Lara K. Mahal

Post-traumatic osteoarthritis is a common sequela of joint injury in humans and companion animals, yet glycosylation changes associated with osteoarthritis remain poorly characterized. Lectin microarray analysis enables high-throughput profiling of glycan features in complex biofluids such as synovial fluid. Here, we used lectin microarrays to characterize synovial fluid glycosylation patterns in healthy and osteoarthritis-affected joints from horses, dogs, and humans. Comparative lectin-binding profiles within and across species revealed both conserved and species-specific glycomic signatures associated with osteoarthritis. Supervised machine learning distinguished osteoarthritis from control joints within a pooled multi-species framework and identified the lectins and their glycan epitopes contributing most strongly to classification, though cross-species generalization was limited and depended on the composition of each species’ control cohort. Elevated high-mannose emerged as the shared glycomic signature across species. Together, these findings support the feasibility of synovial fluid glycomics, integrated with machine learning, for identifying conserved osteoarthritis-associated glycan signatures; and inform the design of glycan-based diagnostic and translational strategies in both veterinary and human medicine.

Glycans form the outermost molecular interface of cells across all kingdoms of life, mediating recognition between organisms and their immune systems. Hypothetical mirror organisms would display glycans of reversed chirality, potentially impairing molecular recognition and contributing to proposed immune-evasion risks. Assessing DNA-encoded multivalent glycan arrays in settings ranging from purified proteins to mice, we found widespread recognition of mirror-image glycans by extant glycan-binding proteins, including recognition of L-glucose and L-galactose by the human immune receptor DC-SIGN. Mirror glycans were also selectively recognized by human immune cells and natural IgM antibodies and exhibited stereochemistry-dependent biodistribution in mice. Thus, glycan recognition can cross the chiral divide, revealing an unexpected molecular interface through which existing life could recognize and interact with hypothetical mirror life.

The post-transcriptional regulation of protein expression by miRNA has given rise to new technologies for therapeutic interventions and synthetic biology. To date, these technologies assume that miRNAs are repressors of protein expression; however, emerging evidence has shown that miRNA regulation of proteins is bidirectional. Although it is now clear that upregulation by miRNAs is a common event, the molecular requirements remain poorly characterized. To address this gap, we utilized the miRFluR assay, a genetically encoded dual-fluorescence reporter assay previously developed in our laboratory, to systematically explore upregulation. Using upregulation of the enzyme B3GLCT by miR-891b as a model system, we find that upregulation requires both specific sequence elements and RNA secondary structure. Mutagenesis and truncation analyses revealed that the effect is context-dependent and influenced by the local UTR structure. We also find that sites can be reengineered to be triggered by alternate miRNA, opening the door to synthetic biology manipulations. Our study provides a new perspective on upregulation by miRNA and sets the stage for further exploration of the bidirectional nature of miRNA action.

A central hallmark of Parkinson′s disease (PD) is the spread of α-Synuclein (α-Syn) aggregates, which is thought to contribute to its progressive nature. To better understand the mechanism of cellular internalization of α-Syn fibrils, we conducted a genome wide CRISPR activation (CRISPRa) screen to identify genetic modifiers of fibril uptake. We report here BRCA associated protein 1 (BAP1) as a regulator of fibril uptake into cells and validate its association with the genetic risk of PD. BAP1 regulates fibril entry into cells by acting as a master transcriptional regulator of the cell surface glycoproteome. BAP1 downregulates Heparan Sulfate Proteoglycan (HSPG) and upregulates O-linked glycoprotein expression, the net effect of which is to reduce fibril internalization. The effect of enhancing BAP1 expression is cell-type specific, as it reduces αSyn uptake in iPSC-derived dopaminergic neurons, but not in microglia. Using human midbrain organoids with integrated microglia, we find this effect is mediated through ASXL3 another PD risk gene and a non-obligatory component of the BAP1 Polycomb Repressive-Deubiquitinase Complex (PR-DUB). These findings are consistent with higher BAP1/ASXL3 co-expression in regions of the brain less vulnerable to PD pathology. Together, the work uncovers a novel role for BAP1/ASXL3 in regulating αSyn fibril internalization by remodelling the cell surface glycoproteome.

Native mass spectrometry (nMS) is a powerful label-free method for detecting biomolecular complexes, resolving stoichiometry, and quantifying affinity (Kd). However, signal overlap in heterogeneous systems often limits its accuracy. Charge detection (CD)-nMS, which independently measures the mass-to-charge ratio and charge of individual ions, overcomes this challenge, enabling quantitative analysis of polydisperse and high molecular weight complexes with unresolved charge states. Here, we systematically validate CD-nMS for precise Kd determination using soluble protein–ligand complexes of known Kd and extend its application to quantify glycan ligand binding by a highly glycosylated immune lectin. We then demonstrate the implementation of slow mixing mode (SLOMO), a time-resolved mixing method that corrects for nonuniform response factors of interacting species, using CD-nMS to enable robust quantification of protein–protein interactions. Finally, we apply SLOMO-CD-nMS to directly detect and quantify bacterial toxin binding to glycolipids embedded in membrane-like assemblies, a capability not accessible with conventional nMS. These measurements uncovered previously unrecognized assembly pathways and demonstrate, for the first time, that SLOMO-CD-nMS can resolve and quantify multivalent lectin engagement with glycolipids in a native-like membrane context. Collectively, these results establish CD-nMS, alone or in combination with SLOMO, as a broadly applicable assay for quantitative characterization of complex biomolecular interactions across soluble, glycosylated, and membrane-associated systems.

