Glycobiology

Glycans coat the surface of all cells, and every glycan is recognised by specific glycan-binding pro-teins (GBPs). There are no general tools that can accurately estimate the binding strength between glycan and GBP from the amino acid sequence of the GBP and the molecular structure of the glycan, represented as SMILES string. We describe models for predicting such binding strengths developed as a part of a Capstone Course at the University of Alberta. The models are trained on a dataset that combines BindingDB, a published database of small-molecule protein interactions, and data from glycan arrays measured by Consortium of Functional Glycomics (CFG). In this hybrid dataset of protein-ligand interactions the ligands are both glycans from CFG and small molecules from BindingDB; similarly, proteins include GBP and proteins from BindingDB. Three models are presented (i) ProMax which fuses ESM-2, MolFormer, and MolCLR features; (ii) APEX which constrains learning to a predetermined form, a physical model of binding; (iii) UltraMax adds inter-atomic distances for the ligands. To address the dataset's severe long-tail distribution, the models employ tail-aware losses for rare high-binding instances. Trained and evaluated on approximately one million protein--ligand pairs using hold-out splits for unseen molecules, the three models provide a unified framework for quantitative glycan-protein binding prediction. We observed that learning glycan-protein binding is harder than the similar task of learning small-molecule-protein interactions. Simple mirror-inversion tests led us to postulate that insufficient use of chiral features is an important source of difficulty in learning these interactions.

Cross-reactive antibodies against dengue virus are known to cause antibody-dependent enhancement (ADE) of infection or disease severity under specific conditions. In our previous study, we showed that primary immunization with the COVID-19 vaccine induces induces cross-reactive IgG causing ADE against dengue. In the present study, we investigated the influence of IgG Fc-glycosylation (analyzed by LC-MS/MS) on ADE mediated by cross-reactive IgG against dengue from IgG against SARS-CoV-2. We found a clear correlation between anti-DENV2 E IgG2 galactosylation and the ADE capacity of cross-reactive IgG against dengue in individuals vaccinated against COVID-19. IgG2 sialylation increased over time; however, it was not correlated with ADE capacity. This phenomenon was restricted to IgG2, whereas anti-DENV2 E IgG1 Fc-glycosylation remained stable after COVID-19 vaccination.

CHI3L1 (YKL-40) is a chitinase-like glycoprotein involved in immune regulation, tissue remodeling, and cancer, yet the molecular principles governing its glycan interactions remain incompletely defined. Previous reports suggested that CHI3L1 can bind to chitin oligosaccharides (COS) and glycosaminoglycan (GAG) ligands, however, the molecular basis and binding sites underlying these interactions remain controversial. Here, a combination of biophysical and computational methods is employed to shed light on carbohydrate interactions of the protein and delineate a potential crosstalk between its glycan-binding interfaces. Our results demonstrate that COS and GAGs bind to distinct, non-overlapping sites on CHI3L1. Both ligand classes exhibit a strong dependence of binding affinity on the degree of polymerization. Molecular dynamics simulations, supported by mutational analysis, identify a GAG-binding site centered on residues R144, R145, and K147 and reveal an additional distal interaction site for longer GAG ligands. Biophysical and biochemical assays fail to confirm a previously proposed allo- or orthosteric interaction between both binding sites. However, physiologically relevant protein-protein interactions mediated by the chitin binding site of CHI3L1 are differentially regulated by GAG and COS ligands. COS inhibit binding of galectin-3 to CHI3L1, whereas GAG ligands enhance the affinity between the proteins by ca. 14-fold. Together, these findings establish CHI3L1 as a dual carbohydrate-binding protein with distinct recognition interfaces and reveal a previously unrecognized role for GAGs in modulating CHI3L1-mediated signaling interactions.

