Search Results (1,062)

ATP-binding cassette (ABC) transporters are one of the largest superfamilies of membrane proteins, but little is known about the structural and evolutionary features of their non-domain regions. To clarify the diversity of these non-canonical regions across evolutionary lineages, we performed an analysis of intrinsically disordered regions, site-specific selection and predicted post-translational modification (PTM) sites among five architectural classes involving 1581 prokaryotic and eukaryotic sequences. Linker and flanking regions were more disordered than transmembrane and nucleotide-binding domains in all architectures. Disorder fraction was significantly different between region types after phylogenetic correction (Pagel’s λ ≈ 0.97). Predicted PTM sites are enriched in disordered non-domain segments, with N-linked glycosylation and phosphoserine showing the strongest positive enrichment. A total of 140 sites satisfied a tiered conservation criterion (MusiteDeep score ≥ 0.5; cross-species conservancy ≥ 30%), including 40 high-confidence or moderate-confidence sites (conservancy ≥ 50%) as well as novel phosphotyrosine candidates in half transporters and NBD-only proteins. Site-specific selection analyses showed pervasive purifying selection across domain cores and architecture-dependent enrichment of episodic positive selection in non-domain regions, with significant non-domain enrichment in full reverse and half forward transporters (Fisher’s exact, BH-adjusted p < 0.05). In summary, these findings establish that non-canonical regions of ABC transporters are evolutionarily dynamic and contain conserved predicted modification sites, supporting the idea that these regions are evolutionary dynamic segments that deserve experimental characterization as candidate regulatory interfaces. Full article

Precision nano-fertilization offers transformative potential for sustainable improvement of grape quality, yet the underlying molecular mechanisms remain poorly understood. Here, we investigated the effects of foliar-applied nano zero-valent iron (nZVI) and potassium dihydrogen phosphate (KH₂PO₄), in combination, on berry quality and secondary metabolic reprogramming in Vitis vinifera cv. Marselan. The combined nZVI/KH₂PO₄ treatment improved photosynthetic capacity, Fe/P co-accumulation, and berry quality traits including soluble solid content, sugar–acid ratio, and phenolic and aroma metabolite profiles. Crucially, integrated transcriptomic and metabolomic profiling identified 631 differentially expressed genes and 838 differentially accumulated metabolites, converging on flavonoid biosynthesis and glutathione metabolism as the dominant regulatory axes. Correlation network analysis pinpointed five hub regulatory genes—VvHCT, VvFLS1, VvLAR1/2, VvUGT88F5, and VvODC—as central orchestrators of nanomaterial-driven metabolic reprogramming: VvHCT and VvFLS1 coordinately redirected carbon flux toward hydroxycinnamic acid conjugates and flavonol accumulation, while VvLAR1/2 governed proanthocyanidin polymerization, and VvUGT88F5 modulated glycosylation-dependent metabolite stabilization. Notably, VvODC linked polyamine metabolism to glutathione-mediated stress buffering, revealing a previously uncharacterized crosstalk between nano-iron signaling and antioxidant reprogramming. These findings establish a mechanistic framework in which nZVI and KH₂PO₄ synergistically remodel the secondary metabolome through discrete yet interconnected transcriptional nodes, providing molecular targets for nano-enabled precision viticulture and broader applications of engineered nanomaterials in high-value crop improvement. Full article

Ring chromosome 14 (RC14) syndrome is an ultra-rare disorder characterized by drug-resistant epilepsy, intellectual disabilities, autism, and recurrent infections, suggesting a possible underlying immune dysregulation. We analyzed immunoglobulin G (IgG) N-glycosylation profiles in six RC14 patients and compared them with age-matched healthy controls using ultra-high-performance liquid chromatography (UHPLC) coupled with fluorescence detection (FLR) and high-resolution electrospray ionization mass spectrometry (ESI-MS). Patients showed decreased galactosylation and sialylation, resembling pro-inflammatory patterns observed in autoimmune diseases. These alterations were not observed in total serum glycoproteins, indicating a selective effect on IgG. One patient treated with intravenous immunoglobulin (IVIG) showed clinical improvement, which led us to investigate causality. Full article

