Glycobiology

Glycosylation of proteins is arguably the most diverse post-translational modification that is altered in almost all cancer types, which has been demonstrated to play a crucial role in creating an immunosuppressive microenvironment that promotes immune tolerance and evasion. However, the biosynthesis of N-linked glycan is mediated by a series of enzymatic reactions catalysed by glycosyltransferases and glycosidases in a template-independent manner, hindering our understanding of specific structure-function relationships and roles of specific glycans on specific proteins. Here we use HLA class I-negative cell line K562, a known reference target for NK-mediated cytolysis, to establish a model investigating how cell surface glycan dynamics influence its susceptibility to cytolysis mediated by NK-92 cells. Treatment of K562 cells with kifunensine, swainsonine, 2F-peracetyl-fucose, or 3Fax-peracetyl Neu5Ac, inhibitors of N-linked glycan processing, resulted in drastic alterations in cell surface carbohydrate phenotype, as could be shown by flow cytometric analysis of the lectiNbinding properties of the cells. Despite these clear changes in carbohydrate phenotype, only K562 cells treated with either kifunensine or 3Fax-peracetyl Neu5Ac exhibited higher susceptibility to the cytolysis medidated by NK-92 cells accompanied with an increased CD107a expression by NK-92 cells. Although K562 cells overexpressing gene MGAT3 exhibited a decreased NK-susceptibility, we further found that this decrease was not exclusively determined by the overexpression of gene MGAT3 product bisecting {beta}1,4-GlcNAc, because the treatment of 3Fax-peracetyl Neu5Ac reversed the resistance of K562 cell against NK-92 cell in despite of expressing higher levels of bisecting {beta}1,4-GlcNAc. Expressing HLA-G on cell surface as extravillous trophoblast did not change the NK-susceptibility of K562 cells, despite evidence that HLA-G molecules expressed by K562 cells can bind to inhibitory receptor ILT2 expressed on NK-92 cell surface. These findings suggest that the level of terminal sialylation, outweighing other components in N-linked glycan, determines the NK-susceptibility of K562 cell, offering a new strategy to weaken the resistance of cancer cells so that the immune system can maximise the elimination.

Production of aberrant glycans by cancer cells constitutes a key immunosuppressive strategy to avoid destruction by immune cells. Although sialic acid-containing glycans are known to dampen the activation of lymphocytes, including Natural Killer (NK) cells, the role of fucose-containing glycans remains poorly characterized. In this work, we explored the role of Lewis X (LeX) in cancer cell-NK cell interactions. We induced ectopic expression of FUT9, an 1-3/4-fucosyltransferase, in two colorectal cancer cell lines and showed this enzyme only synthesized LeX structures but not sialyl-LeX. FUT9 introduction was not associated with altered MHC class I surface expression, nor with CD2 (which has been proposed as a receptor for LeX) binding to cancer cells. By inhibiting fucosylation we could demonstrate that CD2 binding was furthermore independent of surface fucosylated glycans in three independent cell lines. Lastly, FUT9/LeX had a limited role in cancer cell destruction and expression of activation markers by NK cells. Overall, our study suggests that, unlike sialylated glycans, 1-3/4-fucosylated glycans have limited impact on cancer cell evasion of NK cell-mediated destruction.