The glycocalyx of red blood cells (RBCs), a negatively charged surface layer rich in sialic acid residues, plays a crucial role in modulating RBC aggregation. In pathological conditions such as diabetes and sepsis, glycocalyx degradation is often observed along with abnormal RBC aggregation. However, the mechanistic relationship between these phenomena remains poorly defined. In this study, we investigate the effects of enzymatic glycocalyx degradation on RBC aggregation under physiologically relevant flow conditions. Using neuraminidase from Clostridium perfringens (C. welchii) at varying concentrations, we selectively removed sialic acid residues from the RBC glycocalyx, simulating different levels of desialylation observed in health and disease. Confocal microscopy confirmed the dose-dependent depletion of membrane sialic acid, while microfluidic experiments revealed a significant increase in both the size and stability of the RBC aggregates after enzymatic treatment. Our findings suggest that glycocalyx integrity is a crucial biophysical determinant of RBC aggregation, likely influencing both electrostatic repulsion and hydrodynamic forces. This study provides new insights into how the enzymatic modification of the glycocalyx contributes to pathological hemorheology and may inform future strategies for the diagnosis or treatment of vascular diseases.

Fc receptor-like protein 5 (FCRL5) is a low-affinity IgG receptor expressed on B cells, with emerging therapeutic relevance due to its expression on multiple myeloma cells, and a potential role in regulating B cell responses. Previous reports on the FCRL5-IgG interaction vary widely in reported affinities, binding differences across IgG subclasses, and molecular requirements for maximal binding. Furthermore, the impact of Fc-engineering strategies, as used in (therapeutic) monoclonal antibodies, remains poorly understood. Here, we provide a comprehensive biochemical analysis of the FCRL5-IgG interaction. We demonstrate that FCRL5 is a true IgG Fc-receptor, binding with very low affinity (60-80 M). FCRL5 binds IgG in a manner involving primarily the two N-terminal domains of FCRL5, and the third domain for maximal binding, but with distinct essential residues in the IgG Fc-tail. Surface plasmon resonance analysis of the binding of FCRL5 to the various IgG subclasses revealed a preference for IgG1 and IgG4. Interestingly, various Fc-engineered IgG variants commonly used for silencing or enhancing of Fc receptor binding do not impact FCRL5 binding. Screening the binding of a set of IgG antibodies carrying defined sets of Fc-mutations to FCRL5 revealed E293 as a key binding determinant and led to the discovery of E293R as a mutation that selectively abrogates FCRL5 binding while preserving binding to other classical Fc{gamma}Rs. Lastly, we show that FCRL5 has considerable preference for binding afucosylated IgG. Together, our results define the essential characteristics of the IgG-FCRL5 interaction and demonstrate the potential of both naturally occurring IgG variants as well as therapeutically explored bioengineered IgG formats to differentially engage FCRL5.

Congenital disorders of glycosylation and deglycosylation are rare, serious, and lethal disorders afflicting humans. CDGs and CDDGs result in loss of function enzymes which fail to build or break down oligosaccharides on proteins. This can produce protein aggregates and, in turn, reactive oxygen species that harm the cell eventually leading to autophagy and apoptosis. Because sperm contain high concentrations of polyunsaturated fatty acids, they are especially sensitive to these effects, which is understood as one of the leading factors in human male infertility. Sperm are developed in zebrafish similarly to humans and are useful models to examine human reproductive health, as well as genetic disorders. The combination of these advantages makes the analysis of sperm from zebrafish with heterozygous ALG1 or DPAGT1 CDGs or the NGLY1 CDDG suitable. Analysis of sperm concentration, motility, status, viability, and hypoosmotic swelling demonstrated the effects of these disorders on sperm quality. Results showed a significant decrease in sperm concentration, motility, and hypoosmotic swelling for all mutant zebrafish compared to the wild type. This suggests that CDGs and CDDGs influence the amount of sperm produced, the percentage of sperm cells that are mobile, and the integrity of the plasma membrane.

