Glycobiology

Monoclonal antibody (mAb) glycosylation is a critical quality attribute that is difficult to rationally engineer and rapidly assess during cell line development. Here, we investigate whether cell-surface glycosylation can serve as a predictive indicator of mAb product glycosylation following targeted glycogene engineering in CHO cells. Five key glycogenes (COSMC, FUT8, B4GALT1, ST3GAL4, ST6GAL1) were investigated in two mAb-producing CHO cell lines. Product glycan analysis revealed consistent, gene-specific effects across hosts, including loss of core fucosylation, and tuneable galactosylation and sialylation. Lectin-based surface profiling reliably reflected product outcomes for COSMC and FUT8 modifications but showed limited predictive power for galactosylation and 2,3-sialylation, highlighting glycosylation pathway redundancy and context dependence. This study provides the first systematic, cross-cell line evaluation of lectin-based cell-surface glycan profiling as a predictor of mAb product glycosylation, establishing its practical utility and inherent limitations for CHO glycoengineering workflows. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/724788v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@6d5cfborg.highwire.dtl.DTLVardef@1f38e0aorg.highwire.dtl.DTLVardef@f25fa2org.highwire.dtl.DTLVardef@64a0dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pertussis pathogenesis is the result of multiple virulence factors. In addition to the secretory proteins of Bordetella pertussis, surface molecules such as adhesins and endotoxin play a role in the pathogenesis of this disease. There are conflicting reports on the existence and nature of the Bordetella capsular polysaccharides or exoglycans. The data concerning the glycome of Bordetellae is incomplete. This conclusion is primarily derived from genomic data, with only limited indications regarding the actual structures. In this study, we present novel data on the exoglycan produced by all strains and species of the investigated bacteria from the genus Bordetella, including Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, and Bordetella holmesii. This is the first time this type of data has been provided. The exoglycan was consistently recovered from the chemically defined culture media of various Bordetellae species and strains. The compound was identified by nuclear magnetic resonance (NMR) as a free hexasaccharide released into the medium and thus received its name, Bordetella oligosaccharide (BOS). The biosynthetic origin of the BOS was confirmed by NMR combined with metabolic labeling in culture, using 13C,15N-L-glutamate as a primary carbon source. The identification of BOS has the potential to enhance our comprehension of the complete array of virulence factors contributing to the pathogenesis of Bordetella pertussis, particularly in regard to their relations with other Bordetella species. In the field of vaccine design, glycan structures are typically of utmost importance; however, they were hardly ever considered in the case of pertussis.

Graduate-level genomics courses require students to integrate dense material across subfields, concepts and methods. In modular, multi-instructor courses, students may struggle because the coherence between lectures can be difficult to navigate, while the course structure may be visible to instructors. We evaluated a 2025 navigation redesign of BIO322, a graduate genomics course at the Norwegian University of Life Sciences, while preserving course content, multi-instructor teaching, modular organization and assessment framework. The redesign includes introducing a standardized self-learning guide, expanded syllabus, enriched online quiz feedback, and added support for a final group research proposal. Using anonymized course evaluation scores from 2021-2025 and aggregated learning management system access data from 2023-2025, we examined student experience and resource use. In 2025, five of six course evaluation items reached their highest observed BIO322 scores, while one, lecture-specific score remained within the previous range. The consolidated self-learning guide was accessed by nearly all students, whereas access to optional readings declined across the course sequence, despite comparatively stable page views per accessing student. These course-level findings are consistent with improved perceived navigability following the introduction of standardized learning support. However, some students continued to report difficulty identifying priorities and connections among course components, indicating that challenges in perceived course coherence remained for part of the cohort despite the redesign. Practitioner PointsO_LIMaking course structure explicit may improve students perceived navigability in multi-instructor graduate genomics courses. C_LIO_LIA centralized self-learning guide can broaden access to preparatory guidance without changing core course content or assessment. C_LIO_LIOptional learning supports may be used unevenly, so resource availability should not be assumed to translate into uniform resource access. C_LI