The sperm surface glycocalyx undergoes extensive remodeling during epididymal maturation, required for sperm to reach and bind the oviductal epithelium. N-acetylglucosamine (GlcNAc)-containing glycans are candidate mediators of these events, however, how these residues are regulated across the reproductive tract, and whether their changes depend on specific epididymal enzymes or functionally contribute to sperm-oviduct epithelial adhesion, remains poorly defined. Here, we addressed this gap by examining how surface GlcNAc changes as sperm mature and become functionally competent, from epididymal maturation through capacitation and the acrosome reaction. We further asked whether these changes relate to the ability of spermatozoa to bind the oviductal epithelium. Surface GlcNAc, assessed by WGA reactivity, increased progressively from caput to cauda epididymal spermatozoa, with a corresponding shift in GlcNAc-bearing protein profiles, while remaining predominantly localized to the acrosomal region throughout maturation. Incubation of caput spermatozoa with cauda epididymal fluid reduced WGA labeling, an effect blocked by the selective {beta}-N-acetylglucosaminidase ({beta}-NAG) thiourea derived hydroxy pyrrolidine inhibitor VP150, identifying luminal {beta}-NAG as an active contributor to GlcNAc remodeling in the epididymis. In ejaculated spermatozoa, capacitation induced minor changes in surface GlcNAc, whereas the calcium ionophore-induced acrosome reaction produced a marked reduction in WGA reactivity and acrosomal labeling, consistent with glycoprotein loss during acrosomal exocytosis. Functionally, spermatozoa that bound to BOEC monolayers were preferentially WGA-positive, and pre-incubation of BOECs with WGA significantly reduced sperm adhesion, implicating surface GlcNAc in sperm-oviduct epithelial recognition. Together, these findings define surface GlcNAc as a dynamically regulated glycan that is progressively established during epididymal transit, partly through luminal {beta}-NAG activity, redistributed during capacitation and acrosomal exocytosis, and functionally engaged during sperm-BOEC adhesion, providing a mechanistic framework for glycocalyx-mediated sperm selection in cattle.

Extravillous trophoblasts (EVTs) are a trophoblast subpopulation critical for feto-maternal tolerance during early pregnancy, primarily using HLA-G to exert immunomodulatory effect, and possessing N-glycomic profiles distinct from other trophoblast subpopulations. However, whether the N-glycosylation confers distinct immunological properties to EVTs remains poorly understood. To investigate this, we employed JEG-3, a human choriocarcinoma cell line having the capacity to produce pregnancy-related hormones and expressing both HLA-C and HLA-G resembling placental EVTs, as an in vitro EVT model, alongside cell line JAR which exhibits villous trophoblast phenotypes distinct from JEG-3. Both cell lines were treated with kifunensine or swainsonine, inhibitors of -mannosidases, to remodel their N-glycosylation patterns. This led to significant remodelling of their N-glycomic profiles, with JEG-3 cells showing an increased level of polylactosamine chains and decreased levels of -2,6-sialylation and core -1,6-fucosylation. Western blot analysis showed that inhibiting -mannosidases altered only the composition of N-glycans on cell-surface HLA-G, without affecting the overall abundance of cell-surface HLA-G. In kifunensine-treated JEG-3 cells that predominantly express oligomannose type N-glycans, an intracellular accumulation of unfolded HLA-G fragments, increased hCG secretion, and down-regulations of EVT markers GATA3 and KRT7 were observed compared to untreated control, while swainsonine treatment did not impact N-glycan expression. Cytotoxicity assays using NK-92 as effector cells showed that the de-sialylation of JEG-3 by neuraminidase treatment led to increased NK-92 mediated killing. JEG-3 cell sustained its EVT immunological properties through generating bioequivalent N-glycans, exemplified by NK-92 cells pre-conditioned with used culture media of kifunensine-treated JEG-3 cells displaying reduced cytotoxicity toward NK-sensitive lymphoblast cell line K562, an effect not observed with swainsonine-treated JEG-3 cells. This model suggests that EVTs immunological properties are dependent on specific N-glycomic profiles that are maintained by unique N-glycosylation homeostasis, and overall improves our understanding of how EVTs maintain their immunomodulatory effect at the maternal-fetal interface.

