Search Results (27)

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

Extracellular vesicles (EVs) are important mediators of cell–cell communication thanks to their ability to transfer their bioactive cargo, thus regulating a variety of physiological contexts. EVs derived from amniotic mesenchymal/stromal cells (eAMC-EVs) are internalized by equine endometrial cells (eECs) with positive effects on regenerative medicine treatments. As the cellular uptake of EVs is influenced by the glycan profile of both EVs and target cells, this study is focused on the role of surface glycans in the uptake of eAMC-EVs by recipient eECs. Equine ECs were obtained by enzymatic digestion of uteri from healthy mares. Equine AMC-EVs were isolated from amniotic cell cultures according to a standardized protocol. The glycan pattern was studied using a panel of lectins in combination with fucosidase and neuraminidase treatment. Both eECs and eAMC-EVs expressed N-linked high mannose glycans, as well as fucosylated and sialylated glycans. All these glycans were involved in the uptake of eAMC-EVs by eECs. The internalization of eAMC-EVs was strongly reduced after cleavage of α1,2-linked fucose and α2,3/α2,6-linked sialic acids. These results demonstrate that surface glycans are involved in the internalization of eAMC-EVs by eECs and that fucosylated and sialylated glycans are highly relevant in the transfer of bioactive molecules with effects on regenerative medicine treatments. Full article

Fucosidosis is a rare lysosomal storage disease caused by α-L-fucosidase deficiency following a mutation in the FUCA1 gene. This enzyme is responsible for breaking down fucose-containing glycoproteins, glycolipids, and oligosaccharides within the lysosome. Mutations in FUCA1 result in either reduced enzyme activity or complete loss of function, leading to the accumulation of fucose-rich substrates in lysosomes. Lysosomes become engorged with undigested substrates, which leads to secondary storage defects affecting other metabolic pathways. The central nervous system is particularly vulnerable, with lysosomal dysfunction causing microglial activation, inflammation, and neuronal loss, leading to the neurodegenerative symptoms of fucosidosis. Neuroinflammation contributes to secondary damage, including neuronal apoptosis, axonal degeneration, and synaptic dysfunction, exacerbating the disease process. Chronic neuroinflammation impairs synaptic plasticity and neuronal survival, leading to progressive intellectual disability, learning difficulties, and loss of previously acquired skills. Inflammatory cytokines and lysosomal burden in motor neurons and associated pathways contribute to ataxia, spasticity, and hypotonia, which are common motor symptoms in fucosidosis. Elevated neuroinflammatory markers can increase neuronal excitability, leading to the frequent occurrence of epilepsy in affected individuals. So, fucosidosis is characterized by rapid mental and motor loss, along with growth retardation, coarse facial features, hepatosplenomegaly, telangiectasis or angiokeratomas, epilepsy, inguinal hernia, and dysostosis multiplex. Patients usually die at an early age. Treatment of fucosidosis is a great challenge, and there is currently no definitive effective treatment. Hematopoietic cell transplantation studies are ongoing in the treatment of fucosidosis. However, early diagnosis of this disease and treatment can be effective. In addition, the body’s immune system decreases due to chemotherapy applied after transplantation, leaving the body vulnerable to microbes and infections, and the risk of death is high with this treatment. In another treatment method, gene therapy, the use of retroviral vectors, is promising due to their easy integration, high cell efficiency, and safety. In another treatment approach, enzyme replacement therapy, preclinical studies are ongoing for fucosidosis, but the blood–brain barrier is a major obstacle in lysosomal storage diseases affecting the central nervous system. Early diagnosis is important in fucosidosis, a rare disease, due to the delay in the diagnosis of patients identified so far and the rapid progression of the disease. In addition, enzyme replacement therapy, which carries fewer risks, is promising. Full article

