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Applied and Environmental Microbiology Mar 2024Galacto--biose (GNB) is an important core structure of glycan of mucin glycoproteins in the gastrointestinal (GI) mucosa. Because certain beneficial bacteria inhabiting...
UNLABELLED
Galacto--biose (GNB) is an important core structure of glycan of mucin glycoproteins in the gastrointestinal (GI) mucosa. Because certain beneficial bacteria inhabiting the GI tract, such as bifidobacteria and lactic acid bacteria, harbor highly specialized GNB metabolic capabilities, GNB is considered a promising prebiotic for nourishing and manipulating beneficial bacteria in the GI tract. However, the precise interactions between GNB and beneficial bacteria and their accompanying health-promoting effects remain elusive. First, we evaluated the proliferative tendency of beneficial bacteria and their production of beneficial metabolites using gut bacterial strains. By comparing the use of GNB, glucose, and inulin as carbon sources, we found that GNB enhanced acetate production in , , , and . The ability of GNB to promote acetate production was also confirmed by RNA-seq analysis, which indicated the upregulation of gene clusters that catalyze the deacetylation of -acetylgalactosamine-6P and biosynthesize acetyl-CoA from pyruvate, both of which result in acetate production. To explore the effect of GNB in promoting acetate production, antibiotic-treated BALB/cA mice were administered with GNB with , resulting in a fecal acetate content that was 2.7-fold higher than that in mice administered with only . Moreover, 2 days after the last administration, a 3.7-fold higher amount of was detected in feces administered with GNB with than in feces administered with only . These findings strongly suggest the prebiotic potential of GNB in enhancing colonization and converting into higher acetate producers in the GI tract.
IMPORTANCE
Specific members of lactic acid bacteria, which are commonly used as probiotics, possess therapeutic properties that are vital for human health enhancement by producing immunomodulatory metabolites such as exopolysaccharides, short-chain fatty acids, and bacteriocins. The long residence time of probiotic lactic acid bacteria in the GI tract prolongs their beneficial health effects. Moreover, the colonization property is also desirable for the application of probiotics in mucosal vaccination to provoke a local immune response. In this study, we found that GNB could enhance the beneficial properties of intestinal lactic acid bacteria that inhabit the human GI tract, stimulating acetate production and promoting intestinal colonization. Our findings provide a rationale for the addition of GNB to lactic acid bacteria-based functional foods. This has also led to the development of therapeutics supported by more rational prebiotic and probiotic selection, leading to an improved healthy lifestyle for humans.
Topics: Humans; Animals; Mice; Prebiotics; Lactobacillales; Disaccharidases; Probiotics; Acetates; Bacteria
PubMed: 38411084
DOI: 10.1128/aem.01445-23 -
Nature Biotechnology Mar 2024Two decades of research on RNA interference (RNAi) have transformed a breakthrough discovery in biology into a robust platform for a new class of medicines that modulate... (Review)
Review
Two decades of research on RNA interference (RNAi) have transformed a breakthrough discovery in biology into a robust platform for a new class of medicines that modulate mRNA expression. Here we provide an overview of the trajectory of small-interfering RNA (siRNA) drug development, including the first approval in 2018 of a liver-targeted siRNA interference (RNAi) therapeutic in lipid nanoparticles and subsequent approvals of five more RNAi drugs, which used metabolically stable siRNAs combined with N-acetylgalactosamine ligands for conjugate-based liver delivery. We also consider the remaining challenges in the field, such as delivery to muscle, brain and other extrahepatic organs. Today's RNAi therapeutics exhibit high specificity, potency and durability, and are transitioning from applications in rare diseases to widespread, chronic conditions.
Topics: RNA Interference; RNA, Small Interfering; Liver; Acetylgalactosamine
PubMed: 38409587
DOI: 10.1038/s41587-023-02105-y -
International Journal of Biological... Apr 2024Polypeptide N-acetylgalactosamine transferase 9 (GALNT9) catalyzes the initial step of mucin-type O-glycosylation via linking N-acetylgalactosamine (GalNAc) to...