Cyclic oligosaccharides possess industrial advantages, including molecular encapsulation capability and high physicochemical stability, owing to the absence of a reducing end. Recently, a novel cyclic tetrasaccharide, cycloisomaltotetraose (CI4), consisting of four -1,6-linked glucose units, and the enzymes responsible for its synthesis, cycloisomaltotetraose glucanotransferases (CI4Tases), were discovered. Unlike known cycloisomaltooligosaccharide glucanotransferases (CITases) that yield a wide distribution of cyclic products with a degree of polymerization (DP) of 7 or higher, CI4Tases strictly produce CI4. To elucidate the molecular mechanism underlying this strict DP4 specificity, we determined the crystal structures of CI4Tase from Agreia sp. D1110, in its ligand-free form, as well as in complex with the linear hydrolysis product isomaltotetraose (IG4) and with CI4. Structural comparisons revealed that a loop (M247 to R251) blocks the region corresponding to the -5 subsite of typical CITases, narrowing the substrate-binding pocket. This "molecular ruler" mechanism ensures that only a glycan chain of exactly four glucose units is accommodated for cyclization. Among mutants of the residue positioned at the center of bound CI4, the formation of by-products other than CI4 was significantly suppressed in F245L, F245A, and F245W. While the cyclization activity of all F245 mutants decreased, the CI4 hydrolysis activity of these three mutants was also significantly reduced, resulting in an increased specificity for cyclic sugar production. These findings elucidate the strict size-control mechanism of CI4Tase and provide a structural foundation for engineering cycloisomaltooligosaccharide-producing enzymes with optimized transglycosylation efficiency and specificity for industrial applications.

Antibodies exhibit species-specific sequence and structural features that influence their antigen-recognition properties. Although several studies have investigated porcine antibodies, their repertoire and structural characteristics remain less well characterized than those of several other mammalian species. In this study, we analyzed public porcine heavy-chain repertoire sequencing data together with available antibody structural data to identify characteristic features of porcine antibodies. We found several residues enriched in porcine antibody framework regions, particularly at the base of heavy-chain complementarity-determining region 3 (CDR-H3). In particular, Arg101 and Glu123 were closely positioned in available structures and may influence CDR-H3 conformation at its base, whereas Pro120 may help constrain local backbone conformation. We also observed non-canonical cysteine usage in both framework region 1 and CDR-H3, which may contribute to structural diversity in the porcine repertoire. Finally, we evaluated the humanization potential of a porcine antibody using a human antibody language model and found that human-likeness increased after model-guided substitutions, although the resulting sequences did not exceed the T20 score threshold. Overall, these results indicate that porcine antibodies possess distinct sequence and structural features that may influence CDR-H3 properties and should be considered in future antibody analysis and engineering.

Dried blood spot (DBS) sampling is becoming a popular alternative to traditional blood sampling approaches, offering advantages such as convenience of collection, transportation, and storage, as well as lower biohazard risk. N-glycosylation, a major post-translational modification of proteins associated with numerous biological and pathological functions, is one area of interest for DBS analysis. In this study, we utilize a protocol for N-glycosylation profiling of DBS by ultra-high-performance liquid chromatography based on hydrophilic interactions and fluorescence detection (HILIC-UHPLC-FLR). The protocol includes DBS cutting, protein extraction and enzymatic digestion, labeling with 2-aminobenzamide, followed by cleanup and HILIC-UHPLC-FLR measurement. We compare DBS with plasma and demonstrate the stability of DBS N-glycosylation profile when DBS are prepared from fresh blood, frozen whole blood, or a combination of separated frozen blood cells and corresponding frozen plasma. Additionally, we compared DBS N-glycans from pre- and diabetic subjects. Fucosylation, bisection, and galactosylation showed a statistically non-significant increasing trend in diabetes, whereas sialylation showed a statistically non-significant decreasing trend in diabetes. The main advantage of this method is the ability to repurpose samples, which were initially not intended for biomarker N-glycan analysis, such as frozen whole blood. Additionally, DBS N-glycan profiling is the easier, cheapest and the least invasive approach to conventional plasma in pre-diabetes and diabetes patients' diagnostics and monitoring.

Mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE) is a recently recognized epilepsy-associated lesion frequently linked to brain-restricted somatic variants in SLC35A2, a gene encoding the Golgi UDP-galactose transporter. Although previous studies demonstrated altered glycosylation in SLC35A2-mutated MOGHE tissue, the spatial relationship between glycosylation defects and histopathological abnormalities remains poorly understood. We applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) using formalin-fixed paraffin-embedded brain tissue from six histologically confirmed MOGHE cases (three pediatric and three adult) and three temporal lobe epilepsy with hippocampal sclerosis (TLE-HS). We spatially evaluated N-glycan profiles across diagnostic tissue groups, with particular attention to molecular differences between lesional and perilesional regions and to recurrent abundance trends. All MOGHE cases harboured somatic SLC35A2 variants. Histologically, oligodendroglial hyperplasia and heterotopic neurons were present in all cases, while patchy hypomyelination was restricted to pediatric cases. Unsupervised spatial segmentation, integrated with neuropathological evaluation, revealed marked molecular heterogeneity in pediatric MOGHE. In these cases, lesional and perilesional regions were clearly distinguishable in both white matter (WM) and overlying grey matter (GM) boundaries patterns, whereas adult MOGHE and TLE-HS mainly showed a clearcut separation between WM and GM. Spatial analysis confirmed enrichment of the previously reported aberrant N-glycan species m/z 2094 and, to a lesser extent, m/z 2297 within MOGHE tissue, particularly in pediatric lesional WM. Notably, the distribution of m/z 2094 closely overlapped with areas of hypomyelination. Quantitative trajectory analysis of 151 detected N-glycan ions identified recurrent abundance profiles. Three representative spatial patterns emerged: pediatric lesion-enriched, pediatric perilesion-enriched, and TLE-HS-enriched profiles. Pediatric lesions were characterized by increased abundance of multiantennary glycans lacking terminal galactose residues and reduced abundance of galactosylated biantennary and multiantennary structures, consistent with defective UDP-galactose transport. In contrast, adult lesional and perilesional tissues exhibited largely overlapping glycomic profiles. These findings provide the first spatially resolved evidence that glycosylation abnormalities in SLC35A2-mutated MOGHE are closely associated with lesional pathology, particularly hypomyelination, and are substantially more pronounced in pediatric than adult cases. Spatial glycomics may therefore offer new insights into MOGHE pathophysiology and support the development of targeted therapeutic approaches aimed at correcting galactosylation defects.

Syndecans are transmembrane proteoglycans that regulate angiogenesis through both their glycosaminoglycan chains and core proteins. While roles for all four mammalian syndecans in new blood vessel formation are well established, it has more recently emerged that their extracellular core proteins contain discrete bioactive regulatory sequences capable of influencing cellular processes, including angiogenesis. We previously demonstrated that the syndecan-3 (SDC3) ectodomain possesses anti-angiogenic activity independent of its heparan sulphate chains. Here, we identified and characterised a novel anti-angiogenic sequence within the SDC3 ectodomain. Using recombinant truncation mutants, endothelial migration assays and peptide mapping, we localised activity to a discrete region of the extracellular domain and subsequently defined a conserved minimal nine amino acid peptide, QM111, that retained full biological activity. QM111 inhibited endothelial cell migration and angiogenic sprouting in both rat aortic ring and mouse choroidal explant models. Intrinsic disorder analysis revealed that QM111 resides within a region of comparatively reduced disorder, consistent with other syndecan regulatory sequences. This supports the concept that syndecan ectodomains contain conserved functional modules embedded within intrinsically disordered extracellular domains. QM111 did not induce inflammatory chemokine production, exhibited no detectable cytotoxicity, and retained substantial stability in human serum and vitreous humour. Finally, QM111 displayed anti-angiogenic activity comparable to the previously described syndecan-2-derived peptide QM107, with combination treatment producing more robust inhibition of angiogenesis. These findings identify QM111 as a novel endogenous anti-angiogenic peptide and support the concept that syndecan ectodomains are reservoirs of biologically active regulatory sequences with therapeutic potential. The work further establishes syndecan-derived peptides as a promising platform for the development of next-generation anti-angiogenic therapies.