Glycosylation is essential for hematopoietic cell homeostasis and malignant transformation. Dysregulated expression of glycosylation genes in leukemia cells accelerates disease progression and fosters drug resistance. Therefore, targeting these genes offers a promising avenue for anti-leukemic therapy. In this study, we explore the roles of N-glycans in acute promyelocytic leukemia (APL) differentiation using the ATRA-induced wild-type NB4 (WT/ATRA) or HL-60 cell model. We found that expression of N-acetylglucosaminyltransferase IVa (GnT-IVa, encoded by the MGAT4A gene) and its product (β1,4-GlcNAc-branched N-glycan) increased significantly during differentiation, as evaluated by lectin blot, real-time PCR, and flow cytometry. Interestingly, analysis of the Gene Expression Omnibus (GEO) public data showed that MGAT4A expression is significantly lower in APL patients, and higher MGAT4A expression was associated with favorable survival in AML cohorts. To address the role of GnT-IVa in differentiation, we established MGAT4A- and MGAT4B-knockout (KO) NB4 cell lines using CRISPR/Cas9. Compared to WT/ATRA cells, MGAT4A KO, but not MGAT4B KO, markedly suppressed ATRA-induced differentiation, as evidenced by reduced expression of CD11b and CD11c. We found that CD11b is a major glycoprotein carrying β1,4-GlcNAc-branched N-glycans. This modification enhanced CD11b stability, as CD11b expression declined more rapidly in MGAT4A KO cells in the presence of cycloheximide. In addition, MGAT4A KO suppressed ERK/MAPK signaling, which contributed to differentiation. Our study highlights the critical role of GnT-IVa in regulating APL differentiation, which may provide a basis for developing new differentiation therapies for APL. Full article

Background: Antigenic drift limits the protective efficacy of influenza vaccine. Glycosylation of hemagglutinin (HA) represents a promising immunofocusing strategy that enhances neutralizing antibody responses by masking immunodominant non-neutralizing epitopes. Methods: B-cell epitopes of influenza viruses were retrieved from the Immune Epitope Database and were mapped onto the HA structure of A/Puerto Rico/8/1934 (H1N1). Structure-guided analysis identified residues 136 and 137 as candidate sites for N-linked glycosylation (NLG). Single-site mutants (136NLG and 137NLG) were generated using reverse genetics and evaluated for stability, receptor binding, viral replication, and immunogenicity in a murine model with inactivated whole-virus vaccines. Results: Both mutants exhibited increased thermostability at 42 °C. Glycosylation reduced the HA–sialic acid affinity, resulting in decreased viral adsorption and internalization efficiency in MDCK cells, and delayed viral replication at low multiplicity of infection (MOI). In vivo, all vaccine groups provided complete protection against lethal challenge; notably, the 136NLG group exhibited reduced weight loss, indicating improved protective efficacy compared with wild-type (WT). Conclusions: Targeted glycosylation at residue 136 in the HA head domain effectively enhances the viral stability and elicits a 1.78-fold increase in hemagglutination inhibition titer (GMT) relative to the WT, thereby improving vaccine performance. These findings establish a rational and structure-based design strategy for developing more stable and effective influenza vaccines. Full article

The hemagglutinin (HA) of influenza A/H3N2 virus evolves rapidly, with glycosylation driving immune evasion. However, how host microenvironmental cues influence this process remains poorly understood. We identified a novel N-linked glycosylation site at position 110 (N110) in contemporary H3N2 viruses (NSS genotype) that enhances viral fitness by increasing receptor-binding signal, HA cleavage, and replication. Remarkably, hypoxia, which mimics the respiratory tract microenvironment, significantly augments N110 glycosylation. Mechanistically, we identified the B4GAT1-B4GALT1 complex as the key mediator of this modification. Hypoxia upregulates their expression and strengthens their interaction with HA. In ferret models, N110-glycosylated viruses exhibit heightened pathogenicity and evade ancestral antibodies. Furthermore, immunization with N110-containing HA confers broad-spectrum protection, whereas reciprocal immunization is ineffective. Our findings reveal hypoxia-driven glycosylation as a previously unrecognized mechanism of H3N2 adaptation, providing critical insights for vaccine efficacy and highlighting the importance of integrating microenvironmental factors into future antiviral strategies. Full article