Polypeptide N-acetylgalactosamine transferase 9 (GALNT9) catalyzes the initial step of mucin-type O-glycosylation via linking N-acetylgalactosamine (GalNAc) to serine/threonine in a protein. To unravel the association of GALNT9 with Parkinson's disease (PD), a progressive neurodegenerative disorder, GALNT9 levels were evaluated in the patients with PD and mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and statistically analyzed based on the GEO datasets of GSE114918 and GSE216281. Glycoproteins with exposing GalNAc were purified using lectin affinity chromatography and identified by LC-MS/MS. The influence of GALNT9 on cells was evaluated via introducing a GALNT9-specific siRNA into SH-SY5Y cells. Consequently, GALNT9 deficiency was found to occur under PD conditions. GALNT9 silencing contributed to a causative factor in PD pathogenesis via reducing the levels of intracellular dopamine, tyrosine hydroxylase and soluble α-synuclein, and promoting α-synuclein aggregates. MS identification revealed 14 glycoproteins. 5 glycoproteins, including ACO, ATP5B, CKB, CKMT1A, ALDOC, were associated with energy metabolism. GALNT9 silencing resulted in mitochondrial dysfunctions via increasing ROS accumulation, mitochondrial membrane depolarization, mPTPs opening, Ca releasing and activation of the CytC-related apoptotic pathway. The dysfunctional mitochondria then triggered mitophagy, possibly intermediated by adenine nucleotide translocase 1. Our study suggests that GALNT9 is potentially developed into an auxiliary diagnostic index and therapeutic target of PD.
Topics: Humans; Mice; Animals; Parkinson Disease; alpha-Synuclein; Acetylgalactosamine; Transferases; Chromatography, Liquid; Neuroblastoma; Tandem Mass Spectrometry; Peptides; Glycoproteins; N-Acetylgalactosaminyltransferases; Mitochondrial Diseases; Creatine Kinase
PubMed: 38401583
DOI: 10.1016/j.ijbiomac.2024.130347 -
Glycobiology Apr 2024Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological...
Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological processes. Two prominent glycosaminoglycans, heparan sulfate and chondroitin sulfate, are covalently linked to proteoglycan core proteins through a common tetrasaccharide linker comprising glucuronic acid, galactose, galactose, and xylose moities. This tetrasaccharide linker is meticulously assembled step by step by four Golgi-localized glycosyltransferases. The addition of the fifth sugar moiety, either N-acetylglucosamine or N-acetylgalactosamine, initiates further chain elongation, resulting in the formation of heparan sulfate or chondroitin sulfate, respectively. Despite the fundamental significance of this step in glycosaminoglycan biosynthesis, its regulatory mechanisms have remained elusive. In this study, we detail the expression and purification of the four linker-synthesizing glycosyltransferases and their utilization in the production of fluorescent peptides carrying the native tetrasaccharide linker. We generated five tetrasaccharide peptides, mimicking the core proteins of either heparan sulfate or chondroitin sulfate proteoglycans. These peptides were readily accepted as substrates by the EXTL3 enzyme, which adds an N-acetylglucosamine moiety, thereby initiating heparan sulfate biosynthesis. Importantly, EXTL3 showed a preference towards peptides mimicking the core proteins of heparan sulfate proteoglycans over the ones from chondroitin sulfate proteoglycans. This suggests that EXTL3 could play a role in the decision-making step during glycosaminoglycan biosynthesis. The innovative strategy for chemo-enzymatic synthesis of fluorescent-labeled linker-peptides promises to be instrumental in advancing future investigations into the initial steps and the divergent step of glycosaminoglycan biosynthesis.
Topics: Chondroitin Sulfates; Acetylglucosamine; Galactose; Glycosaminoglycans; Heparitin Sulfate; Chondroitin Sulfate Proteoglycans; Oligosaccharides; Peptides; Glycosyltransferases
PubMed: 38401165
DOI: 10.1093/glycob/cwae016 -
International Journal of Molecular... Feb 2024Runx2 (runt related transcription factor 2) is an essential transcription factor for osteoblast proliferation and differentiation. Uridine diphosphate...
Runx2 (runt related transcription factor 2) is an essential transcription factor for osteoblast proliferation and differentiation. Uridine diphosphate (UDP)-N-acetylgalactosamine (GalNAc): polypeptide GalNAc-transferase 3 (Galnt3) prevents proteolytic processing of fibroblast growth factor 23 (Fgf23), which is a hormone that regulates the serum level of phosphorus. and were expressed in osteoblasts and osteocytes, and expression was restricted to osteocytes in bone. Overexpression and knock-down of upregulated and downregulated, respectively, the expressions of and , and Runx2 directly regulated the transcriptional activity of in reporter assays. The expressions of and in osteoblast-specific knockout () mice were about half those in mice. However, the serum levels of phosphorus and intact Fgf23 in mice were similar to those in mice. The trabecular bone volume was increased during aging in both male and female mice, but the osteoid was reduced. The markers for bone formation and resorption in mice were similar to the control in both sexes. mice exhibited hyperphosphatemia and hypercalcemia, and the intact Fgf23 was about 40% that of wild-type mice. These findings indicated that Runx2 regulates the expressions of and and that Galnt3 decelerates the mineralization of osteoid by stabilizing Fgf23.