Protein engineering and precision fermentation provide an opportunity to increase the value of food proteins by improving their solubility, stability, functionality, or nutritional composition. Here, we use {beta}-lactoglobulin ({beta}LG) as a model protein to investigate how state-of-the-art computational protein design approaches affect these properties. First, the deep learning-based design tool ProteinMPNN was used to alter up to 20% of {beta}LG residues for increased stability. Second, the physics-based modeling platform PyRosetta was used to find positions in {beta}LG accommodating increased branched-chain amino acid (BCAA) content and up to 10 residues were simultaneously exchanged. Experimental characterisation of ProteinMPNN and stabilised BCAA-enriched variants showed similar secondary structure and oligomeric state as native {beta}LG. ProteinMPNN variants gave increased titers and increased thermal stability up to 15 {degrees}C, and this correlated with changes in the rate of surface pressure in droplet tensiometry. Stabilized BCAA-enriched mutants had altered acid solubility. Correlations between computationally derived biophysical metrics and experimental properties are presented and suggest some predictive power for surface hydrophobicity on protein yield.

Metaproteomics aims to capture a taxonomically comprehensive snapshot of proteins in a sample. Design of reference databases is a key aspect of metaproteomic workflows, as these define what is ultimately seen. Databases tailored to focal biomes offer optimal performance, yet their construction often requires drawing on heterogeneous data sources, posing a challenge to reproducibility and documentation. Here we present maniFasta, a tool enabling users to generate standardized, reproducible, and robustly documented protein reference sets from diverse input sources and datatypes. Users provide information on their desired input types and sources, and the output is an integrated database comprising a protein sequence file (FASTA), with harmonized identifiers and standardized headers, and an associated provenance metadata table (manifest). We highlight the value of maniFasta in the context of salivary metaproteomics, addressing the need for a taxonomically comprehensive reference database. The AllOralsDB resource includes human proteins, as well as proteins from bacteria and archaea, fungi and other microeukaryotes, viruses and viroid-like elements, dietary sources, and common contaminants. Together, this work provides a community resource for oral and salivary metaproteomics (https://www.homd.org/ftp/AllOralsDB/), and a versatile and accessible tool for constructing protein databases for metaproteomics generally (https://github.com/KauffmanLab/maniFasta).

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.

BackgroundSevere dengue remains difficult to predict because patients with different clinical trajectories may present with overlapping features, and conventional severity classifications may not fully capture underlying biological heterogeneity. In this study, we applied an integrated clinical and proteomic endotyping approach to dissect dengue disease heterogeneity and identify molecular signatures associated with severity. MethodsPlasma proteomic profiles were analyzed together with detailed clinical, biochemical, hematological, coagulation, and immunological parameters from healthy controls and dengue patients classified according to WHO 2009 severity criteria. High-throughput proteomic analysis, unsupervised clustering, pathway enrichment, and machine-learning-based classification were used to identify dengue endotypes and define molecular features associated with predicted severe disease. ResultsIncreasing dengue severity was associated with progressive abnormalities in liver function, coagulation parameters, hematological indices, and inflammatory mediators, including IL-6, IL-15, HGF, and MUC-16. However, proteomic profiling revealed substantial overlap across conventional severity categories, indicating that clinical classification alone does not fully resolve dengue host-response heterogeneity. Integrated clinical-proteomic clustering identified distinct dengue endotypes, including a predicted severe endotype enriched for inflammatory, antiviral, and cytotoxic lymphocyte-associated pathways. This high-risk endotype was characterized by elevated IL-15, IFN-{gamma}, and granzymes, consistent with coordinated activation of cytotoxic lymphocyte-associated antiviral responses. Machine-learning analysis further showed that proteomic features were strong discriminators of this endotype, supporting their potential utility as biomarkers of severe host-response states. ConclusionIntegrated clinical-proteomic endotyping provides molecular resolution beyond conventional severity grading and identifies immune pathways associated with severe dengue. This framework may improve biological understanding of dengue progression and support future risk stratification and biomarker development.

