Search Results (182)

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene plays a pivotal role in the glycolysis/gluconeogenesis process, contributing significantly to glycosyl donor synthesis, plant growth and development, and stress responses. Gastrodia elata Bl., a heterotrophic plant in the Orchidaceae family, has its dried tubers used as the traditional Chinese medicine. This study identified three GeGAPDH genes in G. elata, all encoding basic, stable, hydrophilic proteins. Phylogenetic analysis and subcellular localization predictions categorized GeGAPDH1 as a plastid subtype, while GeGAPDH2 and GeGAPDH3 were classified as cytoplasmic subtypes. Prokaryotic expression experiments demonstrated successful expression of the GeGAPDH1 protein in Escherichia coli, which exhibited significant GAPDH enzymatic activity. Subcellular localization experiments showed that GeGAPDH1 was localized in the plastid. Expression analysis indicated that the three GeGAPDH genes were predominantly expressed in tubers. Under low-temperature stress, although the total GAPDH enzyme activity in tubers did not change significantly, the expression of GeGAPDH1 was significantly up-regulated, while GeGAPDH2 and GeGAPDH3 were significantly down-regulated. This suggests that different subtypes of GeGAPDH may regulate cold resistance through different pathways. Upon pathogen infection, the GeGAPDH gene family exhibited pathogen-specific regulatory patterns. During infection by Fusarium oxysporum, both the expression levels of all three GeGAPDH genes and the total GAPDH enzyme activity in tubers increased significantly; however, F. solani infection induced a significant increase in total GAPDH enzyme activity without significant changes in gene expression. These results suggest that the GeGAPDH gene family may respond to different pathogen infections through transcriptional or translational regulation mechanisms. This study systematically identified and characterized the GeGAPDH gene family in G. elata, providing a theoretical foundation for understanding the functional differentiation of GAPDH in heterotrophic plants. Full article

Phosphomannomutase 2 (PMM2) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate, a key step in the biosynthesis of GDP-mannose for N-glycosylation. Its deficiency is the most common cause of congenital disorders of glycosylation (CDGs), accounting for the subtype known as PMM2-CDG. PMM2-CDG is a rare autosomal recessive disease characterized by multisystemic dysfunction, including cerebellar atrophy, peripheral neuropathy, developmental delay, and coagulation abnormalities. The disease is associated with a spectrum of pathogenic missense mutations, particularly at residues involved in dimerization and catalytic function (i.e., p.Phe119Leu and p.Arg141His). The dimerization of PMM2 is considered essential for enzymatic activity, although it remains unclear whether this supports structural stability alone, or whether both subunits are catalytically active—a distinction that may affect how mutations in each monomer contribute to overall enzyme function and disease phenotype. PMM2 has a paralog, phosphomannomutase 1 (PMM1), which shares substantial structural similarity—including obligate dimerization—and displays mutase activity in vitro, but does not compensate for PMM2 deficiency in vivo. To investigate potential heterodimerization between PMM1 and PMM2 and the effect of interface mutations over PMM2 dimer stability, we first assessed the likelihood of their co-expression using data from GTEx and the Human Protein Atlas. Building on this expression evidence, we modeled all possible dimeric combinations between the two paralogs using AlphaFold3. Models of the PMM2 and PMM1 homodimers were used as internal controls and aligned closely with their respective reference biological assemblies (RMSD < 1 Å). In contrast, the PMM2/PMM1 heterodimer model, the primary result of interest, showed high overall confidence (pLDDT > 90), a low inter-chain predicted alignment error (PAE∼1 Å), and robust interface confidence scores (iPTM = 0.80). Then, we applied PISA, PRODIGY, and mmCSM-PPI to assess interface energetics and evaluate the impact of missense variants specifically at the dimerization interface. Structural modeling suggested that PMM2/PMM1 heterodimers were energetically viable, although slightly less stable than PMM2 homodimers. Interface mutations were predicted to reduce dimer stability, potentially contributing to the destabilizing effects of disease-associated variants. These findings offer a structural framework for understanding PMM2 dimerization, highlighting the role of interface stability, paralogs co-expression, and sensitivity to disease-associated mutations. Full article