Topics: Animals; Female; Male; Mice; Calcinosis; Core Binding Factor Alpha 1 Subunit; Fibroblast Growth Factors; N-Acetylgalactosaminyltransferases; Osteoblasts; Phosphorus; Polypeptide N-acetylgalactosaminyltransferase; Calcification, Physiologic
PubMed: 38396954
DOI: 10.3390/ijms25042275 -
Journal of Biochemistry Feb 2024Cancer antigen 125 (CA125) is a serum marker associated with ovarian cancer. Despite its widespread use, CA125 levels can also be elevated in benign conditions. Recent...
Cancer antigen 125 (CA125) is a serum marker associated with ovarian cancer. Despite its widespread use, CA125 levels can also be elevated in benign conditions. Recent reports suggest that detecting serum CA125 that carries the Tn-antigen, a truncated O-glycan containing only N-acetylgalactosamine on serine or threonine residues, can improve the specificity of ovarian cancer diagnosis. In this study, we engineered cells to express CA125 with a Tn-antigen. To achieve this, we knocked out C1GALT1 and SLC35A1, genes encoding Core1 synthase and a transporter for cytidine-5'-monophospho-sialic acid respectively, in human embryonic kidney 293 (HEK293) cells. In ClGALT1-SLC35A1-knockout (KO) cells, the expression of the Tn-antigen showed a significant increase, whereas the expression of the T-antigen (galactose-β1,3-N-acetylgalactosamine on serine or threonine residues) was decreased. Due to the inefficient secretion of soluble CA125, we employed a glycosylphosphatidylinositol (GPI) anchoring system. This allowed for the expression of GPI-anchored CA125 on the cell surface of ClGALT1-SLC35A1-KO cells. Cells expressing high levels of GPI-anchored CA125 were then enriched through cell sorting. By knocking out the PGAP2 gene, the GPI-anchored form of CA125 was converted to a secretory form. Through the engineering of O-glycans and the use of a GPI-anchoring system, we successfully produced CA125 with Tn-antigen modification.
PubMed: 38382634
DOI: 10.1093/jb/mvae019 -
Acta Histochemica Feb 2024Sialic acids (Sias) are a family of electronegatively charged nine-carbon monosaccharides containing a carboxylic acid, mostly found as terminal residues in glycans of... (Review)
Review
Sialic acids (Sias) are a family of electronegatively charged nine-carbon monosaccharides containing a carboxylic acid, mostly found as terminal residues in glycans of glycoproteins and glycolipids. They are bound to galactose or N-acetylgalactosamine via α2,3 or α2,6 linkage, or to other Sias especially via α2,8 linkage, which results in monomeric, oligomeric, and polymeric forms. Sias play determinant roles in a multitude of biological processes in human tissues from development to adult life until aging. In this review, we summarized the current knowledge on the sialylation status in the human testis with a main focus on sexually mature life and aging, when this organ shows significant morphofunctional changes resulting into variations of hormonal levels, as well as changes in molecules involved in mitochondrial function, receptors, and signaling proteins. Evidence suggests that Sias may have crucial morphofunctional roles in the different testicular components during the sexually mature age. With advancing age, significant loss of Sias and/or changes in sialylation status occur in all the testicular components, which seems to contribute to morphofunctional changes characteristic of the aging testis. Based on the current knowledge, further in-depth investigations will be necessary to better understand the mechanistic role of Sias in the biological processes of human testicular tissue and the significance of their changes during the aging process. Future investigations might also contribute to the development of novel prophylactic and/or therapeutic approaches that, by maintaining/restoring the correct sialylation status, could help in slowing down the testis aging process, thus preserving the testicular structure and functionality and preventing age-related pathologies.
Topics: Adult; Humans; Male; Testis; Aging; Carboxylic Acids; Galactose; Mitochondria
PubMed: 38382219
DOI: 10.1016/j.acthis.2024.152143 -
Glycobiology Apr 2024The glycoprotein-N-acetylgalactosamine β1,3-galactosyltransferase, known as T-synthase (EC 2.4.1.122), plays a crucial role in the synthesis of the T-antigen, which is...
The glycoprotein-N-acetylgalactosamine β1,3-galactosyltransferase, known as T-synthase (EC 2.4.1.122), plays a crucial role in the synthesis of the T-antigen, which is the core 1 O-glycan structure. This enzyme transfers galactose from UDP-Gal to GalNAc-Ser/Thr. The T-antigen has significant functions in animal development, immune response, and recognition processes. Molluscs are a successful group of animals that inhabit various environments, such as freshwater, marine, and terrestrial habitats. They serve important roles in ecosystems as filter feeders and decomposers but can also be pests in agriculture and intermediate hosts for human and cattle parasites. The identification and characterization of novel carbohydrate active enzymes, such as T-synthase, can aid in the understanding of molluscan glycosylation abilities and their adaptation and survival abilities. Here, the T-synthase enzymes from the snail Pomacea canaliculata and the oyster Crassostrea gigas are identified, cloned, expressed, and characterized, with a focus on structural elucidation. The synthesized enzymes display core 1 β1,3-galactosyltransferase activity using pNP-α-GalNAc as substrate and exhibit similar biochemical parameters as previously characterised T-synthases from other species. While the enzyme from C. gigas shares the same structural parameters with the other enzymes characterised so far, the T-synthase from P. canaliculata lacks the consensus sequence CCSD, which was previously considered indispensable.