The endoplasmic reticulum (ER)-Golgi interface is a dynamic trafficking hub maintained in part by TANGO1, a scaffolding protein that coordinates proteins and membranes at ER exit sites (ERES). TANGO1 has two isoforms: TANGO1L, which has a lumenal SH3 domain, and TANGO1S, which lacks this domain but retains the transmembrane and cytoplasmic coiled-coil (CC), TEER, and PRD domains common to both forms. We showed previously that loss of both isoforms disrupts ER-Golgi organization more severely than TANGO1L loss alone, indicating TANGO1S is functional and can compensate. Here we dissect the role of each TANGO1 cytoplasmic domain in maintaining secretory pathway organisation by expressing TANGO1S domain-deletion mutants in TANGO1L-/S-knockout cells. We show that TANGO1 loss causes cis-Golgi vesiculation that cannot be rescued by TANGO1S, suggesting the lumenal domain of TANGO1L is essential in supporting Golgi architecture. Meanwhile, the TEER domain is essential for the organisation of the ER, whilst the TEER, CC2 and PRD domain are required for a defined ERGIC. All constructs partially rescue COPII recruitment. This study represents an advance towards a domain-level resolution of TANGO1S function. Summary statementIn this study we perform rescue experiments in TANGO1 knockout cells to dissect the role of the TANGO1 cytoplasmic domains in maintaining the ER-ERGIC-Golgi continuum.

Our current understanding of carbohydrate-binding module (CBM) function is limited by the fact that most CBM research has focused on single-binding-site modules. CBM family 92 (CBM92) is a recently characterized family of predominantly trivalent proteins that bind {beta}-1,3- and {beta}-1,6-glucans with high specificity. CpCBM92A from Chitinophaga pinensis stands out as the first trivalent member of the family to be structurally determined. Multivalent CBM families are rare, and the way in which the three binding sites cooperate in ligand recognition remains unclear. Here, we use NMR spectroscopy to demonstrate how each of the proteins binding sites plays distinct roles in ligand binding. One binding site, referred to as the {beta} site, can be identified as the primary attachment point because of its higher affinity for all tested ligands, consistent with previous biochemical data suggesting it is the strongest binding site on CpCBM92A. The other two binding sites, referred to as and {gamma}, preferentially bind longer segments of {beta}-1,3- and {beta}-1,6-glucan chains, respectively. We further show that the glycosidic bond position and anomeric configuration of the binding glucosyl unit strongly affects protein affinity due to a preferred ligand pose in the binding sites. Our results provide insight into how the trivalent architecture of CBM92 might enable cross-linking of scleroglucan chains, which may guide the development of new applications for CBMs in biotechnology.

During maturation of a Golgi cisterna, multiple vesicular transport pathways recycle resident Golgi proteins. Recycling vesicles are captured by Golgi-associated tethers. To assign individual tethers to specific recycling pathways in Saccharomyces cerevisiae, we examined tether arrival and departure using kinetic mapping, and we examined tether function using an ectopic tether localization assay. Those approaches yielded mutually consistent results. Our analysis focused on two coiled coil golgin tethers and the multi-subunit tether GARP. At an intermediate stage of cisternal maturation, the golgin Sgm1 tethers proteins that follow an intra-Golgi recycling pathway dependent on COPI. At a late stage of cisternal maturation, GARP and the golgin Imh1 tether trans- Golgi network (TGN) proteins that follow an intra-Golgi recycling pathway dependent on the AP-1 and Ent5 clathrin adaptors. This involvement of GARP in intra-Golgi recycling had not previously been documented. Imh1 also tethers proteins that recycle from prevacuolar endosome compartments to the TGN. Our findings contribute to an integrated model of Golgi membrane traffic.