Glycosaminoglycans (GAGs) are linear, negatively charged, polysaccharides that mediate a wide variety of biologically critical interactions with proteins, underpinning growth factor signalling, extracellular matrix assembly and numerous disease processes. However, GAG-protein interactions remain under characterised, in part because of the lack of high-throughput tools to systematically profile binding across the GAG interactome. In this paper we present a novel Surface Plasmon Resonance-based array methodology utilising 16 commonly sourced GAG preparations (including chondroitin sulphate (CS), dermatan sulphate (DS), heparan sulphate, heparin, hyaluronan and keratan sulphate) allowing the specificity and affinity of GAG-binding proteins to be determined. As proof of principle, we have validated the array using four established GAG-binding proteins (antithrombin III, CD44, heavy chain 1 from inter--inhibitor and Slit2), generating data consistent with the known binding specificities and quantifying affinities for many of the interactions. The array also reveals previously unreported GAG interactions, including Slit2 binding to CS and DS, and CD44 binding to chondroitin sulphate E.

Peptidoglycan is an essential component of the bacterial cell wall, providing mechanical strength and maintaining cell shape. It consists of glycan chains crosslinked by short peptide stems, resulting in a chemically heterogeneous macromolecule that remains challenging to study in a well-defined form. Access to discrete peptidoglycan fragments has therefore been critical for advancing biochemical and structural studies of cell wall-active enzymes. However, current synthetic, semi-synthetic, and cell wall extraction approaches remain limited by the complexity of carbohydrate chemistry and the difficulty of isolating pure, well-defined material. Here, we report a facile enzymatic approach for generating defined, denuded peptidoglycan oligosaccharides from the cell walls of two Staphylococcus species. These oligosaccharides, which terminate in N-acetylglucosamine and range from two to five disaccharide units in length, serve as substrates for a diverse panel of peptidoglycan-active enzymes that cleave or chemically modify the glycan backbone. We further show that these denuded oligosaccharides can be used in lysozyme-catalyzed transglycosylation reactions to generate p-nitrophenyl derivatives, enabling continuous colorimetric monitoring of peptidoglycan-cleaving enzymes. This method provides a practical route to defined peptidoglycan glycans and establishes a platform for further structural diversification, including stem peptide reattachment, quantitative enzyme assays, and structural characterization of peptidoglycan-binding proteins.

The extracellular matrix (ECM) is a complex network of ubiquitously present acellular material that plays a critical role in cell proliferation, migration, invasion, and tissue morphogenesis. Non-enzymatic glycation of ECM modifies the structure and function of ECM proteins and can support a pro-inflammatory milieu in the tumor microenvironment. However, the impact of glycated ECM on cancer cell growth remains underexplored despite its importance in facilitating disease progression. Here, we investigate the effect of ECM glycation on cancer cell morphology and migration behavior. We used methylglyoxal (MG) as a glycation agent and collagen as our ECM model protein. For in vitro growth analysis, breast cancer cells were seeded on growth surfaces coated with both non-glycated and glycated collagen. Cell behavior was monitored for 24 hours using a real-time holographic imaging system. Holographic image analysis revealed significant differences between non-glycated and glycated growth substrates in cell spreading area, eccentricity, perimeter length, optical thickness, and optical volume, as well as cell migration and motility, which directly influence cell adhesion and proliferation. These changes were found to be cell line biased. Overall, our findings suggest that ECM glycation has a significant effect on cell morphology, migration and cell growth. Holographic live cell imaging was determined to be an excellent method to monitor cells without the need for any labeling and with minimal perturbations.

Blood group antigens, defined by epitopes on the erythrocyte surface, are central to transfusion safety and maternal-fetal compatibility. While the genetic basis of many clinically relevant blood group antigens is well established, which structural and biophysical parameters determine whether a single-nucleotide variant gives rise to an antigenic phenotype remains unclear. Here, we integrate structural, biophysical, and evolutionary analyses to systematically evaluate features associated with single amino acid substitutions across 24 human protein-based blood group systems. We analyse 319 variants with curated phenotypic annotations alongside 481 control variants, identifying key determinants of null and antigenic phenotypes. Null variants are characterized by high evolutionary conservation, burial within the protein core, loss of hydrophobicity, increased polarity, and a propensity for arginine substitutions. Antigenic variants are also enriched in arginine; however, in contrast to null variants, they tend to occur at less conserved, more solvent-accessible, and structurally flexible sites. Supervised machine learning models trained on structural and biophysical descriptors were applied to distinguish (i) null and (ii) antigenic variants from controls, achieving balanced accuracies of 0.82 and 0.63, respectively. Feature importance analysis identified predicted pathogenicity, solvent accessibility, and evolutionary conservation as the most predictive determinants of null variants, whereas hydrophobicity, conservation, and flexibility dominated antigen prediction. This work establishes a framework linking molecular variation to blood group phenotypes and provides a foundation for predicting the impact of novel missense mutations in transfusion medicine and beyond.

