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Marine Drugs Feb 2024Sulfation is gaining increased interest due to the role of sulfate in the bioactivity of many polysaccharides of marine origin. Hence, sulfatases, enzymes that control...
Sulfation is gaining increased interest due to the role of sulfate in the bioactivity of many polysaccharides of marine origin. Hence, sulfatases, enzymes that control the degree of sulfation, are being more extensively researched. In this work, a novel sulfatase (SulA1) encoded by the gene was characterized. The -gene is located upstream of a chondroitin lyase encoding gene in the genome of the marine Arthrobacter strain (MAT3885). The sulfatase was produced in . Based on the primary sequence, the enzyme is classified under sulfatase family 1 and the two catalytic residues typical of the sulfatase 1 family-Cys57 (post-translationally modified to formyl glycine for function) and His190-were conserved. The enzyme showed increased activity, but not improved stability, in the presence of Ca, and conserved residues for Ca binding were identified (Asp17, Asp18, Asp277, and Asn278) in a structural model of the enzyme. The temperature and pH activity profiles (screened using -nitrocatechol sulfate) were narrow, with an activity optimum at 40-50 °C and a pH optimum at pH 5.5. The was significantly higher (67 °C) than the activity optimum. Desulfation activity was not detected on polymeric substrates, but was found on GalNAc4S, which is a sulfated monomer in the repeated disaccharide unit (GlcA-GalNAc4S) of, e.g., chondroitin sulfate A. The position of the sulA1 gene upstream of a chondroitin lyase gene and combined with the activity on GalNAc4S suggests that there is an involvement of the enzyme in the chondroitin-degrading cascade reaction, which specifically removes sulfate from monomeric GalNAc4S from chondroitin sulfate degradation products.
Topics: Sulfates; Acetylgalactosamine; Arthrobacter; Sulfatases; Escherichia coli; Galactosamine; Chondroitin Lyases; Cloning, Molecular
PubMed: 38535445
DOI: 10.3390/md22030104 -
CPT: Pharmacometrics & Systems... May 2024The objective of this study was to compare the efficacy of short interfering RNA therapeutics (siRNAs) in reducing hepatitis B surface antigen (HBsAg) levels in... (Meta-Analysis)
Meta-Analysis Review
The objective of this study was to compare the efficacy of short interfering RNA therapeutics (siRNAs) in reducing hepatitis B surface antigen (HBsAg) levels in hepatitis B-infected (HBV) mice across multiple siRNA therapeutic classes using model-based meta-analysis (MBMA) techniques. Literature data from 10 studies in HBV-infected mice were pooled, including 13 siRNAs, formulated as liposomal nanoparticles (LNPs) or conjugated to either cholesterol (chol) or N-acetylgalactosamine (GalNAc). Time course of the baseline- and placebo-corrected mean HBsAg profiles were modeled using kinetics of drug effect (KPD) model coupled to an indirect response model (IRM) within a longitudinal non-linear mixed-effects MBMA framework. Single and multiple dose simulations were performed exploring the role of dosing regimens across evaluated siRNA classes. The HBsAg degradation rate (0.72 day) was consistent across siRNAs but exhibited a large between-study variability of 31.4% (CV%). The siRNA biophase half-life was dependent on the siRNA class and was highest for GalNAc-siRNAs (21.06 days) and lowest for chol-siRNAs (2.89 days). ID estimates were compound-specific and were lowest for chol-siRNAs and highest for GalNAc-siRNAs. Multiple dose simulations suggest GalNAc-siRNAs may require between 4 and 7 times less frequent dosing at higher absolute dose levels compared to LNP-siRNAs and chol-siRNAs, respectively, to reach equipotent HBsAg-lowering effects in HBV mice. In conclusion, non-clinical HBsAg concentration-time data after siRNA administration can be described using the presented KPD-IRM MBMA framework. This framework allows to quantitatively compare the effects of siRNAs on the HBsAg time course and inform dose and regimen selection across siRNA classes. These results may support siRNA development, optimize preclinical study designs, and inform data analysis methodology of future anti-HBV siRNAs; and ultimately, support siRNA model-informed drug development (MIDD) strategies.