Signaling pathways often share components yet produce highly specialized biological responses. How signaling specificity is achieved between pathways utilizing common components is a fundamental question. In budding yeast, the same transmembrane mucin, Msb2, regulates two Mitogen-Activated Protein Kinase (MAPK) pathways controlling filamentous growth (fMAPK) and the response to osmotic stress (HOG). How this shared sensor distinguishes between stimuli and regulates different pathways is not clear. Using structure-guided analysis, we identified a conserved SEA (Sea urchin sperm protein, Enterokinase, Agrin) domain in fungal mucins and found that mutations disrupting protein folding selectively impair one pathway (fMAPK) but were tolerated by another (HOG). Mechanistically, these differences revealed distinct modes of signal transmission. The fMAPK pathway required an intact SEA domain and the cytosolic tail, consistent with a cis signaling mechanism that required structural coupling across the membrane. In contrast, the HOG pathway functioned independently of the cytosolic tail and tolerated misfolded SEA domain variants, consistent with trans signaling mediated by extracellular domains of interacting partners. The HOG pathway may detect misfolding as part of its sensing mechanism, as stressors that induce protein misfolding required Msb2 for survival. This work reveals how differential tolerance to protein deformation confers signaling specificity and identifies sensor deformation as a general feature of mechanosensory pathways that respond to environmental stress. HIGHLIGHTSO_LISignaling pathways differ in tolerance to misfolding of a sensory domain C_LIO_LIMisfolded SEA domains retain function in a stress pathway (HOG) pathway but not a cell differentiation pathway (fMAPK) O_LIMisfolded SEA domain variants showed altered protein levels, mis-localization in the secretory pathway, and turnover by ERAD C_LIO_LINon-functional variants lacked residues that stabilize the structure through intramolecular bonds C_LI C_LIO_LIDifferential tolerance for misfolding revealed distinct modes of signaling O_LITrans signaling predominated in the HOG pathway and did not require proper SEA domain folding or the mucin cytosolic tail O_LIA dominant hyperactive variant next to the SEA domain revealed basal interactions with the CR domain of tetraspanin C_LIO_LIAlphaFold modeling showed distinct interactions occur between the SEA domain and tetraspanin in the basal and activated states C_LI C_LIO_LICis signaling predominated in the fMAPK pathway O_LIRequired a properly folded SEA domain and conformational coupling to the cytosolic tail C_LIO_LIYapsin processing was required for SEA domain activation and turnover of the mucin cytosolic tail C_LI C_LI C_LIO_LIHOG pathway may sense protein misfolding as part of its activation mechanism. C_LIO_LISEA domains are conserved throughout fungal mucins and mammalian glycoprotein sensors suggesting a generalizable mechanism C_LIO_LIProtein deformation may provide information to survival pathways about environmental stress. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/723240v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1cd30f3org.highwire.dtl.DTLVardef@48c96corg.highwire.dtl.DTLVardef@9fffc2org.highwire.dtl.DTLVardef@504b1d_HPS_FORMAT_FIGEXP M_FIG C_FIG Signaling pathways often share components yet activate different effector processes through mechanisms that remain unclear. The same mucin regulates two MAPK pathways (red and green), and the discovery of a conserved SEA domain provided insights into specificity mechanisms. In the fMAPK pathway that regulates filamentous growth, the mucin works in a classical manner, where an external signal (in this case underglycosylation by glucose limitation) transduces a signal to the cytosolic domain in cis. By comparison, the HOG pathway that responds to osmotic stress displayed a remarkable tolerance for mucin and SEA domain deformation. Protein variants that caused SEA domain misfolding, mislocalization, and degradation by ERAD retained function in the HOG pathway. Truncations that removed the cytosolic tail and transmembrane anchor were also functional. These phenotypes support a trans activation mechanism with external partners that was preferential for activation of the HOG pathway. SEA domain deformation may be induced by environmental stress as a trigger for the HOG pathway. Cells may detect misfolding of protein domains to gain information about environmental stress.

