Biology subjects

Mahoney, K. E.

Publications and source records attributed to Mahoney, K. E..

12 recordsLinked to original sources

bioRxiv2026

N-glycoproteomic profiling of the hookworm Ancylostoma ceylanicum reveals heterogeneity and phosphorylcholine-modified glycans in larval and adult parasites.

Glycans of parasitic worms (helminths) have been previously described with roles in immunomodulation and the recognition of helminth proteins by the host immune system. Despite their significance in other systems, little is known about the glycosylation patterns of the hookworm Ancylostoma ceylanicum, an intestinal parasite of animals and humans, due to challenges in sample collection and structural complexity of many nematode glycans. Herein, we describe results of a released N-glycan analysis of adult hookworms, which revealed oligomannose, hybrid, and complex structures, including those with non-mammalian features. Next, we report the results of glycoproteomic analyses to identify hookworm glycoproteins and their associated glycan structures. We compared three enrichment methods and found that strong anion-exchange electrostatic repulsion liquid chromatography (SAX-ERLIC) led to the identification of the most N-glycopeptides. Analyses of larval and adult extracts revealed 82 larval and 157 adult N-glycoproteins, the majority of which were stage-specific. Of particular interest, phosphorylcholine (PC), a known immunomodulatory glycan modification, was detected in both stages, and several unique PC-modified structures were identified in each. Collectively, these results provide the first site-specific characterization of intact N-glycopeptides in A. ceylanicum and, with an adult-stage released glycan analysis, establish a method for glycoproteomic characterizations of other helminths.

microbiology↗

bioRxiv2026

Site-specific O-glycans influence lacritin structure and multimerization in tears

Lacritin is an abundantly expressed glycoprotein in tear fluid and plays key roles in immune response, tear secretion, and bacterial killing. These biological functions are tightly regulated through several biochemical mechanisms including multimerization, proteolysis, and alternative splicing, especially within its C-terminal domain. Given its critical role at the ocular surface, lacritin is currently under investigation as a diagnostic biomarker and therapeutic candidate for dry eye disease (DED). However, despite over three decades since its initial discovery, the functional significance of the O-glycans that comprise more than 50% of its molecular weight remain largely unknown. To address this gap, we leveraged mass spectrometry (MS)-based glycoproteomics and molecular dynamics (MD) to explore the structural role of site-specific O-glycans on C-terminal lacritin. In doing do, we identified distinct glycosylation profiles between monomeric and multimeric lacritin, particularly at glycosites located near crosslinking residues (Lys101 and Lys104) that modulate multimer formation. Building on our glycoproteomics data, we performed MD simulations on monomer and multimer glycoforms and revealed that O-glycans participate in intra-glycan-protein interactions, thereby affecting the conformational flexibility of lacritin and the spatial arrangement of Lys101 and Lys104. Finally, we quantified the solvent-accessible surface area (SASA) of Lys101 and Lys104, highlighting that proximal O-glycosylation is predicted to affect the propensity of these residues to participate in crosslinking. Taken together, these findings underscore a central role for lacritin O-glycans in affecting structural topology with implications for its downstream biological activity.

biophysics↗

bioRxiv2025

Mapping galectin-3 ligands in tear fluid establishes spliceoform-dependent lacritin binding

Galectin-3 (Gal-3) is a carbohydrate-binding protein which plays crucial roles in inflammation, immune response, cell migration, autophagy, and signaling. At the ocular surface, Gal-3 is also known to crosslink transmembrane mucins across the epithelial cell glycocalyx, forming lattice structures important for barrier function. However, the biological role of Gal-3 in circulating tear fluid remains largely unexplored. Similarly, whether Gal-3 engages extracellular glycoproteins in tears to affect downstream biological processes has yet to be investigated. As increased Gal-3 levels in tears are known to correlate with ocular pathologies such as dry eye disease (DED), we sought to elucidate the Gal-3 interactome in tear fluid and uncover biological insights into the function of Gal-3 beyond adhesion to the epithelial cell surface. Here, we combined ELISA, lectin affinity enrichment, mass spectrometry (MS)-based glycoproteomics, and lectin blotting to uncover Gal-3 interactors and their associated glycoepitopes. Overall, we report nearly 100 proteins enriched from tear fluid across 3 different patients, identifying proteins involved in immune response, inflammation, and antimicrobial activity. Most notably, we report lacritin as a novel ligand for Gal-3 and demonstrate that specific glycoforms of lacritin bearing core 2 O-glycans preferentially engage with Gal-3. Lastly, we show that the Gal-3-lacritin axis is spliceoform-specific and dependent on lacritin multimerization. Taken together, this study elucidates new ligands for Gal-3 in tear film and reveals mRNA splicing and multimerization as new biochemical mechanisms that fine-tune Gal-3 binding events.