Topics: Animals; RNA, Small Interfering; Hepatitis B Surface Antigens; Mice; Hepatitis B; Disease Models, Animal; Acetylgalactosamine; Liposomes; Models, Biological; Nanoparticles; Hepatitis B virus
PubMed: 38522000
DOI: 10.1002/psp4.13129 -
Frontiers in Microbiology 2024Mucins are large glycoproteins whose degradation requires the expression of several glycosil hydrolases to catalyze the cleavage of the oligosaccharide chains and...
Mucins are large glycoproteins whose degradation requires the expression of several glycosil hydrolases to catalyze the cleavage of the oligosaccharide chains and release monosaccharides that can be assimilated. In this study, we present a characterization on the strains WC0700, WC0709, and WC0705. These three strains were previously isolated from enrichment cultures on mucin of fecal samples from healthy subjects and can use mucin as sole carbon and nitrogen source. Genome analysis and functional analysis of these strains elucidated their physiological and biochemical features. WC0700 harbored the highest number of glycosyl hydrolases specific for mucin degradation, while WC0705 had the least. These predicted differences were confirmed growing the strains on 5 mucin-decorating monosaccharides (L-fucose, N-Acetylneuraminic acid, galactose, N-acetylgalactosamine, and N-acetylglucosamine) as only source of carbon. Fermenting mucin, they all produced formic, acetic, propionic, butyric, isovaleric, and lactic acids, and ethanol; acetic acid was the main primary metabolite. Further catabolic capabilities were investigated, as well as antibiotic susceptibility, biofilm formation, tolerance to oxygen and temperature. The potential pathogenicity of the strains was evaluated through research of virulence factors. The merge between comparative and functional genomics and biochemical/physiological characterization provided a comprehensive view of these mucin degraders, reassuring on the safety of these species and leaving ample scope for deeper investigations on the relationship with the host and for assessing if some relevant health-promoting effect could be ascribed to these SCFA producing species.
PubMed: 38511005
DOI: 10.3389/fmicb.2024.1359726 -
British Journal of Clinical Pharmacology Jun 2024The aim of this study was to characterize the population pharmacokinetics of AZD8233, an antisense oligonucleotide (ASO) that targets the PCSK9 transcript to reduce... (Randomized Controlled Trial)
Randomized Controlled Trial
AIMS
The aim of this study was to characterize the population pharmacokinetics of AZD8233, an antisense oligonucleotide (ASO) that targets the PCSK9 transcript to reduce hepatocyte PCSK9 protein production and plasma levels. AZD8233 utilizes generation 2.5 S-constrained ethyl motif (cET) chemistry and is conjugated to a triantennary N-acetylgalactosamine (GalNAc3) ligand for targeted hepatocyte uptake.
METHODS
A non-linear mixed-effect modelling approach utilizing NONMEM software was applied to AZD8233 concentration-time data from 3416 samples in 219 participants from four phase 1-2 studies, one in healthy volunteers (NCT03593785) and three in patients with dyslipidaemia (NCT04155645, NCT04641299 and NCT04823611).
RESULTS
The final model described the AZD8233 plasma concentration-time profile from four phase 1-2 studies in healthy volunteers or participants with dyslipidaemia, covering a dose range of 4 to 120 mg. The pharmacokinetics of AZD8233 were adequately described by a two-compartment model with first-order absorption. The supra-proportional increase in maximum plasma concentration (C) across the observed dose range was described by non-linear Michaelis-Menten elimination (maximum elimination rate, 9.9 mg/h [12% relative standard error]; concentration yielding half-maximal elimination rate, 4.8 mg/L [18% relative standard error]). Body weight, sex, estimated glomerular filtration rate and disease status (healthy participant vs. patient with dyslipidaemia) were identified as factors affecting exposure to AZD8233.
CONCLUSIONS
Covariate analysis showed body weight to be the main factor affecting exposure to AZD8233, which largely explained the higher C observed in the Asian population relative to non-Asians.