Glycopeptide enrichment remains a cornerstone in glycoproteomics, but bias and reproducibility issues continue to hinder biological insight and clinical translation. Using curated glycoproteomics datasets and machine learning, we trained a glycopeptide classifier to promptly recognize N-glycopeptide precursor ions in peptide mixtures through mass defect signatures. Integration of the classifier into a data-dependent acquisition framework facilitated efficient and unbiased real-time prediction of N-glycopeptides directly from serum opening avenues for enrichment-free glycoproteomics.

Glycoside hydrolases (GHs) exhibit remarkable specificity dictated by the structural configuration of their target glycosidic linkages. While enzymes that process -1,4- and -1,6-linkages in starch or glycogen are well-characterized, those acting on less common bonds, such as -1,2-glucosidic linkages, remain largely underexplored. In this study, we report the discovery and structural elucidation of a novel -1,2-glucosidase from Arthrobacter humicola A8F5 (A8F5 glucosidase), representing a newly uncovered activity within the poorly characterized GH176 family. Biochemical characterizations revealed that A8F5 glucosidase exclusively cleaves -1,2-linkages via an anomer-inverting mechanism, with a distinct preference for short kojioligosaccharides. To circumvent crystallization obstacles caused by high loop flexibility and translational non-crystallographic symmetry, we engineered a loop-truncated variant. This strategy enabled the determination of high-resolution (up to 1.79 [A]) crystal structures of the enzyme in its ligand-free form and in complex with kojibiose, kojitriose, and selaginose. A8F5 glucosidase adopts a (/{beta})6-barrel fold characteristic of clan GH-G. Complementing the crystal structures with AlphaFold3 prediction demonstrated that two prominent active-site loops (loops 3 and 4) adopt a closed conformation that constricts the catalytic pocket, rendering the architecture suitable for short oligosaccharide recognition while restricting access to larger polymers. Furthermore, sequence similarity network analysis highlights vast, uncharacterized functional diversity within the GH176 family. These findings revealed that the GH176 enzyme recognizes and hydrolyses -1,2-glucosidic bonds through a structural framework distinct from that of the previously known clan GH-L GH65 kojibiose hydrolase, expanding the known functional landscape of this enzyme group toward rare -glucans.

BackgroundDysregulated cell migration leading to metastasis remains the primary cause of cancer-related mortality. It has been challenging to understand how cells regulate migration. We have previously created the first selective inhibitor of cell migration, KBU2046. Here, we use it as a probe to identify regulatory processes. MethodsMetastatic and primary human prostate cancer cells were treated for different times and at different concentrations with KBU2046. Immunofluorescent microscopy examined protein localization in cells. Label-free mass spectrometry (MS) was performed on total cell proteins, Tandem Mass Tag (TMT) labeling MS was used on membrane fractions, and temporal phosphoproteomic profiling. Results were analyzed with a suite of bioinformatic tools. ResultsKBU2046-induced migrastasis is associated with the accumulation of activated integrin {beta}1 into focal adhesions. Whole-cell proteomics demonstrated suppression of processes that mediate intracellular protein trafficking and increases in mitochondrial energy-generation signatures. Evaluation of the membrane fraction identified increases in membrane repair and maintenance processes and decreases in those that drive motility. Temporal- and concentration-dependent phosphoproteomic profiling revealed that KBU2046 initiates a dynamic, cascading sequence of transient signaling waves rather than a static block. ConclusionsKBU2046-induced migrastasis appears to operate through spatial decoupling rather than structural degradation. By restricting the intracellular trafficking machinery required for receptor recycling, KBU2046 limits focal adhesion turnover, providing a correlative framework to inhibit metastatic dissemination independent of direct cytotoxicity. O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/736165v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1cc69d3org.highwire.dtl.DTLVardef@137b843org.highwire.dtl.DTLVardef@1225e50org.highwire.dtl.DTLVardef@15dd8d2_HPS_FORMAT_FIGEXP M_FIG Graphic Abstract C_FIG