β-Galactosidase, a glycoside hydrolase enzyme, also possesses glycosyl transferase activity and can glycosylate various aglycones, including tyrosol, a phenylethanoid with antioxidant and health-promoting effects. This study examines the effect of lactose, tyrosol and deep eutectic solvents (DESs) as co-solvents on the stability and activity of Aspergillus oryzae β-galactosidase during the enzymatic synthesis of tyrosol β-d-galactoside (TG). The enzyme’s thermal stability was assessed using nanoDSF and circular dichroism spectroscopy, while the enzyme’s activity and specificity toward different glycosyl acceptors were investigated using the initial rate method. The effects of tyrosol and DESs on tyrosol galactoside synthesis over a 6 h period were also studied. Lactose and glycerol were found to stabilize the enzyme. Among the DESs tested, those containing betaine showed the highest stabilizing effect. The presence of DESs not only affected the overall enzyme activity but also changed the enzyme specificity, most frequently in favor of lactose hydrolysis. Components of DESs containing alcohol groups (polyols) also acted as transglycosylation acceptors. However, both glycerol and tyrosol were found to inhibit overall enzyme activity and TG synthesis. Overall, our findings provide new and valuable insights into the influence of reaction conditions on the stability and specificity of β-galactosidase. Full article

Aberrant glycosylation, especially sialylation, on cell surface is often associated with cancer progression and immunosuppression. Over-sialylation of stage-specific embryonic antigen-4 (SSEA-4) to generate disialylGb5 (DSGb5) was reported to trigger Siglec-7 recognition and suppress NK-mediated target killing. In this study, efficient chemo-enzymatic and programmable one-pot methods were explored for the synthesis of DSGb5 and related sialosides for assembly of glycan microarrays and evaluation of binding specificity toward Siglecs-7, 9, 10, and 15 associated with immune checkpoint inhibition. The result showed weak binding of DSGb5 to these Siglecs; however, a truncated glycolyl glycan was identified to bind Siglec-10 strongly with a dissociation constant of 50 nM and exhibited a significant inhibition of Siglec-10 interacting with breast cancer cells. Full article

In the field of biotechnology, natural compounds isolated from medicinal plants are highly valued; however, their discovery, purification, biofunctional characterization, and biochemical validation have historically involved time-consuming and laborious processes. Two innovative approaches have emerged to more efficiently discover new bioactive substances: the predicted data mining approach (PDMA) and biotransformation-guided purification (BGP). The PDMA is a computational method that predicts biotransformation potential, identifying potential substrates for specific enzymes from numerous candidate compounds to generate new compounds. BGP combines enzymatic biotransformation with traditional purification techniques to directly identify and isolate biotransformed products from crude extract fractions. This review examines recent research employing BGP or the PDMA for novel compound discovery. This research demonstrates that both approaches effectively allow for the discovery of novel bioactive molecules from natural sources, the enhancement of the bioactivity and solubility of existing compounds, and the development of alternatives to traditional methods. These findings highlight the potential of integrating traditional medicinal knowledge with modern enzymatic and computational tools to advance drug discovery and development. Full article

Background: Inhibition of intestinal α-glucosidase is a key strategy for controlling postprandial hyperglycemia in diabetes. Astragali Radix (AR), a traditional medicinal and dietary herb widely consumed in China, is rich in flavonoids that are believed to exhibit hypoglycemic properties. Methods: A total of 29 AR-related flavonoids, including both original constituents and metabolites, were screened for α-glucosidase inhibitory activity using in vitro enzymatic assays. Mechanistic investigations were conducted through enzyme kinetics, circular dichroism (CD) spectroscopy, surface plasmon resonance (SPR), and molecular docking. The in vivo hypoglycemic effects were assessed using a postprandial hyperglycemic mouse model. Additionally, potential mixture effects of flavonoid combinations were evaluated. Results: Of the 29 flavonoids, 16 demonstrated significant α-glucosidase inhibitory activity, with five (C3, C17, C19, C28, and C29) identified as novel inhibitors. Structure–activity relationship (SAR) analysis revealed that hydroxylation, particularly at the C-3 position, enhanced activity, while glycosylation and methoxylation reduced it. Mechanistic studies demonstrated that these compounds bind to distinct amino acid residues within the active site of α-glucosidase, inducing conformational changes and exerting different types of inhibition, leading to varying inhibitory mechanisms. Additionally, 15 compounds reduced postprandial blood glucose levels, with C3, C16, C17, C19, and C28 confirmed as novel in vivo inhibitors. Notably, two compositions of flavonoids combined at their individually ineffective concentrations exhibited significant inhibitory effects. Conclusions: This study provides a comprehensive evaluation of AR-related flavonoids as α-glucosidase inhibitors and offers valuable insights for the development of highly effective, low-toxicity, flavonoid-based, antidiabetic therapeutics and functional foods. Full article