Topics: Humans; Male; Female; Middle Aged; Adult; Dyslipidemias; Oligonucleotides, Antisense; Proprotein Convertase 9; Young Adult; Healthy Volunteers; Models, Biological; Aged; Dose-Response Relationship, Drug; Adolescent
PubMed: 38504437
DOI: 10.1111/bcp.16046 -
Aging and Disease Mar 2024UDP-GalNAc polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) catalyze mucin-type O-glycosylation by transferring α-N-acetylgalactosamine (GalNAc) from... (Review)
Review
UDP-GalNAc polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) catalyze mucin-type O-glycosylation by transferring α-N-acetylgalactosamine (GalNAc) from UDP-GalNAc to Ser or Thr residues of target proteins. This post-translational modification is common in eukaryotes, yet its biological functions remain unclear. Recent studies have identified specific receptors in the heart and vascular wall cells that can be mucin-type O-glycosylated, and there is now substantial evidence confirming that patients with various cardiovascular diseases (CVDs), such as heart failure, coronary artery disease, myocardial hypertrophy, and vascular calcification, exhibit abnormal changes in GalNAc-Ts. This review aims to highlight recent advances in GalNAc-Ts and their roles in the cardiovascular system, intending to provide evidence for clinical treatment and prevention of CVDs.
PubMed: 38502587
DOI: 10.14336/AD.2024.0308 -
Angewandte Chemie (International Ed. in... May 2024Protein O-GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes....
Protein O-GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes. Investigation of O-GlcNAcylation by metabolic glycoengineering (MGE) has been carried out for two decades with peracetylated N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine derivatives modified with varying reporter groups. Recently, it has been shown that these derivatives can result in non-specific protein labeling termed S-glyco modification. Here, we report norbornene-modified GlcNAc derivatives with a protected phosphate at the anomeric position and their application in MGE. These derivatives overcome two limitations of previously used O-GlcNAc reporters. They do not lead to detectable S-glyco modification, and they efficiently react in the inverse-electron-demand Diels-Alder (IEDDA) reaction, which can be carried out even within living cells. Using a derivative with an S-acetyl-2-thioethyl-protected phosphate, we demonstrate the protein-specific detection of O-GlcNAcylation of several proteins and the protein-specific imaging of O-GlcNAcylation inside living cells by Förster resonance energy transfer (FRET) visualized by confocal fluorescence lifetime imaging microscopy (FLIM).
Topics: Glycosylation; Humans; Acetylglucosamine; Protein Processing, Post-Translational; Norbornanes; Proteins
PubMed: 38501674
DOI: 10.1002/anie.202320247 -
Current Atherosclerosis Reports May 2024Here, we summarize the key findings from preclinical studies that tested the concept that editing of hepatic genes can lower plasma low-density lipoprotein... (Review)
Review
PURPOSE OF REVIEW
Here, we summarize the key findings from preclinical studies that tested the concept that editing of hepatic genes can lower plasma low-density lipoprotein (LDL)-cholesterol levels to subsequently reduce atherosclerotic cardiovascular disease risk.
RECENT FINDINGS
Selective delivery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing tools targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) to hepatocytes, i.e., through encapsulation into N-acetylgalactosamine-coupled lipid nanoparticles, is able to induce a stable ~ 90% decrease in plasma PCSK9 levels and a concomitant 60% reduction in LDL-cholesterol levels in mice and non-humane primates. Studies in mice have shown that this state-of-the-art technology can be extended to include additional targets related to dyslipidemia such as angiopoietin-like 3 and several apolipoproteins. The use of gene editors holds great promise to lower plasma LDL-cholesterol levels also in the human setting. However, gene editing safety has to be guaranteed before this approach can become a clinical success.
Topics: Gene Editing; Humans; Animals; Hypercholesterolemia; Genetic Therapy; Proprotein Convertase 9; Cholesterol, LDL; CRISPR-Cas Systems
PubMed: 38498115
DOI: 10.1007/s11883-024-01198-3 -
Chemical Science Mar 2024Glycan oxidation on the cell surface occurs in many specific life processes including pathogen-cell interactions. This work develops a surface-enhanced Raman scattering...