Background: Tumour glycosylation regulates immune modulation and progression, but whether the CRC sialylome—the complete repertoire of sialylated glycans—defines a biologically distinct subtype remains unclear. We investigated how the “sugar code” shapes CRC biology, immunity, and therapeutic response. Methods: Transcriptomic data from three CRC cohorts (TCGA, Sidra-LUMC, and CPTAC-2; n = 988) were batch-corrected and integrated. Single-sample gene set enrichment analysis (ssGSEA) quantified sialyltransferase expression, sialic acid metabolism, EMT, MDR mechanisms, immune phenotypes, and Siglec-associated transcriptional signatures. GSEA, gene ontology enrichment analysis (GOEA), and drug ontology enrichment analysis (DOEA) characterised pathways and identified drug response-associated transcriptional signatures. Results: High sialylome activity defined a genomically stable but clinically advanced CRC subset enriched for left-sided tumours, mucinous histology, MSI, and BRAF mutations. At the transcriptional level, Sialyl-High tumours were associated with a mesenchymal, stromal-remodelling programme accompanied by reduced proliferative activity. They demonstrated enrichment of vesicular trafficking-related pathways alongside reduced representation of canonical efflux-associated programmes. Critically, the sialylome was associated with Siglec-related immune signatures, with sialylated glycan-related gene expression correlating with Siglec receptor expression (CD33 and SIGLEC7/9/10), consistent with an immune-inflamed yet structurally excluded microenvironment. DOEA identified selective enrichment of drug-response signatures related to sialic acid metabolism inhibitors (oseltamivir and Neu5Ac) and glycocalyx-disrupting agents (ginsenosides and soyasaponins). Conclusions: The CRC sialylome is associated with tumour phenotypic variation, including immune-excluded states linked to Siglec-associated transcriptional signatures and patterns consistent with non-canonical drug resistance programmes. These findings position the “sugar code” as a central organising principle in CRC and identify glycan-directed therapies as a promising strategy for the targeting of this aggressive subtype. Full article

Introduction: Intermittent fasting (IF) is becoming increasingly popular as a method of weight management, but it is unknown whether it affects plasticiser intake with resultant changes in glycaemic control in diabetes and vitamin D (VitD) levels; therefore, this study was undertaken in a cohort of control and type-2 diabetic (T2D) subjects during Ramadan time-restricted feeding (TRF). Methods: In T2D subjects (n = 19) and controls (n = 31) undertaking TRF, 24 h urinary levels of phthalate metabolites, bisphenols and serum VitD were determined pre- and post-TRF by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Anthropometric data and glycosylated haemoglobin (HbA1c) were measured. Results: T2D subjects were older (52 versus 36.73 years, p < 0.001), and had higher BMI (36.54 versus 27.67 kg/m², p < 0.001), body weight (101.77 versus 80.36 kg, p < 0.001), and HbA1c (8.38 versus 5.46%, p < 0.001) compared to controls, while VitD levels did not differ (60.43 versus 63.95 nmol/L, p > 0.05). Post-TRF, HbA1c was unchanged in T2D subjects and there was no difference in weight, BMI or VitD. Increased mono-iso-butyl phthalate (MiBP) in T2D subjects (10 versus 6.1 ng/mL, p = 0.001) and mono-n-butyl phthalate (MnBP) in T2D subjects (37 versus 13 ng/mL, p = 0.018) and controls (8.3 versus 5.4 ng/mL, p = 0.007) were observed post-TRF; however, significance was lost after adjusting for baseline differences in age, BMI, and HbA1c using a general linear model (GLM) repeated-measures ANOVA. Despite having no median differences in DEHP (di-2-ethylhexyl phthalate) metabolites pre- and post-TRF, analyses revealed a significant time × HbA1c interaction for [mono(2-ethyl-5-carboxypentyl) phthalate, MECPP: F(1,42) = 4.79, p = 0.03, mono(2-ethyl-5-hydroxyhexyl) phthalate, MEHHP: F(1,42) = 8.56, p = 0.006, mono(2-ethylhexyl) phthalate, MEHP: F(1,42) = 4.64, p = 0.03 and mono(2-ethyl-5-oxohexyl) phthalate, MEOHP: F(1,42) = 8.19, p = 0.007] and time × group interactions [MEHHP: F(1,42) = 14.27, p < 0.001, MEHP: F(1,42) = 6.35, p = 0.01 and MEOHP: F(1,42) = 10.30, p = 0.003]. Estimated marginal means (adjusted for age, BMI, HbA1c, and VitD) further confirmed higher concentrations of DEHP metabolites [MECPP, MEHHP, MEHP, and MEOHP] in T2D participants over time compared with controls. Additionally, monomethyl phthalate (MMP) trajectories were significantly influenced by the time × group interaction (F(1,42) = 4.28, p = 0.04), with post-TRF elevations observed in T2D subjects. Vitamin D status was observed to modify MCPP and MEP trajectories over time. Conclusion: Ramadan TRF is associated with changes in plasticiser metabolite levels, with estimated increased levels in T2D subjects versus healthy controls. Metabolite levels were influenced by HbA1c and vitamin D, though BMI was not observed to be a contributing factor. Full article