In various life forms, fucose-containing glycans play vital roles in immune recognition, developmental processes, plant immunity, and host-microbe interactions. Together with glucose, galactose, N-acetylglucosamine, and sialic acid, fucose is a significant component of human milk oligosaccharides (HMOs). Fucosylated HMOs benefit infants by acting as prebiotics, preventing pathogen attachment, and potentially protecting against infections, including HIV. Although the need for fucosylated derivatives is clear, their availability is limited. Therefore, synthesis methods for various fucosylated oligosaccharides are explored, employing enzymatic approaches and α-L-fucosidases. This work aimed to characterise α-L-fucosidases identified in an alpaca faeces metagenome. Based on bioinformatic analyses, they were confirmed as members of the GH29A subfamily. The recombinant α-L-fucosidases were expressed in Escherichia coli and showed hydrolytic activity towards p-nitrophenyl-α-L-fucopyranoside and 2′-fucosyllactose. Furthermore, the enzymes’ biochemical properties and kinetic characteristics were also determined. All four α-L-fucosidases could catalyse transfucosylation using a broad diversity of fucosyl acceptor substrates, including lactose, maltotriose, L-serine, and L-threonine. The results contribute insights into the potential use of α-L-fucosidases for synthesising fucosylated amino acids. Full article

Breastfeeding is essential in the first months of a newborn’s life. Breast milk is a source of crucial macronutrients, prebiotic oligosaccharides, and potential probiotic strains of bacteria. Oligosaccharides from breast milk (HMOs) are a significant part of the composition of breast milk and represent a complex of digestible sugars. This study aims to elucidate the enzymatic hydrolysis of these oligosaccharides and other prebiotics by the bacteria present in breast milk. We used modified methods to isolate oligosaccharides (HMOs) from human milk. Using unique techniques, we isolated and identified different bacteria from breast milk, mainly Lactobacillus fermentum. Using enzymatic analyses, we established the participation of α-fucosidase, α-glucosidase, β-galactosidase, and β-glucosidase from breast milk bacteria in the hydrolysis of prebiotic sugars. We also optimized the scheme for isolating oligosaccharides from breast milk by putting the lyophilized product into different food media. We found that the oligosaccharides from breast milk (HMOs) are a potent inducer for the secretion of the studied bacterial enzymes. Also, we found that all the lactobacilli strains we studied in detail could digest mucin-linked glycans. The degradation of these sugars is perhaps a built-in defense mechanism in cases where other sugars are lacking in the environment. We also determined fucosidase activity in some of the isolated strains. We recorded the highest values (2.5 U/mg in L. fermentum ss8) when the medium’s oligosaccharides isolated from breast milk were present. Lactobacilli and Bifidobacteria supplied with breast milk are the first colonizers in most cases in the gastrointestinal tract of the newborn. The presence and study of different genes for synthesizing other enzyme systems and transporters of various sugars in this type of bacteria are essential. Full article

2′-fucosyllactose (2′FL) is an important nutrient in human milk that stimulates beneficial microbiota and prevents infection. α-L-fucosidase is a promising component for 2′FL synthesis. In this study, a soil-oriented α-L-fucosidase-producing strain from Enterococcus gallinarum ZS1 was isolated. Escherichia coli was employed as a host for cloning and expressing the α-L-fucosidase gene (entfuc). The EntFuc was predicted as a member of the GH29 family with a molecular mass of 58 kDa. The optimal pH and temperature for the activity of EntFuc were pH 7.0 and 30 °C, respectively. The enzyme exhibited a strictly specific activity for 4-Nitrophenyl-α-L-fucopyranoside (pNP-Fuc) and had a negligible effect on hydrolyzing 2′FL. EntFuc could catalyze the synthesis of 2′FL via transfucosylation action from pNP-Fuc and lactose. The yield of 2′FL reached 35% under optimal conditions. This study indicated that EntFuc with a high conversion rate is a promising enzyme source for the biosynthesis of 2′FL. Full article