Symptoms of Clostridioides difficile infection (CDI) are primarily caused by two major protein toxins, toxin A (TcdA) and toxin B (TcdB). In addition, approximately 5-30% of C. dif[fi]cile strains produce a third toxin, C. difficile binary toxin (CDT), which is has been associated with enhanced virulence and severe disease. CDT consists of an enzymatic component CDTa, and a binding and translocation component CDTb, which mediates the delivery of CDTa into host cells. CDTb contains two receptor-binding domains, RBD1 and RBD2. Recent structural studies suggest that RBD2 plays a critical role in the formation and stabilization of the di-heptameric CDTb assembly required for efficient intoxication of host cells. In this study, we evaluated the immunogenicity and protective potential of RBD1 and RBD2 using in silico, in vitro and in vivo approaches. Sequence analysis demonstrated that RBD2 is highly conserved among diverse CDT-producing C. difficile ribotypes and toxinotypes. Immunization of mice with RBD2, but not RBD1 conferred effective protection against direct CDT challenge. Moreover, RBD2 immunization protected hamsters against infection with a CDT-only-producing C. difficile strain (DSM 101085; TcdA-TcdB-CDT). Mechanistically, anti-RBD2 serum, but not anti-RBD1 serum, effectively neutralized CDT-mediated cytotoxicity, as demonstrated by inhibition of cell rounding in Vero cells. Collectively, these findings identify RBD2 as a promising vaccine antigen targeting CDT and provide functional evidence supporting its critical role in CDT-mediated host-cell intoxication. Incorporation of RBD2 into multivalent C. difficile vaccines may broaden protection against hypervirulent, CDT-producing strains.

Glycan identification has advanced, but glycan structures remain difficult to translate into reproducible biomedical context because reusable glycan-level annotations are sparse. We present GlycoMeSH, a resource that links glycans to Medical Subject Headings (MeSH) through an inference model, a traceable association database and a glycan-set enrichment workflow. GlycoMeSH-BERT recovered ~60% of literature-derived associations at recall@30 and expanded open-vocabulary MeSH coverage beyond closed-label baselines, without higher per-prediction accuracy. At matched candidate counts, its predictions showed motif-level semantic agreement comparable to those baselines, independently of the training labels. GlycoMeSH-DB contains 789,627 associations between 26,954 glycans and 20,302 MeSH terms. GlycoMeSH-EA returned enriched MeSH terms for glycan sets from glycomics and glycoproteomics datasets. Each association represents a biomedical context rather than a validated mechanism, and retains its source PMID or prediction score for audit. GlycoMeSH supplies the missing, evidence-traceable annotation layer that makes glycan sets directly analyzable by enrichment across glycoscience datasets.

The perilipins generally represent the most abundant lipid droplet (LD) surface proteins in mammalian cells and can target LD subpopulations within the same cell. They are characterized by a conserved organization of disordered and folded regions, as well as a number of divergent features, which contribute to differences in perilipin function and LD targeting. Here, we focus on the C-terminal 4-helix bundle (4HB) domain that is present in all perilipins except for PLIN1. Using biochemical and in silico approaches, we show that the 4HB of PLIN3 is a stably folded domain and interacts with lipid surfaces in vitro and with LDs in model cells. The {beta}-subdomain at the bottom of the helical bundle is required for the binding to LDs, but not for the 4HB stability, suggesting that this region may promote direct interaction with the LD surface. In agreement, the 4HB of PLIN4, which does not contain an {beta}- subdomain, does not bind to LDs. Overall, our work shows that small differences in perilipin structural features impact their differential targeting to LDs.