Adipogenesis is the process of adipose-derived stem cells (ADSCs) responding to extracellular signals from the stem cell niche to differentiate into adipocytes (fat cells) and may be studied in vitro using a cocktail of chemicals that promote adipogenic differentiation to produce differentiated ADSCs (dADSCs). The global membrane N- and O-glycosylation changes of this process have been previously analysed and compared to native adipocytes as a benchmark for a true adipocyte profile, and revealed that bisecting GlcNAc type N-glycans are characteristic of adipogenesis. As stem cell differentiation has been widely reported to result in cellular protein changes, the same cells (ADSCs, dADSCs and mature adipocytes) were characterised for their membrane proteome here using label-free quantitative shotgun proteomics analysis. The membrane proteome displayed more differences in protein numbers between the cell types compared to the previously reported N-glycome which had shown high identical glycomes between stem cells and in vitro dADSCs, suggesting that the proteome is more dynamic during in vitro adipogenesis. Following the global shotgun proteomics analysis, a more targeted approach of carrying out proteomic analysis of de-N-glycosylated peptides of gel-separated proteins unearthed new glycoproteins not detected in the shotgun proteomic analysis. This approach identified the adipogenic marker, CD36, to be under-represented in the shotgun proteome analysis, but as the dominant (glyco)protein in the adipocyte membrane proteome that was also up-regulated at the mRNA transcript level in both the in vitro differentiated ADSCs (7.1-fold increase) and mature adipocytes (102.9-fold increase). A comparison of CD36 sequence coverage in the global shotgun analysis with the de-N-glycosylated CD36 revealed a 41% increase when N-glycans were removed prior to trypsin digestion, explaining its observed increased abundance and highlights the crucial need for de-N-glycosylation of proteins in proteomics experiments for increased identification of glycoproteins. The systems glycobiology approach by the integration of previously reported glycomics data and the proteomics and transcriptomics analyses in this work extended the investigation of membrane protein glycosylation changes in adipose-derived stem cell differentiation. The work provides a framework for future glycoproteomics-based investigations into the differentiation of stem cells into adipocytes, and will allow their related pathologies and potential therapeutic applications to be discovered. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/722121v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@189a786org.highwire.dtl.DTLVardef@5563b8org.highwire.dtl.DTLVardef@5cb5borg.highwire.dtl.DTLVardef@69e11f_HPS_FORMAT_FIGEXP M_FIG C_FIG

IgA nephropathy (IgAN) is a common primary glomerulonephritis characterized by glomerular immune-complex deposits with (co)dominant IgA. These deposits are enriched for IgA1 glycoforms with some O-glycans deficient in galactose (Gd-IgA1). Circulating Gd-IgA1 is bound by IgG autoantibodies to form immune complexes, some of which deposit in glomeruli. Genomic and immunologic studies indicate involvement of pro-inflammatory signaling pathways in the production of Gd-IgA1 in IgAN. Genomic studies identified multiple genetic loci associated with IgAN and suggested a convergence on the NF-{kappa}B pathway, including RELA, the gene encoding the NF-{kappa}B subunit p65. However, the mechanisms by which NF-{kappa}B pathways may affect O-glycosylation in IgA1-producing cells are unknown. Using EBV-immortalized B cells derived from peripheral-blood mononuclear cells of IgAN patients and healthy controls that have constitutively activated NF-{kappa}B, we report that inhibition of NF-{kappa}B/p65 by a selective IKK{beta} inhibitor TPCA-1 reduced phosphorylation of NF-{kappa}B/p65 at S536 and decreased production of IgA1 and, conversely, increased Gd-IgA1 production. This was likely related to reduced expression of C1GALT1 gene that encodes the enzyme responsible for galactosylation of IgA1 O-glycans. Flow-cytometry imaging revealed changes in nuclear translocation and co-localization of the NF-{kappa}B/p65 with co-transcriptional factor SP1, a transcriptional activator of C1GALT1, suggesting that NF-{kappa}B pathway affects IgA1 O-glycosylation via SP1 transcriptional control of C1GALT1 expression. Furthermore, prolonged IKK{beta} inhibition altered B cell subpopulations, enhancing generation of cells with a plasmablast-like phenotype, characterized by high SSC MFI and CD138 expression. Together, these findings provide functional evidence for involvement of NF-{kappa}B/p65 and its transcriptional partners in IgA1 O-glycosylation. HighlightsO_LIIKK{beta} inhibition reduced C1GALT1 expression and thereby increased galactose-deficient IgA1 (Gd-IgA1) production in immortalized human B cells. C_LIO_LISP1+ subpopulations, a transcriptional activator of C1GALT1, declined after sustained NF-{kappa}B inhibition. C_LIO_LINF-{kappa}B inhibition shifted a subpopulation of B cells into a plasmablast-like phenotype. C_LIO_LIThis study links NF-{kappa}B signaling with the GWAS-identified RELA susceptibility locus and IgA1 O-glycosylation. C_LI