Botulinum neurotoxin (BoNT) is an etiologic agent of food poisoning caused by Clostridium botulinum. The large progenitor toxin complex (L-PTC) crosses the intestinal epithelial barrier to deliver BoNT to target neurons; however, it is not clearly understood how BoNT enters the host. Here, we identified low-density lipoprotein receptor-related protein 1 (LRP1) as a major enterocyte transcytosis receptor for the hyper-oral-toxic L-PTC serotype B-Okra (L-PTC/BOkra). We found that hemagglutinin (HA), a neurotoxin-associated protein within the L-PTC/BOkra complex, binds to LRP1 via N-glycans. HA/BOkra co-localized with LRP1 within the internalized vesicles in cultured cells and enterocytes. LRP1 deletion inhibited the apical-to-basal transcytosis of L-PTC/BOkra in an intestinal epithelial cell line, and this effect was rescued by LRP1 re-expression. Finally, intestinal epithelial cell-specific LRP1-deficient mice displayed reduced susceptibility to toxicity caused by oral administration of L-PTC/BOkra. Taken together, these results indicate that N-glycosylated LRP1 mediates L-PTC/BOkra transcytosis via enterocytes, enabling BoNT to traverse the intestinal epithelial barrier.

The unique peri-islet and double-layered vascular basement membrane (BM) of the human pancreatic islet are critical regulators of beta cell survival and function. While animal models imply that endothelial cells (ECs) are the exclusive source of islet BM, the precise cellular origins and spatial organization of the human islet matrisome remain poorly defined due to overlap in genes that mark non-epithelial cell populations and loss of spatial context during single-cell dissociation. In this study, we combine computational integration of whole-pancreas single-cell transcriptomes using CONCORD with high-resolution MERFISH spatial genomics to map the extracellular matrix (ECM) landscape across 251,477 spatially resolved cells from seven non-diabetic and five type 2 diabetic human donors. Contrary to an endothelial-centric paradigm, our data support a cooperative division of labor in the provision of BM, where pericytes represent the dominant transcriptional source of structural BM collagens (COL4A1, COL4A2) and ECs selectively express complementary matrix factors (HSPG2, LAMA5). Spatial neighborhood analysis further resolves a specialized population of islet-associated fibroblasts enriched at the islet boundary that are characterized by expression of peri-islet laminin genes. In type 2 diabetes, this homeostatic perivascular niche changes composition, marked by a significant increase in the islet fibroblast-to-pericyte ratio. Concurrently, islet pericytes undergo pro-fibrotic reprogramming characterized by the loss of canonical identity markers (PDGFRB), altered expression of ECM genes including COL1A2 and COL18A1, and upregulation of contractile machinery (MYL9). In the non-diabetic pancreas, pericytes constitute the principal vascular BM-expressing population within islets, whereas type 2 diabetes is associated with coordinated, compartment-specific remodeling of vascular-supportive stromal populations. Research in ContextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIExtracellular matrix (ECM), and in particular basement membrane (BM), are essential structural and signaling components of the pancreatic islet microenvironment that contribute to beta cell function and survival. C_LIO_LIIslet capillaries are closely associated with endocrine cells and are surrounded by specialized BMs; however, the cellular sources of these BM components in the adult human pancreas remain incompletely defined. C_LIO_LIType 2 diabetes is associated with islet fibrosis and vascular dysfunction, but cell type-specific alterations in ECM-producing populations have not been comprehensively characterized in situ. C_LI What is the key question?O_LIWhich cell populations produce the components of ECM, including BM, within the adult human islet, and how are these populations altered in type 2 diabetes? C_LI What are the new findings?O_LISpatial transcriptomics identifies pericytes as the predominant vascular-associated source of ECM, including BM, gene expression in human islets, whereas endothelial cells exhibit complementary but more limited matrix-producing programs. C_LIO_LISpatial transcriptomics identifies an islet-associated fibroblast population enriched for fibrillar collagen and BM-associated genes that localizes preferentially to the islet surface niche. C_LIO_LIType 2 diabetes is associated with remodeling of perivascular ECM programs, including reduced expression of vascular basement membrane genes, a shift from a pericyte to smooth muscle-like identity, and increased expression of matrix-remodeling and fibrosis-associated genes. C_LI How might this impact clinical practice in the foreseeable future?O_LIDefining the cellular sources and disease-associated remodeling of the human islet ECM may inform the development of therapies aimed at preserving or restoring the islet microenvironment in type 2 diabetes. C_LIO_LIIncluding key subtypes of islet-associated ECM-producing cells may be important in improving current protocols to generate replacement islets from human pluripotent stem cells for cell replacement therapy for diabetes. C_LI

