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Journal of the American Chemical Society Aug 2019O-Linked α--acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20...
O-Linked α--acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide -acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate -acetylgalactosamine (UDP-GalNAc) analogs to identify enzymesubstrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzymesubstrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems.
Topics: Acetylgalactosamine; Amino Acid Sequence; Catalytic Domain; Humans; Isoenzymes; Kinetics; Models, Molecular; Mutagenesis; N-Acetylgalactosaminyltransferases; Protein Engineering; Substrate Specificity; Uridine Diphosphate; Polypeptide N-acetylgalactosaminyltransferase
PubMed: 31373799
DOI: 10.1021/jacs.9b04695 -
International Journal of Biological... Jun 2020Lectins are ubiquitous carbohydrate-binding proteins that interact with sugar moieties in a highly specific manner. H-type lectins represent a new group of lectins that... (Review)
Review
Lectins are ubiquitous carbohydrate-binding proteins that interact with sugar moieties in a highly specific manner. H-type lectins represent a new group of lectins that were identified in invertebrates. These lectins share structural homology and bind mainly to N-acetylgalactosamine (GalNAc). Recent structural studies on the H-type lectins provided a detailed description of the GalNAc-lectin interaction that is already exploited in a number of biomedical applications. Two members of the H-type lectin family, Helix pomatia agglutinin (HPA) and Helix aspersa agglutinin (HAA), have already been extensively used in many diagnostic tests due their ability to specifically recognize GalNAc. This ability is especially important because aberrant glycosylation patterns of proteins expressed by cancer cells contain GalNAc. In addition, H-type lectins were utilized in diagnostics of other non-cancer diseases and represent great potential as components of drug delivery systems. Here, we present an overview of the H-type lectins and their applications in diagnostics, analytics and drug delivery.
Topics: Acetylgalactosamine; Agglutinins; Amino Acid Sequence; Animals; Drug Delivery Systems; Glycosylation; Humans; Lectins; Plant Lectins
PubMed: 32119947
DOI: 10.1016/j.ijbiomac.2020.02.320 -
Communications Biology May 2022The genetic capacity to synthesize the biofilm matrix exopolysaccharide Pel is widespread among Gram-negative and Gram-positive bacteria. However, its exact chemical...
The genetic capacity to synthesize the biofilm matrix exopolysaccharide Pel is widespread among Gram-negative and Gram-positive bacteria. However, its exact chemical structure has been challenging to determine. Using a Pseudomonas aeruginosa strain engineered to overproduce Pel, improvements to the isolation procedure, and selective hydrolysis with the glycoside hydrolase PelA, we demonstrate that Pel is a partially de-N-acetylated linear polymer of α-1,4-N-acetylgalactosamine comprised predominantly of dimeric repeats of galactosamine and N-acetylgalactosamine.
Topics: Acetylgalactosamine; Biofilms; Galactosamine; Polymers; Polysaccharides, Bacterial
PubMed: 35618750
DOI: 10.1038/s42003-022-03453-2 -
Clinical Pharmacokinetics Dec 2023Small-interfering ribonucleic acids (siRNAs) with N-acetylgalactosamine (GalNAc) conjugation for improved liver uptake represent an emerging class of drugs that modulate...
Small-interfering ribonucleic acids (siRNAs) with N-acetylgalactosamine (GalNAc) conjugation for improved liver uptake represent an emerging class of drugs that modulate liver-expressed therapeutic targets. The pharmacokinetics of GalNAc-siRNAs are characterized by a rapid distribution from plasma to tissue (hours) and a long terminal plasma half-life, analyzed in the form of the antisense strand, driven by redistribution from tissue (weeks). Understanding how clinical pharmacokinetics relate to the dose and type of siRNA chemical stabilizing method used is critical, e.g., to design studies, to investigate safety windows, and to predict the pharmacokinetics of new preclinical assets. To this end, we collected and analyzed pharmacokinetic data from the literature regarding nine GalNAc-siRNAs. Based on this analysis, we showed that the clinical plasma pharmacokinetics of GalNAc-siRNAs are approximately dose proportional and similar between chemical stabilizing methods. This holds for both the area under the concentration-time curve (AUC) and the maximum plasma concentration (C). Corresponding rat and monkey pharmacokinetic data for a subset of the nine GalNAc-siRNAs show dose-proportional C, supra-dose-proportional AUC, and similar pharmacokinetics between chemical stabilizing methods. Together, the animal and human pharmacokinetic data indicate that plasma clearance divided by bioavailability follows allometric principles and scales between species with an exponent of 0.75. Finally, the clinical plasma concentration-time profiles can be empirically described by standard one-compartment kinetics with first-order absorption up to 24 h after subcutaneous dosing, and by three-compartment kinetics with first-order absorption in general. To describe the system more mechanistically, we report a corrected and unambiguously defined version of a previously published physiologically based pharmacokinetic model.
