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The Journal of Biological Chemistry May 1996
Review
Topics: Acetylgalactosamine; Animals; Carbohydrate Conformation; Glycosylation; Hormones; Humans; Oligosaccharides; Proteins
PubMed: 8647799
DOI: 10.1074/jbc.271.21.12117 -
Advanced Healthcare Materials May 2023Peptide nucleic acids (PNAs) are used/applied in various studies to target genomic DNA and RNA to modulate gene expression. Non-specific targeting and rapid elimination...
Peptide nucleic acids (PNAs) are used/applied in various studies to target genomic DNA and RNA to modulate gene expression. Non-specific targeting and rapid elimination always remain a challenge for PNA-based applications. Here, the synthesis, characterization, in vitro and in vivo study of di lactobionic acid (diLBA) and tris N-acetyl galactosamine (tGalNAc) conjugated PNAs for liver-targeted delivery are reported. For proof of concept, diLBA, and tGalNAc conjugated PNAs (anti-miR-122 PNAs) were synthesized to target microRNA-122 (miR-122) which is over-expressed in the hepatic tissue. Different lengths of anti-miR-122 PNAs conjugated with diLBA and tGalNAc are tested. Cell culture and in vivo analyses to determine biodistribution, efficacy, and toxicity profile are performed. This work indicates that diLBA conjugates show significant retention in hepatocytes in addition to tGalNAc conjugates after in vivo delivery. Full-length PNA conjugates show significant downregulation of miR-122 levels and subsequent de-repression of its downstream targets with no evidence of toxicity. The results provide a robust framework for ligand-conjugated delivery systems for PNAs that can be explored for broader biomedical applications.
Topics: Peptide Nucleic Acids; Acetylgalactosamine; Tissue Distribution; Antagomirs; Hepatocytes
PubMed: 36636995
DOI: 10.1002/adhm.202202859 -
Proceedings of the National Academy of... Oct 2020Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by...
Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe -()-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked -acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched -acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding -acetylglucosamine analog by the epimerase -acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.
Topics: Acetylgalactosamine; Gene Expression Regulation, Enzymologic; Glycoproteins; Glycosylation; Humans; Racemases and Epimerases; Substrate Specificity; Uridine Diphosphate N-Acetylgalactosamine
PubMed: 32989128
DOI: 10.1073/pnas.2007297117 -
Drug Metabolism and Disposition: the... Oct 2019Understanding small interfering RNA (siRNA) fraction unbound ( ) in relevant physiologic compartments is critical for establishing pharmacokinetic-pharmacodynamic...
Understanding small interfering RNA (siRNA) fraction unbound ( ) in relevant physiologic compartments is critical for establishing pharmacokinetic-pharmacodynamic relationships for this emerging modality. In our attempts to isolate the equilibrium free fraction of -acetylgalactosamine-conjugated siRNA using classic small-molecule in vitro techniques, we found that the hydrodynamic radius was critical in determining the size exclusion limit requirements for isolation, largely validating the siRNA "rigid rod" hypothesis. With this knowledge, we developed an orthogonally validated 50 kDa molecular-mass cutoff ultrafiltration assay to quantify in biologic matrices including human, nonhuman primate, rat, and mouse plasma, and human liver homogenate. To enhance understanding of the siRNA-plasma interaction landscape, we examined the effects of various common oligonucleotide therapeutic modifications to the ribose and helix backbone on siRNA in plasma ( ) and found that chemical modifications can alter plasma protein binding by at least 20%. Finally, to gain insight into which specific plasma proteins bind to siRNA, we developed a qualitative screen to identify binding "hits" across a panel of select purified human plasma proteins.
Topics: Acetylgalactosamine; Adult; Animals; Blood Proteins; Female; Humans; Liver; Macaca fascicularis; Mice; Protein Binding; RNA, Small Interfering; Rats
PubMed: 31097425
DOI: 10.1124/dmd.119.086967 -
The Journal of Biological Chemistry Jan 1986The cell surface glycoprotein of Halobacteria contains two different types of sulfated saccharides: hexuronic acid-containing oligosaccharides linked to the protein via...
The cell surface glycoprotein of Halobacteria contains two different types of sulfated saccharides: hexuronic acid-containing oligosaccharides linked to the protein via asparaginylglucose, and a serially repeated saccharide unit containing amino sugars that resembles the animal glycosaminoglycans. Here we report that 1) the sulfated repeating unit saccharide is linked to the cell surface glycoprotein via asparaginyl-N-acetylgalactosamine, 2) the amino acid sequence surrounding this linkage region is -Asn-Ala-Ser-, and thus in agreement with the acceptor sequence ASN-X-Thr(Ser) common to all eucaryotic N-glycosidically bound saccharides determined so far; 3) in addition to galactose, galacturonic acid, N-acetylglucosamine, and N-acetylgalactosamine, the methylated hexuronic acid 3-O-methylgalacturonic acid occurs as a stoichiometric constituent of the sulfated building block of the glycosaminoglycan chain.