Glycation, a non-enzymatic reaction occurring between sugars and biological macromolecules, plays a critical role in ageing and disease pathogenesis. Methylglyoxal (MG) is a highly reactive -oxoaldehyde that leads to the formation of endogenous advanced glycation end products (AGEs). These AGEs are associated with diabetes and many other diseases, including neurodegeneration and cancer. This is often through interactions with the receptor for advanced glycation end products (RAGE). Inhibition of glycation/AGEs formation using natural products to target cancer is an area of recent interest. In vitro AGEs formation was observed by browning of samples, increased fluorescence, and carbonyl stress. MG induced changes in the structure of BSA were analysed using electrophoresis, spectroscopy, TEM, AFM, DLS, and CD spectroscopy. Our results show that AGEs form random structures, oligomeric aggregates, and {beta}-sheets. Thioflavin T and Congo red staining further validated these findings. Galangin and Caffeic acid demonstrated significant antiglycation activity, suppressing AGEs formation in vitro. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/737425v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@113b391org.highwire.dtl.DTLVardef@7208a1org.highwire.dtl.DTLVardef@94c2e1org.highwire.dtl.DTLVardef@867b85_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMethylglyoxal-induced Advanced Glycation End Products were prepared in vitro C_LIO_LIMethylglyoxal -induced structural modifications in BSA C_LIO_LIAGEs were characterised using various parameters C_LIO_LIBoth fluorescent and non-fluorescent AGEs were formed. C_LIO_LIPhytochemical treatment induced inhibition of AGEs formation C_LI

Heparan sulfate proteoglycans (HSPGs) are essential cell surface and extracellular matrix glycoconjugates that mediate diverse biological processes through interactions between their heparan sulfate (HS) chains and extracellular ligands. While HS sulfation patterning is known to dictate ligand specificity, how cells control HS assembly to regulate these interactions remains incompletely understood. To systematically identify genetic modifiers of HS-protein interactions, we performed genome-wide CRISPR activation (CRISPRa) screens in HEK293T cells using binding of antithrombin (AT), which selectively recognizes 3-O-sulfated HS motifs, or the N-sulfation-specific antibody 10E4 as functional readouts. Strikingly, the screens revealed proteoglycan core proteins as key modulators of HS function. In particular, syndecan-1 (SDC1) emerged as a preferential enhancer of AT binding compared to other syndecan family members. Targeted upregulation of syndecan family members increased total HS levels, but only SDC1 enhanced AT binding. Structural and enzymatic analyses demonstrated that SDC1-associated HS chains contain elevated 6-O-sulfation and serve as superior substrates for 3-O-sulfotransferases relative to SDC2-associated HS chains. Additionally, SDC1 exhibited slower cell surface recovery, which was blocked by cycloheximide treatment, consistent with extended trafficking and biosynthetic processing. Overall, these findings indicate that proteoglycan core protein identity influences HS sulfation patterning and ligand-binding specificity and trafficking kinetics may contribute to core protein-dependent regulation of HS modification.