Topics: Humans; Rats; Animals; Acetylgalactosamine; Liver; RNA, Small Interfering; Biological Availability
PubMed: 37824025
DOI: 10.1007/s40262-023-01314-7 -
The Journal of Biological Chemistry Aug 2012We used a comparative genomics approach to reconstruct the N-acetyl-d-galactosamine (GalNAc) and galactosamine (GalN) utilization pathways and transcriptional regulons...
We used a comparative genomics approach to reconstruct the N-acetyl-d-galactosamine (GalNAc) and galactosamine (GalN) utilization pathways and transcriptional regulons in Proteobacteria. The reconstructed GalNAc/GalN utilization pathways include multiple novel genes with specific functional roles. Most of the pathway variations were attributed to the amino sugar transport, phosphorylation, and deacetylation steps, whereas the downstream catabolic enzymes in the pathway were largely conserved. The predicted GalNAc kinase AgaK, the novel variant of GalNAc-6-phosphate deacetylase AgaA(II) and the GalN-6-phosphate deaminase AgaS from Shewanella sp. ANA-3 were validated in vitro using individual enzymatic assays and reconstitution of the three-step pathway. By using genetic techniques, we confirmed that AgaS but not AgaI functions as the main GalN-6-P deaminase in the GalNAc/GalN utilization pathway in Escherichia coli. Regulons controlled by AgaR repressors were reconstructed by bioinformatics in most proteobacterial genomes encoding GalNAc pathways. Candidate AgaR-binding motifs share a common sequence with consensus CTTTC that was found in multiple copies and arrangements in regulatory regions of aga genes. This study provides comprehensive insights into the common and distinctive features of the GalNAc/GalN catabolism and its regulation in diverse Proteobacteria.
Topics: Acetylgalactosamine; Biological Transport, Active; Carboxylic Ester Hydrolases; Escherichia coli K12; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Regulon; Repressor Proteins; Shewanella
PubMed: 22711537
DOI: 10.1074/jbc.M112.382333 -
Nucleic Acids Research Apr 2019For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro...
For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.
Topics: Acetylgalactosamine; Animals; Deoxyribonucleotides; Female; Fluorine; Humans; Male; RNA, Small Interfering; Rats; Rats, Sprague-Dawley
PubMed: 30820542
DOI: 10.1093/nar/gkz140 -
Journal of the Indian Society of... 2023The frequency of adenotonsillar hypertrophy in mouth-breathing children when compared to the average found in the general population is considered to be higher. Mouth...
Assessment of changes in levels using N-acetylgalactosamine-6-sulfatase marker and pharyngeal airway space with appliance therapy in mouth breathers - An ELISA-based study.
BACKGROUND
The frequency of adenotonsillar hypertrophy in mouth-breathing children when compared to the average found in the general population is considered to be higher. Mouth breathing is considered as one of the causative factors for tonsillitis in children. Through continuous irritation on tonsillar wall, tonsils swell up and inflammation develops.
PURPOSE
The purpose of the study is to evaluate Streptococcus pyogenes count using colony-forming units (CFUs) and N-acetylgalactosamine-6-sulfatase side chain marker on ELISA (enzyme linked immunosorbent assay) in mouth breathers and to establish its correlation with pharyngeal airway space pre- and post-oral screen appliance therapy.
MATERIALS AND METHODS
A total number of 24 (n) mouth breathers aged between 5 and 12 years were included in the study and given oral screen appliance therapy. The subjects were evaluated for the various parameters before the delivery of a habit-breaking appliance and then reevaluated for the same parameters (presence of S. pyogenes and its counts, size of tonsils, and pharyngeal airway space dimensions) after 6 months of appliance usage.