biochemistry↗

bioRxiv2025

In-depth analysis of the tear fluid glycoproteome reveals diverse lacritin glycosylation and spliceoforms

Tear fluid comprises a diverse group of extracellular glycoproteins which are critical for ocular homeostasis. Within the tear fluid glycoproteome, lacritin is highly expressed and plays a key role in immune response, tear secretion, and antimicrobial activity. Importantly, glycosylation constitutes over 50% of lactritins molecular weight. However, despite this fact, nothing is known about the specific glycan structures on lacritin and how they influence its protein folding, function, or downstream biological processes. Similarly, it remains completely unknown whether alterations to lacritin glycans are correlated with ocular pathologies. To address this gap in knowledge, we harnessed mass spectrometry (MS) to conduct the first O-glycoproteomic study of tear fluid. Here, we report unprecedented coverage of lacritin glycosylation, detailing 19 O-glycosites bearing a myriad of glycan structures. Further, we leveraged Alphafold 3.0 and GlycoShape to visualize the impact of these glycans on its structure, demonstrating that O-glycosylation renders the protein backbone rigid and extended. Surprisingly, we also detected protein-level evidence of two lacritin spliceoforms, representing the first observation of these isoforms by MS. Simultaneously, we describe the most comprehensive characterization of the tear fluid glycoproteome to date, elucidating the glycosylation profile of Immunoglobulin A (IgA), lactoferrin, and other glycoproteins with demonstrated clinical relevance as diagnostic biomarkers. Overall, this study lays critical groundwork for future biochemical investigation of tear fluid glycoproteins and their application as diagnostic or therapeutic tools for ocular diseases.

biochemistry↗

bioRxiv2024

Glycosite Mapping and in situ Mass Spectrometry Imaging of MUC2 Glycopeptides via On-slide Digestion with Mucinase StcE

Many cancers are characterized by altered mucin expression and glycosylation, although the mechanistic relationship between tumor glycosylation and disease progression is not well-defined. Herein, our goal was to map specific mucin glycoforms in diseased tissue, enabling correlation of the tumor glycan profile with malignant features. To this end, we developed a workflow implementing on-tissue digestion with mucinase StcE, followed by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and liquid chromatography coupled to mass spectrometry (LC-MS). To optimize our workflow, we analyzed four different mucinous carcinomas derived from colon, esophageal, and salivary gland tissue. Using this technique, we deduced the spatial distribution of StcE-generated O-glycopeptides within mucinous tumors using MALDI-IMS. Subsequent LC-MS analyses revealed the identity of different species detected in imaging experiments, in addition to comprehensively characterizing the mucinome and proteome of each tissue. Our coupled MS approach unveiled a striking mucin 2 (MUC2) expression pattern in two colorectal mucinous adenocarcinomas, in which different glycoforms clearly stratified regions within the tumor. Notably, our LC-MS experiments obtained near-complete sequence coverage over the mucin domains of MUC2, enabling glycoproteomic mapping of this canonical mucin in unprecedented depth. MUC2 glycosylation was dominated by the T and Tn antigens, with surprisingly little sialylation detected. However, O-glycans containing mono- and di-O-acetylated sialic acid were detected in low abundance. Finally, we obtained spectral evidence for an endogenous O-acetylated GalNAc, an O-glycan structure not previously reported in the literature. Overall, this proof-of-concept work underscores the potential of this technique to generate new research avenues in oncology and beyond. Significance StatementAberrant mucin expression and glycosylation are hallmarks of cancer, but how these changes promote malignant processes are not well understood. Solid tumors are highly heterogeneous in their cellular and molecular composition, and many advanced spatial techniques have emerged in recent years to study the tumor microenvironment (TME) for better understanding disease progression. Spatially resolved glycoprotein analyses typically detect either the protein or glycan components, but not both. We developed a workflow using a dual mass spectrometry approach to map the location of intact glycopeptides in mucinous tumors, enabled by on-tissue digestion with the mucin-specific protease StcE. Future applications of this method on larger patient cohorts will enhance our understanding of glycans in malignancy, identify disease biomarkers, and define therapeutic targets.