Muco-obstructive lung diseases, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and bronchiectasis, are characterized by the accumulation of highly viscoelastic mucus that compromises mucociliary clearance and fosters infection and inflammation. Mucoactive therapy, encompassing both true mucolytics and non-cleaving agents, seeks to restore airway patency by altering mucus structure, hydration, and transport properties, yet its clinical impact remains variable. This narrative review provides a critical reappraisal of the pharmacological actions and therapeutic outcomes of the main mucolytic agents: N-acetylcysteine (NAC), erdosteine, carbocisteine, bromhexine, ambroxol, and dornase alfa. Beyond their classical role in reducing mucus viscosity, these drugs exhibit pleiotropic effects, including antioxidant, anti-inflammatory, and immunomodulatory activities. Specifically, for thiol-based compounds, the action consists of breaking the disulfide bonds that stabilize the mucin network; for carbocisteine, it lies in modulating mucin glycosylation and chloride transport. Ambroxol and bromhexine act by stimulating surfactant secretion and enhancing mucociliary clearance. Finally, dornase alfa exerts an enzymatic effect on extracellular DNA, a key contributor to the tenacity of mucus in cystic fibrosis. Clinical evidence indicates that NAC and erdosteine can reduce exacerbation rates in COPD, carbocisteine shows benefit with prolonged administration, and dornase alfa remains a cornerstone in CF management. However, therapeutic efficacy is constrained by heterogeneous mucus composition, pharmacokinetic limitations, and disease-specific variability. A key interpretative message is that clinical benefit appears greatest when the dominant biophysical determinant of mucus pathology is specifically targeted, supporting a transition from broad disease-label prescribing to mechanism-informed, phenotype-aware mucolytic therapy. Emerging strategies, such as agents targeting mucin–DNA interactions and advanced inhalation delivery systems, promise improved specificity and durability. By integrating mechanistic insights with clinical data, this review underscores the need for personalized mucolytic therapy and innovative approaches to overcome current challenges in managing muco-obstructive lung diseases. Full article

Glycosylation is one of the most prevalent post-translational modifications of membrane proteins and plays a central role in regulating their structure and function. Unlike many existing reviews that address glycosylation in a system-wide context, this review focuses specifically on membrane proteins and examines how glycosylation shapes their functional behavior and clinical relevance. Because membrane proteins are exposed to the extracellular environment, glycans on their surface directly influence protein folding, receptor organization, and interactions with ligands and immune components. These diverse effects can be understood within a common mechanistic framework in which glycosylation modulates protein conformation, receptor clustering, and membrane organization, thereby altering signaling, adhesion, transport, and immune recognition. We discuss how N-linked and O-linked glycosylation regulate major classes of membrane proteins across these processes. Particular attention is given to disease-associated alterations in glycosylation, especially in cancer, immune and inflammatory disorders, and metabolic disease. For instance, glycosylation-dependent stabilization of PD-L1 and modulation of receptor signaling, such as EGFR, illustrate how glycan modifications contribute to immune evasion and therapeutic response. We further consider the clinical implications of membrane protein glycosylation, including its roles in biomarker development and as a potential target for therapeutic intervention. Advances in glycoproteomic technologies have enabled increasingly detailed characterization of site-specific glycosylation, although significant analytical challenges remain, particularly for membrane proteins. Overall, this review highlights membrane protein glycosylation as a dynamic regulatory layer that links molecular mechanisms to functional outcomes and clinical applications. Full article