Human RNase 2 (eosinophil-derived neurotoxin, EDN) is a major eosinophil granule protein of the vertebrate-specific RNase A superfamily and is involved in antiviral response and inflammation. Identifying ligand-binding pockets in EDN is thus relevant to structure-based drug design. In our laboratory we identified by protein crystallography a conserved site at the protein surface binding to carboxylic anion molecules (malonate, tartrate and citrate). Searching for potential biomolecules rich in anion groups and considering previous report of EDN binding to glycosaminoglycans, we explored the protein binding to saccharides. Next, EDN crystals were soaked with mono- and disaccharides, and the 3D structures of ten complexes were solved by X-ray crystallography at atomic resolution. We identified protein binding pockets to glucose, fucose, mannose, sucrose, galactose, trehalose, N-acetyl-D-glucosamine, N-acetylmuramic acid, and the sialic acid N-acetylneuraminic acid. A main site for glucose, fucose, and galactose was located adjacent to the spotted carboxylic anion site. Secondarily, N-acetylneuraminic acid, N-acetylmuramic acid, sucrose, galactose, and mannose shared another protein surface region. Overall, the saccharides clustered into seven defined sites, outlining a conserved recognition pattern, which was further analysed by molecular modelling. Interestingly, within the RNase A family, we find amphibian RNases that were initially isolated as carbohydrate binding proteins and named as leczymes, combining enzymatic and lectin properties. The present data is the first systematic structural characterization of a mammalian sugar-binding RNase within the family. The results highlight unique EDN residues that mediate its sugar specific interactions, of particular interest for a better understanding of the protein physiological role. HighlightsO_LIstructure of RNase 2 in complex with mono and disaccharides at atomic resolution C_LIO_LIidentification of RNase 2 unique sugar binding sites C_LIO_LIcharacterization of a mammalian RNase A family enzyme with lectin properties C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/713198v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1d805f7org.highwire.dtl.DTLVardef@16fcc49org.highwire.dtl.DTLVardef@ccfd92org.highwire.dtl.DTLVardef@1b8f1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biodesign is an interdisciplinary research domain that incorporates principles from design and the life sciences to develop new systems, processes, and objects. Collegiate biodesign educators face unique pedagogical challenges, including an absence of relevant scholarship on curriculum design and instructional best practices for cultivating student scientific literacy. These difficulties may be overcome with newly available technologies, like generative AI systems, that enable personalized learning through domain-specific semantic spaces. This article examines the instructional value of one such domain-specific LLM, Biodesign Buddy, through a mixed-methods analysis of an eight-week study involving 64 students participating in an international biodesign competition. Results indicate strong support for integrating AI into biodesign coursework. Surveys captured attitudes toward AI, scientific literature, and learning experiences to assess AIs impact on learning outcomes. Findings suggest that integrating AI into biodesign pedagogy can meaningfully redress conceptual issues in biodesign while informing broader debates on AIs role in higher education. Impact StatementThis article introduces Biodesign Buddy, a domain-specific generative AI system for collegiate biodesign education, and reports on its exploratory deployment, offering design principles and preliminary findings to inform the development of AI-supported pedagogies for interdisciplinary biodesign instruction.

