Biology subjects

Chang, V.

Publications and source records attributed to Chang, V..

9 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

Substrate Stiffness Dictates Unique Doxorubicin-induced Senescence-associated Secretory Phenotypes and Transcriptomic Signatures in Human Pulmonary Fibroblasts

Cells are subjected to dynamic mechanical environments which impart forces and induce cellular responses. In age-related conditions like pulmonary fibrosis, there is both an increase in tissue stiffness and an accumulation of senescent cells. While senescent cells produce a senescence-associated secretory phenotype (SASP), the impact of physical stimuli on both cellular senescence and the SASP is not well understood. Here, we show that mechanical tension, modeled using cell culture substrate rigidity, influences senescent cell markers like SA-{beta}-gal and secretory phenotypes. Comparing human primary pulmonary fibroblasts (IMR-90) cultured on physiological (2 kPa), fibrotic (50 kPa), and plastic (approximately 3 GPa) substrates, followed by senescence induction using doxorubicin, we identified unique high-stiffness-driven secretory protein profiles using mass spectrometry and transcriptomic signatures, both showing an enrichment in collagen proteins. Consistently, clusters of p21+ cells are seen in fibrotic regions of bleomycin induced pulmonary fibrosis in mice. Computational meta-analysis of single-cell RNA sequencing datasets from human interstitial lung disease confirmed these stiffness SASP genes are highly expressed in disease fibroblasts and strongly correlate with mechanotransduction and senescence-related pathways. Thus, mechanical forces shape cell senescence and their secretory phenotypes.

cell biology↗

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↗

bioRxiv2022

Investigating the Consistency of Extracellular Vesicle Production from Breast Cancer Subtypes Using CELLine Adherent Bioreactors

Extracellular vesicle (EV) research has grown rapidly in recent years, largely due to the potential use of EVs as liquid biopsy biomarkers or therapeutics. However, in-depth characterisation and validation of EVs produced using conventional in vitro cultures can be challenging due to the large area of cell monolayers and volumes of culture media required. To overcome this obstacle, multiple bioreactor designs have been tested for EV production with varying success, but the consistency of EVs produced over time in these systems has not been reported previously. In this study, we demonstrate that several breast cancer cell lines of different subtypes can be cultured simultaneously in space, resource, and time efficient manner using CELLine AD 1000 systems, allowing the consistent production of vast amounts of EVs for downstream experimentation. We report an improved workflow used for inoculating, maintaining, and monitoring the bioreactors, their EV production, and the characterisation of the EVs produced. Lastly, our proteomic analyses of the EVs produced throughout the lifetime of the bioreactors show that core EV-associated proteins are relatively consistent, with few minor variations over time, and that tracking the production of EVs may be a convenient method for indirectly monitoring the bioreactors health. These findings will aid future studies requiring the simultaneous production of large amounts of EVs from several cell lines of different subtypes of a disease and other EV biomanufacturing applications.

cancer biology↗

bioRxiv2019

Cytochrome P450 26b1-mediated specification of vestibular striola and central zones is required for transient responses in linear acceleration

Each vestibular sensory epithelia of the inner ear is divided into two zones, the striola and extrastriola in maculae of otolith organs and the central and peripheral zones in cristae of semicircular canals, that differ in morphology and physiology. We found that formation of striolar/central zones during embryogenesis requires Cytochrome P450 26b1 (Cyp26b1)-mediated degradation of retinoic acid (RA). In Cyp26b1 conditional knockout mice, the identities of the striolar/central zones were compromised, including abnormal innervating neurons and otoconia in otolith organs. Vestibular evoked potentials (VsEP) in response to jerk stimuli were largely absent. Vestibulo-ocular reflexes and standard motor performances such as forced swimming were unaffected, but mutants had head tremors and deficits in balance beam tests that were consistent with abnormal vestibular input. Thus, degradation of RA during embryogenesis is required for patterning highly specialized regions of the vestibular sensory epithelia that may provide acute feedback about head motion.

neuroscience↗