RESULTS
A statistically significant difference was seen in levels of S. pyogenes using ELISA and CFUs. Furthermore, statistically significant difference was observed in Friedman tonsil scoring and pharyngeal airway space and pre- and post-oral screen appliance therapy.
CONCLUSION
Oral screen appliance therapy reduced the frequency of occurrence of tonsillitis in mouth breathers by decreasing the counts of S. pyogenes bacteria. Upper and lower pharyngeal airway space dimensions were increased after 6 months of appliance therapy in mouth breathers.
Topics: Child; Humans; Child, Preschool; Streptococcus pyogenes; Acetylgalactosamine; Mouth Breathing; Tonsillitis; Sulfatases
PubMed: 37635469
DOI: 10.4103/jisppd.jisppd_105_23 -
Infection and Immunity Jun 2016Kingella kingae is a common cause of invasive disease in young children and was recently found to produce a polysaccharide capsule containing N-acetylgalactosamine...
Kingella kingae is a common cause of invasive disease in young children and was recently found to produce a polysaccharide capsule containing N-acetylgalactosamine (GalNAc) and β-3-deoxy-d-manno-octulosonic acid (βKdo). Given the role of capsules as important virulence factors and effective vaccine antigens, we set out to determine the genetic determinants of K. kingae encapsulation. Using a transposon library and a screen for nonencapsulated mutants, we identified the previously identified ctrABCD (ABC transporter) operon, a lipA (kpsC)-like gene, a lipB (kpsS)-like gene, and a putative glycosyltransferase gene designated csaA (capsule synthesis type a gene A). These genes were found to be present at unlinked locations scattered throughout the genome, an atypical genetic arrangement for Gram-negative bacteria that elaborate a capsule dependent on an ABC-type transporter for surface localization. The csaA gene product contains a predicted glycosyltransferase domain with structural homology to GalNAc transferases and a predicted capsule synthesis domain with structural homology to Kdo transferases, raising the possibility that this enzyme is responsible for alternately linking GalNAc to βKdo and βKdo to GalNAc. Consistent with this conclusion, mutation of the DXD motif in the GalNAc transferase domain and of the HP motif in the Kdo transferase domain resulted in a loss of encapsulation. Examination of intracellular and surface-associated capsule in deletion mutants and complemented strains further implicated the lipA (kpsC)-like gene, the lipB (kpsS)-like gene, and the csaA gene in K. kingae capsule production. These data define the genetic requirements for encapsulation in K. kingae and demonstrate an atypical organization of capsule synthesis, assembly, and export genes.
Topics: Acetylgalactosamine; Bacterial Capsules; Bacterial Proteins; DNA Transposable Elements; Escherichia coli; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Genome, Bacterial; Glycosyltransferases; Kingella kingae; Mutation; Operon; Polysaccharides, Bacterial; Protein Domains; Recombinant Proteins; Sugar Acids
PubMed: 27045037
DOI: 10.1128/IAI.00128-16 -
International Journal of Molecular... Oct 2022β--Acetylhexosaminidase from (Hex; EC 3.2.1.52) is an -glycosidase with dual activity for cleaving -acetylglucosamine (GlcNAc) and -acetylgalactosamine (GalNAc) units...
β--Acetylhexosaminidase from (Hex; EC 3.2.1.52) is an -glycosidase with dual activity for cleaving -acetylglucosamine (GlcNAc) and -acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast expression system, which is required for the correct folding of tetrameric fungal β--acetylhexosaminidases. While the wild-type Hex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr Hex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers - they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.
Topics: beta-N-Acetylhexosaminidases; Substrate Specificity; Acetylglucosamine; Acetylgalactosamine; Kinetics; Acetylglucosaminidase; Mutation
PubMed: 36293310
DOI: 10.3390/ijms232012456 -
Nucleic Acids Research Jun 2024Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited...
Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.
Topics: Animals; Acetylgalactosamine; RNA, Small Interfering; Administration, Oral; Mice; Rats; RNA Interference; Male; Biological Availability; Humans; Rats, Sprague-Dawley; Macaca fascicularis; Liver; Macaca mulatta
PubMed: 38742636
DOI: 10.1093/nar/gkae350