Xanthan gum is a widely used industrial polysaccharide employed as a thickening and stabilizing agent in food, pharmaceutical, and technological applications. Its biosynthesis involves membrane-associated glycosyltransferases that assemble the repeating unit at the cytoplasmic side of the inner membrane. Among them, GumH and GumI catalyze consecutive reactions using the same donor substrate, guanosine 5-diphospho-alpha-D-mannose, but with opposite stereoselectivity. Despite their biochemical characterization, structural insights into their catalytic mechanisms and membrane interactions remain limited, hindering a detailed understanding of their function and future engineering efforts. In this work, we combined artificial intelligence-based structure prediction with atomistic molecular dynamics simulations to investigate the structural organization and substrate-binding modes of GumH (family GT4) and GumI (family GT94). The predicted apo structures exhibit a conserved GT-B fold but differ in interdomain flexibility and membrane-anchoring strategies. GumH displays a more structured interdomain linker and a defined clamp-like region in the acceptor-binding domain, consistent with stable membrane interaction, whereas GumI shows a more flexible linker and an open groove architecture. Modeling of the donor-bound complexes reveals distinct substrate-binding modes. In GumH, it adopts a geometry consistent with its retaining stereochemical outcome, positioning the sugar close to the conserved catalytic residue. In contrast, GumI exhibits a different donor orientation, lacking a clearly positioned catalytic base near the reactive center, suggesting a substrate-assisted catalytic mechanism. Although the predicted ternary complexes show limited stability in our simulations, they provide chemically reasonable conformations and offer structural insights into substrate recognition, membrane association, and stereochemical control in these two glycosyltransferase families. Significance statementXanthan gum is an industrially important polysaccharide widely used in food and other technological products. Although several enzymes in its biosynthetic pathway have been studied, structural information remains limited. Using AI-based structure predictions and molecular simulations, we revealed how these enzymes sit in the membrane and bind sugar substrates. These structural insights clarify xanthan biosynthesis and could help improve or engineer its production.

O-fucosylation plays an essential role in controlling protein folding, secretion and protein-protein interactions within the extracellular space. Recently, we identified a new form of protein O-fucosylation occurring on the N-terminal Elastin Microfibril Interaction (EMI) domain of several secreted proteins, mediated by two previously uncharacterized protein O-fucosyltransferases, POFUT3 (FUT10) and POFUT4 (FUT11). As all POFUT enzymes (POFUT1-4) are highly specific for the three-dimensional (3D) structure of their substrate protein domains, we postulated that structural homologues of these domains in other proteins may also be O-fucosylated. Here, we employed iterative protein structural homology searches as a novel strategy for identifying EMI-like domains that may serve as potential substrates for POFUT3/4. We discovered that microfibrillar-associated protein 2 and 5 (MFAP2/MFAP5) contain EMI-like domains and are O-fucosylated at high stoichiometry in human tissues. Unexpectedly, we showed that only POFUT3 is both necessary and sufficient for MFAP2/MFAP5 O-fucosylation, despite POFUT4 also having strong protein-protein interactions with MFAP2/MFAP5. Finally, we determined that O-fucosylation of MFAP2/MFAP5 is required for their efficient secretion, similar to other EMI domain-containing proteins. Together, these data demonstrate the power of sensitive structural homology analysis in identifying new enzyme-substrate relationships and protein-protein interactions.

The extracellular matrix (ECM) is a meshwork of proteins that orchestrates a broad range of cellular phenotypes, including proliferation, adhesion, migration, and differentiation. SNED1 is a newly characterized ECM glycoprotein that promotes cell adhesion and is essential for embryonic development. Its upregulation is also associated with breast cancer metastasis and poor prognosis for breast cancer patients. We recently showed that SNED1 assembles into fibrillar structures, but the mechanisms guiding its incorporation into the ECM scaffold remain unknown. Combining biochemical assays and confocal immunofluorescence imaging, we found that SNED1 assembly in the ECM occurs early in the process of ECM building and is concomitant and overlaps with the deposition of fibronectin and collagen I, two major ECM proteins. By knocking down fibronectin or destabilizing collagen I fibers, we further demonstrate that SNED1 requires the presence of these proteins for its assembly. Last, using biolayer interferometry, we identify collagen I as the first direct binding partner of SNED1. Altogether, our results lay the foundation for future studies aimed at determining the mechanisms by which SNED1 fibers contribute to SNED1 pathophysiological functions. SUMMARY STATEMENTThe novel protein SNED1 requires the presence of fibronectin and collagen I to assemble into fibrillar structures in the extracellular matrix scaffold.