cancer biology↗

bioRxiv2024

Quantification and site-specific analysis of co-occupied N- and O-glycopeptides

Protein glycosylation is a complex post-translational modification that is generally classified as N- or O-linked. Site-specific analysis of glycopeptides is accomplished with a variety of fragmentation methods, depending on the type of glycosylation being investigated and the instrumentation available. For instance, collisional dissociation methods are frequently used for N-glycoproteomic analysis with the assumption that one N-sequon exists per tryptic peptide. Alternatively, electron-based methods are indispensable for O-glycosite localization. However, the presence of simultaneously N- and O-glycosylated peptides could suggest the necessity of electron-based fragmentation methods for N-glycoproteomics, which is not commonly performed. Thus, we quantified the prevalence of N- and O-glycopeptides in mucins and other glycoproteins. A much higher frequency of co-occupancy within mucins was detected whereas only a negligible occurrence occurred within non-mucin glycoproteins. This was demonstrated from analyses of recombinant and/or purified proteins, as well as more complex samples. Where co-occupancy occurred, O-glycosites were frequently localized to the Ser/Thr within the N-sequon. Additionally, we found that O-glycans in close proximity to the occupied Asn were predominantly unelaborated core 1 structures, while those further away were more extended. Overall, we demonstrate electron-based methods are required for robust site-specific analysis of mucins, wherein co-occupancy is more prevalent. Conversely, collisional methods are generally sufficient for analyses of other types of glycoproteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/602348v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b14908org.highwire.dtl.DTLVardef@131e812org.highwire.dtl.DTLVardef@17b07cborg.highwire.dtl.DTLVardef@3cb199_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Xylosyltransferase Bump-and-hole Engineering to Chemically Manipulate Proteoglycans in Mammalian Cells

Mammalian cells orchestrate signalling through interaction events on their surfaces. Proteoglycans are an intricate part of these interactions, carrying large glycosaminoglycan polysaccharides that recruit signalling molecules. Despite their importance in development, cancer and neurobiology, a relatively small number of proteoglycans have been identified. In addition to the complexity of glycan extension, biosynthetic redundancy in the first protein glycosylation step by two xylosyltransferase isoenzymes XT1 and XT2 complicates annotation of proteoglycans. Here, we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. By employing a tactic termed bump- and-hole engineering, we engineer the two isoenzymes XT1 and XT2 to specifically transfer a chemically modified xylose analogue to target proteins. The chemical modification contains a bioorthogonal tag, allowing the ability to visualise and profile target proteins modified by both transferases in mammalian cells. The versatility of our approach allows pinpointing glycosylation sites by tandem mass spectrometry, and exploiting the chemical handle to manufacture proteoglycans with defined GAG chains for cellular applications. Engineered XT enzymes permit a view into proteoglycan biology that is orthogonal to conventional techniques in biochemistry.

biochemistry↗

bioRxiv2023

Optimized mucin-selective enrichment strategy to probe the mucinome

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of healthy and diseasedriven biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to solid support. While this method was effective, it suffered from low throughput and high sample requirements. Further, the elution step required boiling in SDS, thus necessitating an in-gel digest with trypsin. Here, we optimized our previous enrichment method to include elution conditions amenable to mucinase digestion and downstream analysis with mass spectrometry. This increased throughput and lowered sample input while maintaining mucin selectivity and enhancing glycopeptide signal. We then benchmarked this technique against different O-glycan binding moieties for their ability to enrich mucins from various cell lines and human serum. Overall, the new method outperformed our previous procedure and all other enrichment techniques tested. This allowed for effective isolation of more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, thus enhancing our ability to analyze the mucinome.