Uridine diphosphate glycosyltransferase (UGT) is a core protein for glycosylation of plant natural products and other small molecules. Although many studies on functional identification of UGTs are now available, analysis of UGTs in Platycodon grandiflorus is still relatively scarce. We identified 107 PgUGTs genome-wide from P. grandiflorus and investigated their phylogenetic relationships, chromosomal localisation, collinearity, cis-regulatory elements, motifs, domains, and gene structures. PgUGT29 and PgUGT72 were two putative glycosyltransferases for platycodins biosynthesis in P. grandiflorus according to our previous study and bioinfornatical analyses. In vitro enzyme activity showed that PgUGT29 can catalyse the glycosylation of the C3 position of Platycodin D (PD) to generate Platycodin D3 (PD3), while candidate enzyme PgUGT72 does not function as a glycosyltransferase. Molecular docking indicated that T145, D392, Q393, and N396 may be the crucial residues for PgUGT29 to catalyse the generation of PD3 from UDP-Glc and PD. In this study, we identified and cloned PgUGT29, elucidated its catalytic function in converting PD to PD3, and predicted key residues critical for its enzymatic activity. These findings provide a theoretical foundation and technical framework for future targeted metabolic engineering and directional regulation of medicinal components in Platycodon grandiflorus. Full article

Glycation, or non-enzymatic glycosylation, has recently attracted increasing interest in the context of its impact on aging. Advanced glycation end products (AGEs) contribute to various age-related pathological conditions such as inflammation, fibrosis, and vascular calcification. However, the molecular mechanisms underlying glycation-induced disruption of cell–matrix interactions during cellular senescence are not fully understood. The aim of this study was to investigate transcriptomic changes in young and senescent dermal fibroblasts (HdFbs) cultured in 3D post-glycated collagen type I matrices after 10 and 17 days. Our findings indicate that D-ribose-mediated glycation increases the accumulation of fluorescent AGEs and the stiffness of matrices in a dose-dependent manner. The transcriptome alterations in cells encompassed the modulation of age-related genes and signaling pathways, including activation of genes related to senescence-associated secretory phenotype (SASP). Notably, the alterations in the transcriptome profiles due to glycation were more pronounced (in terms of both the number of genes and their fold changes) after 10 days of culture compared to day 17 in both passages. These findings suggest that cellular responses to glycation and resulting stiffness depend on both the concentration of reducing sugar and the time spent under those conditions. Full article

Background: N-glycosylation is a post-translational modification involving the attachment of oligosaccharides to proteins and is known to influence immunoglobulin G (IgG) effector functions and even antigen binding. IgG contains an evolutionarily conserved N-glycosylation site in its fragment crystallizable (Fc) region, while during V-D-J recombination and somatic hypermutation processes it can also obtain N-glycosylation sites in its antigen binding fragment (Fab). Our previous study demonstrated altered IgG N-glycosylation in children at type 1 diabetes (T1D) onset, with the most prominent changes involving sialylated glycans, hypothesized to mainly come from the Fab region, however, the analytical method used could not distinguish between Fc and Fab. Methods: IgG was isolated from plasma from 118 children with T1D and 98 healthy controls from the Danish Registry of Childhood and Adolescent Diabetes. Isolated IgG was cleaved into Fc and Fab fragments using IdeS enzyme. N-glycans were enzymatically released from each fragment, fluorescently labelled with procainamide, and analyzed separately using the UPLC-MS method. Structural annotation of resulting chromatograms was performed using MS/MS. Results: T1D related N-glycosylation changes were more pronounced in the Fab glycans compared to Fc glycans, with five Fab glycans (Man5, Man7, FA2BG1S1, A2G2S2, FA2BG2S1) being significantly altered compared to only one in the Fc region (FA2[3]BG1). Comparing Fc and Fab glycosylation overall reveals stark differences in the types of glycans on each region, with a more diverse and complex repertoire being present in the Fab region. Conclusions: These findings suggest that N-glycosylation changes in early onset T1D predominantly originate from the Fab region, underscoring their potential role in modulating (auto)immunity and highlighting distinct glycosylation patterns between Fc and Fab. Full article