This study aims to explore the intra-species distribution of genetic characteristics that favor the persistence in the gastrointestinal tract (GIT) and host interaction of bacteria belonging to species of the Lacticaseibacillus genus. These bacterial species comprise commercial probiotics with the widest use among consumers and strains naturally occurring in GIT and in fermented food. Since little is known about the distribution of genetic traits for adhesion capacity, polysaccharide production, biofilm formation, and utilization of substrates critically important for survival in GIT, which influence probiotic characteristics, a list of genetic determinants possibly involved in such functions was created by a search for specific genes involved in the above aspects in the genome of the extensively characterized probiotic L. rhamnosus GG. Eighty-two gene loci were retrieved and their presence and variability in other Lacticaseibacillus spp. genomes were assessed by alignment with the publicly available fully annotated genome sequences of L. casei, L. paracasei, L. rhamnosus, and L. zeae. Forty-nine of these genes were found to be absent in some strains or species. The remaining genes were conserved and covered almost all the functions considered, indicating that all strains of the genus may exert some probiotic effects. Among the variable loci, a taurine utilization operon and a α-L-fucosidase were examined for the presence/absence in 26 strains isolated from infant feces by PCR-based tests. Results were variable among the isolates, though their common origin indicated the capacity to survive in the intestinal niche. This study indicated that the capacity to exert probiotic actions of Lacticaseibacillus spp. depends on a conserved set of genes but variable genetic factors, whose role is only in part elucidated, are more numerous and can explain the enhanced probiotic characteristics for some strains. The selection of the most promising probiotic candidates to be used in food is feasible by analyzing the presence/absence of a set of variable traits. Full article

Glycans function as valuable markers of stem cells but also regulate the ability of these cells to self-renew and differentiate. Approximately 2% of the human genome encodes for proteins that are involved in the biosynthesis and recognition of glycans. In the present study, we evaluated the expression of a small subset of glycogenes in human limbal epithelial cells with distinct clonogenic potential. Individual clones were classified as abortive or clonogenic, based on the fraction of the terminal colonies produced; clones leading exclusively to terminal colonies were referred to as abortive while those with half or fewer terminal colonies were referred to as clonogenic. An analysis of glycogene expression in clonogenic cultures revealed a high content of transcripts regulating the galactose and mannose metabolic pathways. Abortive clones were characterized by increased levels of GCNT4 and FUCA2, genes that are responsible for the branching of mucin-type O-glycans and the hydrolysis of fucose residues on N-glycans, respectively. The expansion of primary cultures of human limbal epithelial cells for 10 days resulted in stratification and a concomitant increase in MUC16, GCNT4 and FUCA2 expression. These data indicate that the clonogenic potential of human limbal epithelial cells is associated with specific glycosylation pathways. Mucin-type O-glycan branching and increased fucose metabolism are linked to limbal epithelial cell differentiation. Full article

Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria. Full article

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species—Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked β1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology. Full article

Little is known about the extent of variation and activity of naturally occurring milk glycosidases and their potential to degrade milk glycans. A multi-omics approach was used to investigate the relationship between glycosidases and important bioactive compounds such as free oligosaccharides and O-linked glycans in bovine milk. Using 4-methylumbelliferone (4-MU) assays activities of eight indigenous glycosidases were determined, and by mass spectrometry and ¹H NMR spectroscopy various substrates and metabolite products were quantified in a subset of milk samples from eight native North European cattle breeds. The results showed a clear variation in glycosidase activities among the native breeds. Interestingly, negative correlations between some glycosidases including β-galactosidase, N-acetyl-β-d-glucosaminidase, certain oligosaccharide isomers as well as O-linked glycans of κ-casein were revealed. Further, a positive correlation was found for free fucose content and α-fucosidase activity (r = 0.37, p-value < 0.001) indicating cleavage of fucosylated glycans in milk at room temperature. The results obtained suggest that milk glycosidases might partially degrade valuable glycans, which would result in lower recovery of glycans and thus represent a loss for the dairy ingredients industry if these activities are pronounced. Full article