T cells are important components of the adaptive immune system and develop through a selection process regulated by signaling through the T-cell receptor (TCR). Thymocyte-Expressed Molecule Expressed in Selection (THEMIS) is a TCR-proximal protein that modulates the activity of Shp1 phosphatase to influence TCR signaling during development. THEMIS has been shown to both activate and inhibit Shp1, but the molecular mechanisms of these functions are poorly understood. THEMIS contains two rare Cysteine All-Beta In THEMIS (CABIT) domains, the N-terminal of which interacts with Shp1 and is likely responsible for modulation of its phosphatase activity. Herein, we report the first crystal structure of the THEMIS CABIT1 domain. While a portion of the CABIT1 domain is poorly resolved, it appears to share the same overall fold observed in our recent CABIT2 crystal structure and AlphaFold predictions. We show that phosphorylation of the CABIT1 domain by LCK is required for association with SHP1 and that phosphorylated CABIT1 can protect Shp1 from oxidation and inhibition by reactive oxygen species (ROS), which may serve as a mechanism by which THEMIS enhances Shp1 activity.

The AMY2B gene encodes pancreatic amylase, a critical enzyme for starch digestion. While previous studies have examined AMY2B copy number variation (CNV) in domestic and some wild animals, less is known about wild carnivores inhabiting regions with limited anthropogenic starch exposure. We analyzed blood samples for serum amylase activity and copy number variation in AMY2B gene from 8 wolves (Canis lupus), 11 brown bears (Ursus arctos), and 3 red foxes (Vulpes vulpes) from Bosnia and Herzegovina. AMY2B gene copy number was assessed using droplet digital PCR (ddPCR), and serum amylase activity and glucose levels were quantified. Although the number of fox samples was limited, foxes and wolves consistently harbored two copies of AMY2B, while brown bears exhibited higher CNV (3.67-8.40, mean 5.88). Serum amylase activity was highest in foxes, moderate in wolves, and variable but lower in bears. Despite differences in AMY2B copy number and serum amylase activity, circulating glucose concentrations did not differ significantly among species. Our findings suggest that variation in AMY2B copy number among wild carnivores may be associated with species-specific evolutionary histories and dietary adaptations, providing insight into genomic mechanisms underlying carbohydrate utilization in natural populations.

Core fucosylation of the IgG1 Fc N297 glycan is known to reduce binding affinity to the Fc{gamma}RIIIa (CD16a) receptor and attenuate antibody-dependent cellular cytotoxicity (ADCC), yet the structural mechanisms underlying this effect remain incompletely understood. Here, we use extensive all-atom molecular dynamics simulations to systematically investigate how Fc glycosylation modulates the structural, energetic, and dynamical landscape of the IgG1 Fc-CD16a complex across multiple systems with fucosylation and galactosylation. Relative binding free energy calculations reproduce experimentally established trends, showing that afucosylation consistently strengthens Fc-CD16a interactions. Mechanistically, dual fucosylation (on both Fc arms) increases inter-glycan packing between the Fc N297 glycans, restricts Fc glycan conformational sampling, and destabilizes the conformational organization of the CD16a N162 glycan. These glycan-mediated perturbations propagate to the protein interface. The result is reduced Fc-CD16a contact persistence, redistribution of energetically important residues away from the canonical binding interface, and broader, less stable receptor-bound conformational states. Dynamic cross-correlation analysis further reveals that afucosylated systems maintain substantially stronger coordinated motions across the Fc-CD16a assembly, whereas fucosylation disrupts long-range dynamic coupling between the receptor and antibody domains. Across these different energetic, structural, conformational, and dynamical readouts, fucosylation systematically shifts the Fc-CD16a assembly from a compact, interface-stabilized binding mode toward a more heterogeneous and weakly coupled receptor-bound ensemble. Together, our findings set forth a mechanistic basis for Fc glycosylation regulating receptor engagement through ensemble-level conformational and dynamical reorganization rather than simple local steric effects. These results provide mechanistic design principles for rational Fc glycoengineering and the development of therapeutic antibodies with enhanced effector functions. More broadly, this work highlights how glycan composition can be leveraged as a tunable molecular design parameter for engineering protein recognition, conformational stability, and immune effector function in therapeutic glycoproteins.