The Notch signaling pathway plays a central role in development and cell fate determination. Its function depends on tightly regulated intracellular trafficking of the Notch receptor and the Notch intracellular domain (NICD) after cleavage by {gamma}-secretase. Notch signaling is essential for principal cell differentiation within the renal collecting duct and for proximal-distal patterning during kidney development. Notch activity has also been shown to influence the trafficking of several membrane proteins, including nephrin in kidney cells and monocarboxylate transporter 1 in brain endothelial cells. Aquaporin-2 (AQP2) is the key vasopressin-regulated water channel in the collecting duct, and proper AQP2 trafficking and recycling are required for physiologically appropriate urine concentration. To determine whether and, if so, how Notch signaling modulates AQP2 trafficking, we performed studies using LLCPK1 renal epithelial cells stably expressing AQP2 (LLCPK1-AQP2). Exposing cells to 35 M DAPT (which inhibits y-secretase, preventing cleavage and activation of Notch receptor signaling) for 30 min significantly increased AQP2 membrane accumulation in LLCPK1-AQP2 cells as revealed by immunofluorescence staining. Using a rhodamine-transferrin internalization assay, we found that DAPT reduced clathrin-mediated endocytosis by 60%. This blockade increases AQP2 membrane accumulation by preventing the reinternalization of AQP2 that is delivered to the plasma membrane by exocytosis during its constitutive recycling pathway. Using an F-actin polymerization assay, we then found that Notch inhibition decreases F-actin polymerization by de-activating the small GTPase RhoA, using GSTRBD, a substrate that binds to active RhoA, as seen by western blotting using phospho-specific antibodies. Because actin polymerization is required for AQP2 endocytosis, RhoA inhibition by DAPT would result in the decreased internalization of AQP2 that we observed by immunofluorescence. While the mechanism by which DAPT inhibits RhoA activity remains to be determined, our study shows that AQP2 trafficking is regulated by the Notch signaling pathway in vitro and suggests that modulation of Notch signaling may represent a novel strategy to address water balance disorders that involve defects in the AQP2 trafficking process.

Non-typhoidal Salmonella is a major cause of bacterial foodborne illness leading to acute gastroenteritis. In individuals with type 2 diabetes (T2D), Salmonella infection is more likely to cause life-threatening extraintestinal infections. The mechanism underlying this susceptibility remains unclear. In this study, 8-week-old TALLYHO mice were fed either a chow or high-fat diet (HFD, 45% fat) for 8 weeks to induce the T2D. As expected, HFD-fed mice gained more weight and developed diabetic-range blood glucose levels by 16 weeks of age. Next, mice from each diet group were orally infected with a fully virulent bioluminescent Salmonella Typhimurium to monitor infection spread by in-vivo imaging. Although both groups developed clinical signs of salmonellosis, Salmonella spread was accelerated and followed an unusual pattern in T2D mice compared with healthy animals. Additionally, hyperglycemia increased gut-derived lipopolysaccharide leakage into the bloodstream. Based on the link between T2D and altered levels of butyrate-producing bacteria in the gut, we analyzed the intestinal short-chain fatty acid (SCFA) profiles in the TALLYHO mice. As expected, intestinal SCFA concentrations, including butyrate, were lower in HFD mice than in chow-fed animals. Given butyrate?s role in gut health and its ability to downregulate Salmonella invasion genes, mice received oral butyrate supplementation. We found that butyrate supplementation reduced the extraintestinal spread of Salmonella in normoglycemic chow-fed animals. Unexpectedly, although butyrate improved intestinal health in hyperglycemic mice, it failed to decrease Salmonella spread in diabetic animals. Taken together, these findings provide novel insights into the pathogenesis of enteric salmonellosis in the context of T2D.