Glycan oxidation on the cell surface occurs in many specific life processes including pathogen-cell interactions. This work develops a surface-enhanced Raman scattering (SERS) imaging strategy for quantitative monitoring of protein-specific glycan oxidation mediated pathogen-cell interactions by utilizing Raman reporter DTNB and aptamer co-assembled platinum shelled gold nanoparticles (Au@Pt-DTNB/Apt). Using () and MCF-7 cells as models, Au@Pt-DTNB/Apt can specifically bind to MUC1 protein on the cell surface containing heavy galactose (Gal) and -acetylgalactosamine (GalNAc) modification. When interacts with cells, the secreted galactose oxidase (GO) can oxidize Gal/GalNAc, and the generated reactive oxygen species (ROS) further oxidizes DTNB to produce TNB for greatly enhancing the SERS signal. This strategy can quantitatively visualize for the first time both the protein-specific glycan oxidation and the mediated pathogen-cell interactions, thus providing key quantitative information to distinguish and explore the pathogen-resistance and pharmacological mechanisms of different drugs.
PubMed: 38487245
DOI: 10.1039/d4sc00157e -
European Journal of Medicinal Chemistry Apr 2024Asialoglycoprotein receptor (ASGPR) specifically recognizes glycans terminated with β-d-galactose or N-acetylgalactosamine. Its exclusive expression in mammalian... (Review)
Review
Asialoglycoprotein receptor (ASGPR) specifically recognizes glycans terminated with β-d-galactose or N-acetylgalactosamine. Its exclusive expression in mammalian hepatocytes renders it an ideal hepatic-targeted biomarker. To date, ASGPR-targeted ligands have been actively developed for drug delivery and hepatic imaging. This review provides a comprehensive summary of the progress achieved to-date in the field of developing ASGPR-targeted nuclear medicine imaging (NMI) radiotracers, highlighting the recent advancements over the last decade in terms of structure, radionuclides and labeling strategies. The biodistribution patterns, imaging characteristics, challenges and future prospective are discussed.
Topics: Animals; Asialoglycoprotein Receptor; Hepatocytes; Liver; Mammals; Nuclear Medicine; Tissue Distribution; Acetylgalactosamine
PubMed: 38479165
DOI: 10.1016/j.ejmech.2024.116278 -
Analytical Methods : Advancing Methods... Mar 2024Uridine diphosphate--acetylglucosamine (UDP-GlcNAc) is one of the major nucleotide sugars in living organisms and serves as the key donor substrate for the...
Uridine diphosphate--acetylglucosamine (UDP-GlcNAc) is one of the major nucleotide sugars in living organisms and serves as the key donor substrate for the post-translational modification of protein -GlcNAcylation. It undergoes interconversion to its epimer uridine diphosphate--acetylgalactosamine (UDP-GalNAc), which acts as a sugar donor initiating mucin-type -linked glycosylation. The intracellular levels of the two differ between the cell lines and largely fluctuate in response to metabolic perturbations, and recent studies have focused on the details of their biosynthesis or turnover. However, due to their similar chemical properties, sufficient resolution for the two epimers required non-volatile mobile phases that cannot be applied directly to a mass spectrometer. In this study, to implement simple liquid chromatography-mass spectrometry for UDP-GlcNAc and UDP-GalNAc, we optimized a condition of hydrophilic interaction liquid chromatography-mass spectrometry. We found that the use of ammonium hydroxide and an amide column with an optimized water-acetonitrile ratio, flow rate, and column temperature, provided complete separation of the two. The method allowed the analysis of intracellular levels, a stable isotope-labeled target, and patterns of product ion spectra in a single run with fewer sample preparation steps. The new method can be widely used for mass spectrometric analysis of UDP-GlcNAc and UDP-GalNAc.
Topics: Uridine Diphosphate N-Acetylgalactosamine; Liquid Chromatography-Mass Spectrometry; Nucleotides; Uridine Diphosphate N-Acetylglucosamine; Hydrophobic and Hydrophilic Interactions
PubMed: 38433563
DOI: 10.1039/d4ay00122b