Background/Objective: Immunoglobulin G (IgG) N-glycosylation is an important regulator of immune function and systemic inflammation and has been associated with cardiometabolic diseases. However, little is known about how IgG glycosylation changes during the course of acute coronary syndrome (ACS) and whether these alterations relate to lipid-lowering response after the initiation of statin therapy. The primary aim of this study was to investigate IgG N-glycosylation following ACS and evaluate its association with response to atorvastatin therapy defined as baseline LDL cholesterol reduction of ≥50%. Methods: In this prospective cohort study, 79 statin-naïve patients hospitalized for the first episode of ACS and treated with atorvastatin 80 mg daily after percutaneous coronary intervention were followed longitudinally. Plasma samples were collected at admission (acute phase), discharge (subacute phase), and follow-up (chronic phase). A control group of 21 individuals received atorvastatin for primary prevention. IgG was isolated from plasma, and N-glycans were released, fluorescently labeled with 2-aminobenzamide, and analyzed using hydrophilic interaction-based ultra-high-performance liquid chromatography with fluorescence detection. Derived glycan traits were calculated, including agalactosylated (G0), monogalactosylated (G1), digalactosylated (G2), core fucosylated (F), bisected (B), and sialylated (S) glycans. Results: No significant differences in derived IgG glycan traits were observed between ACS patients and controls at baseline or follow-up. Within the ACS group, a longitudinal analysis revealed significant increases in G0 and F and a decrease in G2 between the acute and chronic phases. A total of 65% of patients achieved ≥50% reduction in LDL cholesterol (LDL-C), whereas only 22% reached the guideline-recommended LDL-C target of <1.4 mmol/L. Patients achieving ≥50% LDL-C reduction exhibited consistently higher G0 and lower G2 and S across disease phases. In a subgroup of patients with baseline LDL-C >3.9 mmol/L, those who failed to achieve ≥50% LDL-C reduction had significantly lower G0 and higher S across all time points. Conclusions: Specific glycan traits are associated with the degree of LDL-C reduction achieved during statin therapy, particularly in patients with high baseline LDL-C. These findings suggest that IgG glycosylation patterns may reflect biological phenotypes associated with differential lipid-lowering responsiveness after ACS. Full article

Seasonal human coronaviruses (HCoVs) continue to undergo adaptive evolution under structural and immune-mediated constraints. We investigated the molecular epidemiology and spike (S) protein structural variation of circulating coronaviruses in Riyadh, Saudi Arabia, during the 2025–2026 winter season, with particular emphasis on genotype persistence and glycosylation architecture in HCoV-OC43. Among 293 nasopharyngeal aspirates (NPAs) collected from hospitalized patients with acute respiratory illness, HCoV-OC43 was detected in 26 cases (8.87%), whereas other seasonal coronaviruses were not identified. Partial sequencing of the S gene revealed 97.84–98.23% nucleotide identity relative to the prototype strain VR-759, with amino acid substitutions distributed at discrete positions rather than within extended variable domains, indicating structural conservation. Phylogenetic reconstruction demonstrated that all Riyadh isolates clustered within genotype C, together with previously circulating local strains, supporting sustained endemic persistence and in situ evolution. In silico analysis of the S protein glycosylation landscape identified four invariant N-linked glycosylation motifs (N-X-S/T) at residues 46, 121, 134, and 190, reflecting strong structural constraints on glycan-dependent folding and antigenic configuration. A genotype-associated K68N substitution generated an additional N-glycosylation motif (68NGTD) in multiple Riyadh isolates, potentially modifying local glycan shielding without disrupting the overall glycosylation framework. The preservation of core glycosylation sites alongside selective motif acquisition suggests evolutionary fine-tuning of S surface topology rather than large-scale structural remodeling. Collectively, these findings indicate that genotype C persistence in Riyadh is accompanied by conserved S architecture and subtle glycosylation adjustments that may modulate immune recognition while maintaining structural integrity. Continued high-resolution molecular surveillance will be critical for defining the functional consequences of S microevolution in endemic HCoVs. Full article