Lawsonia inermis L. is renowned for its hair dyeing properties, with henna quality and safety often regulated by restrictions on the lawsone (2-hydroxy-1,4-naphthoquinone) content. In henna leaves, lawsone exists as glycosylated precursors, hennosides A, B, and C. Aqueous maceration revealed the sensitivity of enzymatic lawsone release, while ethanol extraction inhibited β-glucosidase activity, enabling controlled hennoside extraction. Hennoside A was isolated via RP-column chromatography and characterized using ESI-TOF, ¹H-/¹³C-NMR, COSY, NOESY, HSQC, and HMBC. The purified compound proved suitable as an HPLC reference standard. The acidic hydrolysis of hennoside-rich extracts highlighted the limitations of lawsone-based analysis, underscoring glycosylated precursors as more reliable quality markers. Lawsone quantification via enzymatic or acid catalysis demonstrated varying accuracy in quality control. A hennoside-based approach ensures consistency by estimating the maximum releasable lawsone without inducing its formation, providing a more robust metric for a henna quality assessment. Full article

Floral coloration in Freesia hybrida is predominantly attributed to anthocyanins, with glycosylation playing a critical role in their stability and diversity. This study investigates the molecular mechanisms underlying color variation between F. hybrida cultivars, focusing on anthocyanin 5-O-glucosyltransferases (An5GTs). HPLC analysis revealed that ‘Pink Passion’ petals accumulate 3,5-O-diglucosidic anthocyanins, absent in ‘Red River^®’ and ‘Ambiance’. RNA-seq identified seven candidate Fh5GT genes, with phylogenetic and subcellular localization analyses confirming their classification as cytosolic glycosyltransferases. Expression profiling highlighted elevated transcript levels of Fh5GT1, Fh5GT3, and Fh5GT7 in ‘Pink Passion’, correlating with its di-glucosidic anthocyanin accumulation. In vitro enzymatic assays demonstrated that Fh5GT3 and Fh5GT7 preferentially glucosylate 3-O-monoglucosidic anthocyanins to form stable 3,5-O-diglucosides, with minimal activity on anthocyanidins to generate 5-O-glucosidic anthocyanins. Heterologous expression of Fh5GT3 and Fh5GT7 in Arabidopsis complemented anthocyanin deficiency in 5gt mutants, restoring pigmentation. These findings elucidate the potential role of 5GTs in modulating floral color diversity through anthocyanin modification, providing insights for targeted breeding strategies to enhance ornamental traits in horticultural species. Full article

Glycosylation is a critical enzymatic modification that involves the attachment of sugar moieties to target compounds, considerably influencing their physicochemical and biological characteristics. This review explored the role of two primary enzyme classes—glycosyltransferases (GTs) and glycoside hydrolases (GHs, glycosidases)—in catalyzing the glycosylation of natural products, with a specific focus on Ganoderma triterpenoids. While GTs typically use activated sugar donors, such as uridine diphosphate glucose, certain GHs can leverage more economical sugar sources, such as sucrose and starch, through transglycosylation. This paper also reviewed strategies for producing novel terpenoid glycosides, particularly recently isolated bacterial GTs and GHs capable of glycosylating terpenoids and flavonoids. It summarized the newly synthesized glycosides’ structures and biotransformation mechanisms, enhanced aqueous solubility, and potential applications. The regioselectivity and substrate specificity of GTs and GHs in catalyzing O-glycosylation (glucosylation) at distinct hydroxyl and carboxyl groups were compared. Furthermore, a special case in which the novel glycosylation reactions were mediated by GHs, including the formation of unique glycoside anomers, was included. The advantages and specific capabilities of GT/GH enzymes were evaluated for their potential in biotechnological applications and future research directions. Novel fungal triterpenoid glycosides produced through various glycosidases and sugars is expected to expand their potential applications in the future. Full article