Herpes simplex virus (HSV) fusion and entry is mediated by a cascade of interactions among gB, gD and gH/gL. The gB homotrimer undergoes a conformational transition from a metastable prefusion state to a more stable postfusion form, driving fusion of the viral envelope and a host cell membrane. An H516P mutation in gB domain III restricts formation of the extended core alpha helix and constrains gB in a prefusion state. Several prefusion gB structures have been determined that contain this mutation. We assessed the antigenic reactivity of gB H516P by quantitative immunodotblot using a panel of gB-reactive monoclonal antibodies All antibodies tested bound to both prefusion (H516P) gB and wild type gB. Antibodies tested to gB domains II, IV and V exhibited differential binding to H516P gB compared to wild type gB. The results suggest that gB H516P has a distinct antigenic profile. The antigenic signature of H516P may be useful as a rapid indicator of prefusion forms of gB. The low pH environment of the cellular endosome is a cell-specific factor for HSV entry and triggers antigenic changes in gB. The step at which low pH impacts gB refolding to execute fusion is not well-understood. The results suggest that gB H516P undergoes wild-type-like conformational changes in gB domains I and V triggered by low pH. We propose that pH acts on an early stage of gB fusion function, prior to extension of the domain III core helix.

A human infection with clade 2.3.4.4b H5N1 influenza A virus in Canada revealed minority variants E190D and Q226H in the hemagglutinin (HA) receptor-binding site (RBS). Because mutations at positions 190 and 226 have been associated with altered receptor specificity in other influenza subtypes, we investigated their impact on receptor binding in H5 HA. Using a recombinant protein approach and an ELISA-based glycan-binding assay, we assessed binding to representative avian- and human-type sialylated glycans. Both single mutations and their combination resulted in a complete loss of detectable binding to the tested glycans. To evaluate whether this phenotype was background-dependent, Q226H was additionally introduced into two other H5 HA proteins, each representing a distinct clade. In both cases, the mutation similarly abolished receptor binding. These findings independently validate recent glycan microarray observations and demonstrate that the patient-derived E190D and Q226H substitutions severely impair receptor-binding capacity across multiple H5 backgrounds. Single mutations at key RBS residues in H5 often disrupt receptor binding rather than confer human-type receptor specificity, confirming complex mutational pathways required for adaptation to human-type receptors.

Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 {micro}M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher than that of Fvp (76.5 {micro}M). At nanomolar concentrations, binding was observed only to the Avi-tag-, but not amine-biotinylated RBDs, suggesting better accessibility of their binding sites. The association rates (kon) were faster for Fvc than for Fvp. Similarly, affinities of Fvc_1098 (23.4 nM) and Fvc_58 (4.48 M) for ACE2 were greater than that of Fvp (66.8 M), indicating that Fvc can bind directly to both RBD and ACE2. Fvc demonstrated enhanced inhibitory potency (IC50 = 58 g/mL) compared to Fvp (IC50 > 239 g/mL) in the pseudovirus entry assay and did not induce cytotoxicity in HEK293T cells. In conclusion, crude fucoidan with high fucose content and high molecular weight shows promising antiviral activity.