Astaxanthin (AST), despite its high bioactivity, exhibits poor stability and low bioavailability due to its strong lipophilicity and inherent degradation susceptibility. To overcome such a challenge, we developed a food-grade oleogel delivery system using a soy protein–arabinoxylan (SA) glycosylated complex modulated by different concentrations (0.5–3%) of sucrose ester (SE) or soy lecithin. We show that the emulsifier concentration has a non-linear effect on the oleogel microstructure: an optimal level of 1% had a significant impact on the interfacial compactness and network density, giving rise to improved thermal stability, rheological strength and AST encapsulation efficiency (81.27%). During in vitro digestion, the SA matrix in combination with emulsifiers allowed gastric protection and intestinal-targeted release of AST with a bioaccessibility of up to 88.84% (SAO-SE-AST). This controlled release profile directly translated into enhanced in vivo antioxidant efficacy in wild-type Bristol N2 Caenorhabditis elegans, as evidenced by reduced lipofuscin accumulation, elevated thermotolerance (survival rate: 64.44–73.33%), suppressed reactive oxygen species levels and activation of endogenous antioxidant enzymes (superoxide dismutase as well as glutathione peroxidase). Collectively, this research has uncovered that food-grade emulsifiers are not only stabilizers, but also key regulators of oleogel architecture and bioactive functionality. These results provide a structure–digestion–bioactivity correlation for protein–polysaccharide oleogels, representing a rational design strategy for high-performance delivery systems of lipid-soluble nutraceuticals. Full article

N-glycosylation is a fundamental post-translational modification that contributes to protein folding, stability, and secretion in eukaryotes. The catalytic subunit STT3 of the oligosaccharyltransferase complex mediates the transfer of preassembled oligosaccharides to nascent polypeptides in the endoplasmic reticulum. Here, we identified and functionally characterized PsSTT3B, one of the STT3 paralogs in Phytophthora sojae (P. sojae). PsSTT3B plays an important role in the growth, development, and pathogenicity of P. sojae. CRISPR/Cas9-mediated deletion of PsSTT3B resulted in reduced vegetative growth, sporangia production, and zoospore production in P. sojae. PsSTT3B deletion mutants demonstrated significantly reduced virulence on soybean leaves and etiolated seedlings. Importantly, PsSTT3B deletion mutants also exhibited reduced zoospore germination and diminished chemotaxis toward soybean isoflavones. Moreover, deletion of PsSTT3B increased sensitivity to tunicamycin and dithiothreitol and influenced the ConA-binding glycoprotein profile. Our findings show that PsSTT3B is associated with asexual development, virulence, and sensitivity to ER stress-related conditions of P. sojae. Our study suggests that PsSTT3B represents a potential candidate gene for the prevention and control of P. sojae. Full article

Fabrazyme (agalsidase beta) is a therapeutic enzyme whose clinical efficacy is contingent upon its complex N-glycosylation patterns. Nevertheless, comprehensive glycosylation profiling remains challenging due to high site-specific heterogeneity. To address this, three orthogonal liquid chromatography–mass spectrometry (LC-MS) approaches were employed: (1) released N-glycan analysis with fluorescence detection and MS annotation, (2) site-specific glycopeptide mapping, and (3) intact protein MS. The released glycan profiling method was validated for reproducibility, intermediate precision, and inter-laboratory transferability, thereby enabling reliable separation and quantification of neutral, phosphorylated, and sialylated species. Glycopeptide mapping revealed distinct site-specific distributions: N108 was found to predominantly carry sialylated complex glycans; N161 was enriched in phosphorylated oligomannose structures; and N184 displayed the highest heterogeneity, including bisphosphorylated and sialylated glycans. Intact protein analysis was performed on both intact and desialylated Fabrazyme, thereby enabling confirmation of glycan assignments. Desialylation reduced spectral complexity, thereby facilitating accurate mass matching with a combinatorial library generated from glycopeptide-level data. The complementary use of these three analytical levels provides a comprehensive view of Fabrazyme glycosylation, offering a robust reference for quality control and biosimilar development. Full article