biochemistry↗

bioRxiv2023

False positive glycopeptide identification via in-FAIMS fragmentation

High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates glycopeptides in the gas phase prior to mass spectrometry (MS) analysis, thus offering the potential to analyze glycopeptides without prior enrichment. Several studies have demonstrated the ability of FAIMS to enhance glycopeptide detection but have primarily focused on N-glycosylation. Here, we evaluated FAIMS for O-glycoprotein and mucin-domain glycoprotein analysis using samples of varying complexity. We demonstrated that FAIMS was useful in increasingly complex samples, as it allowed for the identification of more glycosylated species. However, during our analyses, we observed a phenomenon called "in FAIMS fragmentation" (IFF) akin to in source fragmentation but occurring during FAIMS separation. FAIMS experiments showed a 2-5-fold increase in spectral matches from IFF compared to control experiments. These results were also replicated in previously published data, indicating that this is likely a systemic occurrence when using FAIMS. Our study highlights that although there are potential benefits to using FAIMS separation, caution must be exercised in data analysis because of prevalent IFF, which may limit its applicability in the broader field of O-glycoproteomics. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/542648v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@d94300org.highwire.dtl.DTLVardef@1bdc6dforg.highwire.dtl.DTLVardef@4b8ea8org.highwire.dtl.DTLVardef@82beed_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. In particular, the T cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, -3, -4) decorate immune cells and act as key checkpoint inhibitors in cancer. However, their dense O-glycosylation remains enigmatic both in terms of glycoproteomic landscape and structural dynamics, primarily due to the challenges associated with studying mucin domains. Here, we present a mucinase (SmE) and demonstrate its ability to selectively cleave along the mucin glycoprotein backbone, similar to others of its kind. Unlike other mucinases, though, SmE harbors the unique ability to cleave at residues bearing extremely complex glycans which enabled improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we performed molecular dynamics (MD) simulations of TIM-3 and -4 to demonstrate how glycosylation affects structural features of these proteins. Overall, we present a powerful workflow to better understand the detailed molecular structures of the mucinome.

cancer biology↗

bioRxiv2022

Comprehensive analysis of platelet glycoprotein Ibα glycosylation.

BackgroundPlatelet glycoprotein (GP) Ib is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand Factor (VWF). GPIb is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, VWF binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIb is not well characterized. ObjectivesThe aim of this study was to comprehensively analyze GPIb amino acid sites of glycosylation (glycosites) and glycan structures. MethodsGPIb ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry (MS) glycomics, and MS glycoproteomics to define glycosites and the structures of the attached glycans. ResultsWe identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABH blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified at least 48 unique O-glycosites and 1 N-glycosite. The GPIb mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain (MSD). ConclusionsThis comprehensive analysis of GPIb glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIb glycans. Essentials- Glycosylation of glycoprotein Ib (GPIb) is important for platelet function. - We report a comprehensive and site-specific analysis of human GPIb glycosylation. - GPIb carries sialoglycans, Tn antigen, T antigen, and ABO blood group (ABH) antigens. - We experimentally determined 48 O-glycosites and 1 N-glycosite by mass spectrometry.

biochemistry↗

bioRxiv2022

A systematic comparison of current bioinformatic tools for glycoproteomics data

Glycosylation is one of the most common post-translational modifications and generates an enormous amount of proteomic diversity; changes in glycosylation are associated with nearly all disease states. Intact glycoproteomics seeks to determine the site-localization and composition of glycans along a protein backbone via mass spectrometry. Following data acquisition, raw files are analyzed using search algorithms to define peptide sequence, glycan composition, and site localization. Glycoproteomics is rapidly expanding, creating the pressing need to establish bioinformatic community standards. Recently, several new search algorithms were released, many of which vary in terms of search strategy, localization system, score cutoffs, and glycan databases, thus warranting a comprehensive comparison of these new programs along with existing programs. Here, we analyzed three common samples: an enriched cell lysate, a mixture of 6 glycoproteins, and a mucin-domain glycoprotein. All raw files were searched with comparable parameters among software and the results were extensively manually validated to compare accuracy and completion of the output. Our results highlight the continued need for manual validation of glycopeptide spectral matches, especially for O-glycopeptides. Despite this, O-Pair outperformed all other programs in correct identification of O-glycopeptides and its localization system proved to be useful. On the other hand, Byonic and pGlyco performed best for N-glycoproteomics; the former was best for proteome-wide searches, but the latter identified more N-glycosites in less complex samples. Overall, we summarize the strengths, weaknesses, and potential improvements for these search algorithms. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/484528v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@17c371forg.highwire.dtl.DTLVardef@70532org.highwire.dtl.DTLVardef@e640b7org.highwire.dtl.DTLVardef@a2bb26_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