed was replicated by virulent A2 ovine isolates; however colonisation was 10-fold lower in comparison to bovine A1 strains. This investigation provides new insight into the interactions of M. haemolytica with bovine airway epithelial cells which are occurring in vivo during pneumonia pasteurellosis.

microbiology↗