Fucosylated carbohydrates and glycoproteins from human breast milk are essential for the development of the gut microbiota in early life because they are selectively metabolized by bifidobacteria. In this regard, α-L-fucosidases play a key role in this successful bifidobacterial colonization allowing the utilization of these substrates. Although a considerable number of α-L-fucosidases from bifidobacteria have been identified by computational analysis, only a few of them have been characterized. Hitherto, α-L-fucosidases are classified into three families: GH29, GH95, and GH151, based on their catalytic structure. However, bifidobacterial α-L-fucosidases belonging to a particular family show significant differences in their sequence. Because this fact could underlie distinct phylogenetic evolution, here extensive similarity searches and comparative analyses of the bifidobacterial α-L-fucosidases identified were carried out with the assistance of previous physicochemical studies available. This work reveals four and two paralogue bifidobacterial fucosidase groups within GH29 and GH95 families, respectively. Moreover, Bifidobacterium longum subsp. infantis species exhibited the greatest number of phylogenetic lineages in their fucosidases clustered in every family: GH29, GH95, and GH151. Since α-L-fucosidases phylogenetically descended from other glycosyl hydrolase families, we hypothesized that they could exhibit additional glycosidase activities other than fucosidase, raising the possibility of their application to transfucosylate substrates other than lactose in order to synthesis novel prebiotics. Full article

The acidic hydrolase α-fucosidase (AF) is a biomarker for maladies such as cancer and inflammation. The most advanced probes for α-fucosidase are unfortunately constrained to ex vivo or in vitro applications. The in vivo detection and quantification of AF using positron emission tomography would allow for better discovery and diagnosis of disease as well as provide better understanding of disease progression. We synthesized, characterized, and evaluated a radiolabeled small molecule inhibitor of AF based on a known molecule. The radiosynthesis involved the ¹¹C methylation of a phenoxide, which was generated in situ by ultrasonification of the precursor with sodium hydride. The tracer was produced with a decay corrected yield of 41.7 ± 16.5% and had a molar activity of 65.4 ± 30.3 GBq/μmol. The tracer was shown to be stable in mouse serum at 60 min. To test the new tracer, HCT116 colorectal carcinoma cells were engineered to overexpress human AF. In vitro evaluation revealed 3.5-fold higher uptake in HCT116AF cells compared to HCT116 controls (26.4 ± 7.8 vs. 7.5 ± 1.0 kBq/10⁶ cells). Static PET scans 50 min post injection revealed 2.5-fold higher tracer uptake in the HCT116AF tumors (3.0 ± 0.8%ID/cc (n = 6)) compared with the controls (1.2 ± 0.8 (n = 5)). Dynamic scans showed higher uptake in the HCT116AF tumors at all time-points (n = 2). Ex vivo analysis of the tumors, utilizing fluorescent DDK2 antibodies, confirmed the expression of human AF in the HCT116AF xenografts. We have developed a novel PET tracer to image AF in vivo and will now apply this to relevant disease models. Full article

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed “recoding”. In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for −1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed −1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed. Full article

In the field of biocatalysis and the development of a bio-based economy, hemicellulases have attracted great interest for various applications in industrial processes. However, the study of the catalytic activity of the lignocellulose-degrading enzymes needs to be improved to achieve the efficient hydrolysis of plant biomasses. In this framework, hemicellulases from hyperthermophilic archaea show interesting features as biocatalysts and provide many advantages in industrial applications thanks to their stability in the harsh conditions encountered during the pretreatment process. However, the hemicellulases from archaea are less studied compared to their bacterial counterpart, and the activity of most of them has been barely tested on natural substrates. Here, we investigated the hydrolysis of xyloglucan oligosaccharides from two different plants by using, both synergistically and individually, three glycoside hydrolases from Saccharolobus solfataricus: a GH1 β-gluco-/β-galactosidase, a α-fucosidase belonging to GH29, and a α-xylosidase from GH31. The results showed that the three enzymes were able to release monosaccharides from xyloglucan oligosaccharides after incubation at 65 °C. The concerted actions of β-gluco-/β-galactosidase and the α-xylosidase on both xyloglucan oligosaccharides have been observed, while the α-fucosidase was capable of releasing all α-linked fucose units from xyloglucan from apple pomace, representing the first GH29 enzyme belonging to subfamily A that is active on xyloglucan. Full article