Salla disease is caused by a genetic mutation in sialin, a lysosomal membrane transporter, which exports sialic acid from lysosomes. Substrate translocation occurs via a rocker-switch mechanism that alternately exposes the substrate-binding site to the lysosomal lumen and the cytosol. The pathogenic mutation R39C found in most Salla disease patients decreases the lysosomal localisation and the transport activity. In this study, we used computational and mutagenesis approaches to elucidate the molecular effects of the R39C mutation. Using three-dimensional models of human sialin in the lumen-open (LO) and cytosol-open (CO) states combined with the mutagenesis of selected residues, we identify a critical "triplet" motif comprising R39, E194, and E262, which is associated with an ionic lock formed between K197 and D350 in the LO conformation. Molecular dynamics simulations suggest that the electrostatic triplet negatively modulates the ionic lock, and are consistent with a strengthened ionic lock in R39C sialin, potentially favouring the LO state. To assess the global effects of the R39C mutation, we computed dynamic cross-correlation matrices and identified correlation patterns consistent with an allosteric coupling between the ionic lock K197/D350 and the region surrounding the sialic acid binding site in wild-type sialin, whereas in the LO state of R39C sialin, this communication preferentially bypasses this region. Therefore, the R39C mutation may impede the LO to CO conformational transition required for sialic acid transport, providing a plausible mechanistic framework for the decreased transport activity, and possibly the decreased lysosomal localisation, observed in Salla disease. HighlightsO_LIThe R39 residue participates in an interaction triplet, which negatively regulates an ionic lock stabilising the lumen-open conformation C_LIO_LIThe R39C mutation is associated with a stronger ionic lock in the simulations, and may favour the lumen-open state C_LIO_LICorrelation network analysis suggests an allosteric coupling between the ionic lock and the region surrounding the sialic acid binding site C_LIO_LIThe R39C mutation alters the inferred allosteric coupling between the ionic lock and the region surrounding the sialic acid binding site C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/719580v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1bf7144org.highwire.dtl.DTLVardef@1a53ab8org.highwire.dtl.DTLVardef@b2249forg.highwire.dtl.DTLVardef@1827244_HPS_FORMAT_FIGEXP M_FIG C_FIG

Listeria monocytogenes relies on a tightly controlled set of surface-associated and secreted proteins to mediate host interaction and infection. The correct localization and exposure of these proteins at the bacterial surface are critical for virulence, yet the role of cell wall components in organizing this process remains incompletely understood. In particular, wall teichoic acid (WTA) glycosylation has been implicated in anchoring and function of selected surface proteins, but its global impact on protein distribution across the bacterial cell envelope is unclear. Here, we performed a comprehensive proteomic analysis to investigate how WTA glycosylation influences protein distribution in L. monocytogenes. Using isogenic mutants lacking rhamnose ({Delta}rmlT) or GlcNAc ({Delta}lmo1079) WTA glycosylation, we compared the exoproteome, the surface-accessible proteome and the surface-exposed proteome. Loss of WTA glycosylation did not result in a global disruption of the surface proteome but instead induced a redistribution of proteins across extracellular and surface-associated fractions. This effect was dependent on protein anchoring mechanisms, with limited changes observed for LPXTG-anchored proteins, moderate effects on non-covalently associated proteins, and a marked enrichment of lipoproteins in the surface-exposed proteome, particularly in the {Delta}lmo1079 mutant. In parallel, virulence-associated proteins displayed altered accessibility and exposure, with a progressive shift towards increased surface localization and a combination of shared and mutant-specific responses. This global effect was supported by functional annotation, which revealed that the affected proteins were associated with similar biological processes across fractions, highlighting a broad rather than pathway-specific impact of WTA glycosylation loss Together, these findings indicate that WTA glycosylation plays a key role in organizing the bacterial surface by modulating protein retention, exposure and release. Rather than affecting specific proteins, WTA glycosylation broadly shapes the spatial distribution of proteins across the cell envelope, with potential consequences for host- pathogen interactions.

{beta}-glucosidase (BglB) from Paenibacillus polymyxa was mutated (G23S, Rosetta/Foldit numbering; G26S, conventional numbering) to assess structural and functional changes. Foldit modeling and prior Design 2 Data (D2D) database results led us to hypothesize that this mutation would increase substrate binding affinity and catalytic efficiency, with a moderate reduction in thermal stability. The mutant protein was expressed, purified, and analyzed using kinetics and thermal stability assays. Relative to the wild-type (WT), G23S exhibited a similar binding affinity (similar Km), an approximately 2-fold increase in turnover number (kcat) and catalytic efficiency (kcat/Km), an almost 14-fold increase in maximum reaction velocity (Vmax) and a slight decrease in thermostability (T50). The results largely support the hypothesis, indicating that changes in residue 23 can enhance catalytic power while minimally compromising stability.