Topics: Animals; Humans; Cattle; Amino Acid Sequence; Ecosystem; Galactosyltransferases; Cloning, Molecular; Mollusca; Antigens, Viral, Tumor
PubMed: 38366999
DOI: 10.1093/glycob/cwae013 -
Signal Transduction and Targeted Therapy Feb 2024Immunostaining in lungs of patients who died with COVID-19 infection showed increased intensity and distribution of chondroitin sulfate and decline in...
Immunostaining in lungs of patients who died with COVID-19 infection showed increased intensity and distribution of chondroitin sulfate and decline in N-acetylgalactostamine-4-sulfatase (Arylsulfatase B; ARSB). To explain these findings, human small airway epithelial cells were exposed to the SARS-CoV-2 spike protein receptor binding domain (SPRBD) and transcriptional mechanisms were investigated. Phospho-p38 MAPK and phospho-SMAD3 increased following exposure to the SPRBD, and their inhibition suppressed the promoter activation of the carbohydrate sulfotransferases CHST15 and CHST11, which contributed to chondroitin sulfate biosynthesis. Decline in ARSB was mediated by phospho-38 MAPK-induced N-terminal Rb phosphorylation and an associated increase in Rb-E2F1 binding and decline in E2F1 binding to the ARSB promoter. The increases in chondroitin sulfotransferases were inhibited when treated with phospho-p38-MAPK inhibitors, SMAD3 (SIS3) inhibitors, as well as antihistamine desloratadine and antibiotic monensin. In the mouse model of carrageenan-induced systemic inflammation, increases in phospho-p38 MAPK and expression of CHST15 and CHST11 and declines in DNA-E2F binding and ARSB expression occurred in the lung, similar to the observed effects in this SPRBD model of COVID-19 infection. Since accumulation of chondroitin sulfates is associated with fibrotic lung conditions and diffuse alveolar damage, increased attention to p38-MAPK inhibition may be beneficial in ameliorating Covid-19 infections.
Topics: Mice; Animals; Humans; N-Acetylgalactosamine-4-Sulfatase; Chondroitin Sulfates; Spike Glycoprotein, Coronavirus; Carbohydrate Sulfotransferases; Angiotensin-Converting Enzyme 2; p38 Mitogen-Activated Protein Kinases; COVID-19; SARS-CoV-2
PubMed: 38355690
DOI: 10.1038/s41392-024-01741-3 -
Nucleic Acids Research May 2024RNA interference (RNAi) is an endogenous process that can be harnessed using chemically modified small interfering RNAs (siRNAs) to potently modulate gene expression in...
RNA interference (RNAi) is an endogenous process that can be harnessed using chemically modified small interfering RNAs (siRNAs) to potently modulate gene expression in many tissues. The route of administration and chemical architecture are the primary drivers of oligonucleotide tissue distribution, including siRNAs. Independently of the nature and type, oligonucleotides are eliminated from the body through clearance tissues, where their unintended accumulation may result in undesired gene modulation. Divalent siRNAs (di-siRNAs) administered into the CSF induce robust gene silencing throughout the central nervous system (CNS). Upon clearance from the CSF, they are mainly filtered by the kidneys and liver, with the most functionally significant accumulation occurring in the liver. siRNA- and miRNA-induced silencing can be blocked through substrate inhibition using single-stranded, stabilized oligonucleotides called antagomirs or anti-siRNAs. Using APOE as a model target, we show that undesired di-siRNA-induced silencing in the liver can be mitigated through administration of liver targeting GalNAc-conjugated anti-siRNAs, without impacting CNS activity. Blocking unwanted hepatic APOE silencing achieves fully CNS-selective silencing, essential for potential clinical translation. While we focus on CNS/liver selectivity, coadministration of differentially targeting siRNA and anti-siRNAs can be adapted as a strategy to achieve tissue selectivity in different organ combinations.
Topics: Animals; Humans; Male; Mice; Acetylgalactosamine; Antagomirs; Apolipoproteins E; Central Nervous System; Gene Silencing; Liver; Mice, Inbred C57BL; MicroRNAs; RNA Interference; RNA, Small Interfering
PubMed: 38348876
DOI: 10.1093/nar/gkae100