Piezo1 is a mechanically activated cation channel whose N-linked glycans support protein maturation and plasma membrane trafficking, but their contribution to mechanical gating is unknown. We asked whether hypoglycosylation alters Piezo1 mechanosensitivity and cortical neuronal mechanotransduction, with potential relevance to neurological manifestations of congenital disorders of glycosylation (CDG). Human Piezo1 was studied in HEK293 cells after mutation of two conserved cap-domain N-glycosylation sites or inhibition of N-glycan maturation with swainsonine or kifunensine. Mechanically activated currents were recorded by cell-attached patch-clamp during incremental negative-pressure pulses, whereas Ca2+ responses were measured during uniaxial stretch. Piezo1 abundance, synaptic localisation and stretch-evoked Ca2+ signals were also examined in primary mouse cortical neurons. On poly-L-lysine, N2293Q or N2330Q shifted the pressure-response relationship towards lower activating pressures without changing maximal current or inactivation kinetics. This effect was absent on collagen. Swainsonine and kifunensine reduced mature Piezo1 glycosylation and lowered the mechanical activation threshold. Hypoglycosylation enhanced Ca2+ entry during submaximal stretch in HEK293 cells. In cortical neurons, inhibition of glycan maturation increased somatic Piezo1 immunoreactivity without changing its association with synaptic markers, and potentiated Ca2+ responses to both the Piezo1 activator Yoda1 and submaximal stretch. Thus, mature N-glycans and the extracellular adhesive environment jointly set Piezo1s mechanical activation threshold rather than merely regulating biosynthesis and trafficking. These findings establish glycosylation-mechanics coupling as a determinant of neuronal force sensing and suggest that, by facilitating Piezo1 recruitment, defective glycosylation may increase cortical vulnerability to mechanical stress, potentially contributing to head trauma-triggered neurological episodes in phosphomannomutase 2 deficiency (PMM2-CDG). Key pointsO_LIPiezo1 channels convert mechanical forces into electrical and calcium signals. N-linked glycans support channel trafficking to the plasma membrane, but whether they tune the force needed for Piezo1 activation was unknown. C_LIO_LIMutating either of two conserved N-glycosylation sites in Piezo1 cap domain, or pharmacologically restricting N-glycan maturation, lowered channels mechanical activation threshold without changing maximal current or inactivation. C_LIO_LIThis sensitisation depended on the adhesive substrate (occurred on poly-L-lysine but not collagen), and was most evident during submaximal stretch, showing that glycosylation and the extracellular mechanical environment jointly determine Piezo1 force sensing. C_LIO_LIIn mouse cortical neurons, impaired N-glycan maturation increased somatic Piezo1 abundance and enhanced Ca2+ responses to its chemical activator Yoda1 and stretch, without changing synaptic localisation. C_LIO_LIBy allowing weak mechanical inputs to recruit Piezo1 more effectively, defective glycosylation may increase cortical responses to mechanical stress and help explain susceptibility to head trauma-triggered neurological episodes in phosphomannomutase 2 deficiency (PMM2-CDG). C_LI

Background: Tumor necrosis factor- inhibitors such as infliximab and adalimumab have transformed autoimmune disease treatment; however, infliximab is a mouse-human chimeric antibody that remains immunogenic, is associated with self-association/aggregation liability, and requires prolonged intravenous administration. We humanized infliximab and engineered infliximab-derived candidates with improved potency and developability. Methods: Infliximab complementarity-determining regions were grafted onto human germline frameworks to generate humanized infliximab. STage-Enhanced Maturation (STEM) technology produced an affinity-matured clone (hInBG4), followed by targeted amino-acid substitutions in the complementarity-determining regions to generate LW2Y, LW2YR2S, and LW2YHR1K. Variants were evaluated by a cell-based tumor necrosis factor alpha neutralization assay, affinity-capture self-interaction nanoparticle spectroscopy, a baculovirus particle enzyme-linked immunosorbent assay, size-exclusion high-performance liquid chromatography, transient expression in human embryonic kidney 293 cells, and tumor necrosis factor alpha binding kinetics by biolayer interferometry, including dissociation at pH 7.4 and 5.8. Results: All three variants showed two- to three-fold higher neutralization potency than chimeric infliximab and outperformed adalimumab. Affinity-capture self-interaction nanoparticle spectroscopy shifts decreased from double-digit parental values to low single digits, while baculovirus particle binding ratios remained acceptable. Size-exclusion chromatography showed cleaner monomer peaks with reduced tailing, and expression increased relative to humanized infliximab. LW2Y combined very high affinity at pH 7.4 with markedly faster dissociation at pH 5.8, consistent with pH-dependent antigen release. Conclusions: Humanization, affinity maturation, and targeted complementarity-determining region re-engineering generated infliximab-derived candidates with improved potency and developability and identified LW2Y as a lead for further preclinical evaluation.