Background/Objectives: Salivary gland tumors (SGTs) are a rare and histologically heterogeneous group of neoplasms that are challenging to diagnose due to phenotypic heterogeneity and overlapping histomorphological markers. Accurate diagnosis is required for clinical management, particularly in unusual subtypes. The objective of this study was to ascertain whether attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy, in combination with enzymatic deglycosylation, would be useful in SGT classification by detecting glycosylation-related metabolic variations. Methods: 155 tissue sections, consisting of 80 SGTs and 75 controls, were analyzed. ATR-FTIR spectroscopy was used to record the mid-infrared (MIR) spectra (4000–400 cm⁻¹) of enzymatically untreated and deglycosylated samples. Spectral data were preprocessed and analyzed by principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Enzymatic deglycosylation focused on sialic acid and fucose residues with α2-3,6,8 neuraminidase, α1-2,4,6 fucosidase O, and α1-3,4 fucosidase. Results: Tumor and control samples were discriminated with an OPLS-DA model, achieving an accuracy of 81.9% (78.7% for controls and 85.0% for tumors), especially in the glycosylation-relevant spectral range (850–1250 cm⁻¹). Classification between benign and malignant tumors was more challenging, with an accuracy of 70.0% (72.5% for benign and 67.5% for malignant cases). Enzymatic deglycosylation resulted in detectable changes in the MIR spectra, confirming the contribution of glycosylation to tumor-specific signatures. Benign vs. malignant tumor discrimination was still poor and was not much enhanced in the sense of incorporating glycosylation-specific regions. Conclusions: ATR-FTIR spectroscopy coupled with enzymatic deglycosylation can distinguish tumor and control tissues based on glycan-associated spectral differences. Application of the technique to benign/malignant SGT discrimination is hampered by spectral overlap and tumor heterogeneity. Further research will be necessary to explore other clustering algorithms and larger and more homogeneous datasets for improved diagnostic accuracy. Full article

Inherited metabolic disorders (IMDs) are genetic disorders that occur in as many as 1:2500 births worldwide. Nevertheless, they are quite rare individually and even more rare is the co-occurrence of two IMDs in one individual. To better understand the metabolic cross-talk between glycosylation changes and deficient energy metabolism, and its potential effect on outcomes, we evaluated patient fibroblasts with likely pathogenic variants in PGM1 and pathogenic variants in NDUFA13 derived from a patient who passed away at 16 years of age. The patient presented with characteristic of PGM1-CDG including bifid uvula, muscle involvement, abnormal glycosylation in blood, and elevated liver transaminases. In addition, hearing loss, seizures, elevated plasma and CSF lactate and a Leigh-like MRI brain pattern were present, which are commonly associated with Leigh syndrome. PGM1-CDG has been reported in about 70 individuals, while NDUFA13 deficiency has so far only been reported in 13 patients. As abundant energy is essential for glycosylation, and both PGM1 and NDUFA13 are linked to energy metabolism, we sought to better understand the underlying biochemical cause of the patient’s clinical presentation. To do so, we performed extensive investigations including tracer metabolomics, lipidomics and enzymatic studies on the patient’s fibroblasts. We found a profound depletion of UDP-hexoses, consistent with PGM1-CDG. Complex I enzyme activity and mitochondrial function were also impaired, corroborating complex I deficiency and Leigh syndrome. Further, lipidomics analysis showed similarities with both PGM1-CDG and OXPHOS-deficient patients. Based on our results, the patient was diagnosed with both PGM1-CDG and Leigh syndrome. In summary, we present the first case of combined CDG and Leigh syndrome, caused by (likely) pathogenic variants in PGM1 and NDUFA13, and underline the importance of considering the synergistic effects of multiple disease-causing variants in patients with complex clinical presentation, leading to the patient’s early demise. Full article

Lysosomal enzymes are synthesized as N-glycosylated glycoproteins with mannose-6-phosphate (M6P) moieties, which are responsible for their binding to M6P receptors and transporting to the lysosome. In the M6P biosynthetic pathway, a Man₈GlcNAc₂ glycoform is converted to M6P groups through two consecutive enzymatic reactions, including N-acetylglucosamine (GlcNAc)-1-phosphotransferase (GNPT), transferring GlcNAc-1-phosphate from UDP-GlcNAc to the C6 hydroxyl groups of mannose residues, and then, removal of the covering GlcNAc moiety from the GlcNAc-P-mannose phosphodiester was carried out using an α-N-acetylglucosaminidase (referred to as ‘uncovering enzyme’, UCE) in the trans-Golgi network (TGN). Here, we expressed differently tailored versions of the UCE, including four truncated variants, in Escherichia coli. The four variants with the signal peptide, transmembrane domain, propiece and cytoplasmic tail truncated, respectively, were purified by affinity chromatography, and their enzymatic activities were assayed using a UDP-Glo kit. By fusing a maltose-binding protein (MBP) in the N-terminus of the UCE variants, the fusion proteins could be soluble when expressed in E. coli. The highest concentration of the purified enzyme was 80.5 mg/L of fermentation broth. Furthermore, the UCE with the core catalytic domain exhibited the highest uncovering activity. Full article