Altered glucose metabolism is an auspicious feature of solid tumors, but is it an intrinsic property of tumor cells or a metabolic adaptation to the tumor microenvironment? Using normal epithelial cells cultured in a physiological culture medium under conditions that mimic the physical properties of healthy or cancerous tissues, we establish multiomics relationships between the biochemical and physical properties of the microenvironment and its impact on biosynthetic outputs of altered glucose metabolism. We find that microenvironmental properties, such as hyperglycemia, can affect the composition and thickness of the epithelial glycocalyx, in part through the activity of mechanosensitive stress responses associated with Heat Shock Factor 1 (HSF1). Because glycocalyx thickness alters immune surveillance of epithelial-origin tumor cells, we examined the relationship between the HSF1-hyperglycemia axis in human breast tumors and validate it as a druggable vulnerability to license natural killer cell lethality against cancer cells.

C-type lectins (CTLs), many of which bind carbohydrates (glycans) in a Ca2+-dependent manner, are an important family of glycan-binding proteins that play essential roles in immune recognition and regulation. Among them, dendritic cell–specific ICAM-3-grabbing nonintegrin (DC-SIGN) is notable for its ability to recognize a broad range of self- and pathogen-derived glycans. Despite extensive study, the glycan binding specificity of DC-SIGN remains incompletely understood, in part because accurately quantifying intrinsic lectin–glycan binding affinities across diverse glycan repertoires remains challenging. Here, we employ native mass spectrometry (nMS), a label- and immobilization-free approach well suited for probing weak lectin–glycan interactions, to characterize the glycan-binding preferences of the carbohydrate recognition domain (CRD) of DC-SIGN. Native MS, applied directly or via a catch-and-release (CaR) strategy, enabled quantitative measurements and high-throughput screening under physiologically relevant, nonvolatile salt conditions. We further examined the binding of several divalent metal ions to the DC-SIGN CRD and their influence on glycan recognition. Although divalent cations other than Ca2+ bind the CRD with comparable affinity, they exert only modest effects on ligand binding. Screening a defined glycan library, including oligomannoses, human milk oligosaccharides, Lewis and blood group antigens, and ganglioside oligosaccharides, revealed a preference for oligomannoses and Lewis antigens, with affinity positively correlated with the number of mannose or fucose residues. Finally, application of CaR-nMS to a natural N-glycan library identified fucosylated N-glycans as DC-SIGN ligands. Together, these results define the glycan specificity of DC-SIGN and establish nMS as a platform for comprehensive interactome profiling of CTLs.

Recent synthesis of essential enzymes, such as DNA and RNA polymerases with opposite chirality, has boosted the feasibility of creating mirror-image life. Such life, if ever produced, will undoubtedly be coated by a dense display of glycans (glycoproteins, glycolipids, polysaccharides) built from enantiomers of common monosaccharides. Recognition of mirror-image glycans by extant glycan-binding proteins (GBPs) may be critical for colonization by or immune response to mirror life organisms. We evaluated recognition of enantiomers of common glycans by a diverse set of purified GBPs (plant and human derived), antibodies (including IgM from human plasma), mammalian cells (including immune cells), and organs in live animals. We found that GBP binding to enantiomers of naturally prevalent glycans is widespread. Notably, L-glucose and L-galactose interact with fucose-binding lectins, including DC-SIGN, a C-type lectin expressed on immune cells. These interactions can be inhibited by soluble “natural” glycan ligands and enantiomeric ones confirming specificity. Binding of L-glycans to diverse immune cell repertoires revealed preferences for specific glycan enantiomers. IgM antibodies from human serum showed donor-specific recognition of L-glycans. We propose that the recognition of L-glycans by extant GBPs arises from their co-evolution over millennia with the L-glycans that are present in the glycocalyx of many microorganisms.

Cooperative ligand binding is an important determinant of specificity and regulation in biomolecular complexes. Yet, its prevalence and mechanistic basis in glycan-binding proteins (GBPs) remain unclear. Here, we present the first quantitative analysis of the temperature dependence of cooperative glycan binding. Variable-temperature native mass spectrometry (VT-nMS) resolved sequential ligand binding to either the five primary or the five secondary sites of the cholera toxin B subunit homopentamer (CTB₅). Stepwise apparent affinities for the primary sites reveal positive cooperativity that strengthens with both ligand occupancy and temperature. Van’t Hoff analysis shows that this temperature-enhanced cooperativity is predominantly entropy driven. Mechanistic insight from temperature replica exchange molecular dynamics simulations shows that ligand binding at one primary site restrains a loop on an adjacent subunit (counterclockwise), widening its binding site. This prestructuring progressively lowers the unfavorable conformational entropy penalty of binding, independent of the order of subunit occupancy. In contrast, sequential ligand binding at the secondary sites exhibits negligible cooperativity except at the highest temperatures, although the enthalpic and entropic contributions are comparable in magnitude to those for primary-site binding, suggesting a shared energetic framework. Together, these results provide detailed thermodynamic and structural insight into cooperative GBP–glycan interactions and establish an integrated VT-nMS and molecular dynamics framework for quantitatively probing cooperative ligand binding in complex biomolecular systems.