Topics: Acetylgalactosamine; Amino Acids; Asparagine; Carbohydrate Conformation; Chromatography, High Pressure Liquid; Galactosamine; Gas Chromatography-Mass Spectrometry; Glycosaminoglycans; Halobacterium; Hydrolysis; Sulfates
PubMed: 3944078
DOI: No ID Found -
The Journal of Biological Chemistry Mar 2022Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some...
Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrP, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)-galactose-sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrP GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases.
Topics: Acetylgalactosamine; Animals; Brain; Glycosylphosphatidylinositols; Mice; Osteogenesis; Prion Diseases; Prions; Structure-Activity Relationship
PubMed: 35151686
DOI: 10.1016/j.jbc.2022.101720 -
Journal of Enzyme Inhibition and... Jun 2010N-Acetylgalactosamine kinase (GALK2) is a small molecule kinase from the GHMP family which phosphorylates N-acetylgalactosamine at the expense of ATP. Recombinant GALK2...
N-Acetylgalactosamine kinase (GALK2) is a small molecule kinase from the GHMP family which phosphorylates N-acetylgalactosamine at the expense of ATP. Recombinant GALK2 expressed in, and purified from, Escherichia coli was shown to be active with the following kinetic parameters: Michaelis constant for ATP, 14 +/- 3 microM; Michaelis constant for N-acetylgalactosamine, 40 +/- 14 microM; and turnover number, 1.0 +/- 0.1 s(-1). The combination of substrate inhibition by N-acetylgalactosamine and alpha-methylgalactopyranoside acting as an uncompetitive inhibitor with respect to ATP suggested that the enzyme has an ordered ternary complex mechanism in which ATP is the first substrate to bind. The effects of pH on the kinetic parameters provided evidence for ionizable residues playing a role in substrate binding and catalysis. These results are discussed in the context of the mechanisms of the GHMP kinases.
Topics: Acetylgalactosamine; Adenosine Triphosphate; Catalysis; Cloning, Molecular; Galactokinase; Humans; Hydrogen-Ion Concentration; Kinetics; Protein Binding; Substrate Specificity
PubMed: 19874134
DOI: 10.3109/14756360903179492 -
The Journal of Biological Chemistry Nov 1959
Topics: Acetylgalactosamine; Amino Sugars
PubMed: 13828347
DOI: No ID Found -
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 -
Histology and Histopathology Jan 2000The lectin from Helix pomatia, the Roman snail (HPA), recognises terminal alpha N-acetylgalactosamine residues. A large number of lectin histochemical studies have... (Review)
Review
The lectin from Helix pomatia, the Roman snail (HPA), recognises terminal alpha N-acetylgalactosamine residues. A large number of lectin histochemical studies have demonstrated that expression of HPA-binding glycoproteins by cancer cells to be a marker of metastatic competence and poor prognosis in a range of common human adenocarcinomas, including those of breast, stomach, ovary, oesophagus, colorectum, thyroid and prostate. Around 80% of metastases arising from primary breast cancer are predictably HPA positive, but, intriguingly, around 20% do not express HPA binding glycoproteins reflecting the complexity of metastatic mechanisms and the further disruptions in cellular glycosylation that attend tumour progression. HPA binding is not an independent prognostic factor, but is strongly associated with the presence of metastases in local lymph nodes. It does appear to be independent of other clinical features of prognostic importance such as tumour size, histological grade, S-phase fraction, ploidy, and there is little convincing evidence of any association with oncogene expression or hormone receptor positivity. The precise nature of the metastasis-associated HPA binding partner(s) is a question of some interest, but thus far remains unclear. HPA will recognise, for example, the Tn epitope and blood group A antigen, but its prognostic significance appears to be through recognition of a much broader and heterogeneous array of N-galactosaminylated glycoproteins. Their synthesis appears to be mediated through alteration in expression or activity of one or more of the enzymes of glycosylation. The most likely putative roles of HPA-binding ligands in the metastatic cascade may be enhancement of invasive capacity, or interaction with an as yet unidentified lectin-like receptor facilitating adhesion processes. The prognostic information provided by HPA lectin histochemistry may be used clinically to inform the physician and aid treatment decisions; far more interesting is the challenge of further understanding the precise nature of the HPA-binding ligands, and defining their role in the complex mechanisms of metastasis.
Topics: Acetylgalactosamine; Animals; Biomarkers, Tumor; Disease Progression; Female; Helix, Snails; Humans; Lectins; Lymphatic Metastasis; Male; Neoplasm Metastasis; Neoplasms; Polysaccharides
PubMed: 10668205
DOI: 10.14670/HH-15.143