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Bioorganic & Medicinal Chemistry Jun 2019Targeted drug delivery platforms can increase the concentration of drugs in specific cell populations, reduce adverse effects, and hence improve the therapeutic effect...
Targeted drug delivery platforms can increase the concentration of drugs in specific cell populations, reduce adverse effects, and hence improve the therapeutic effect of drugs. Herein, we designed two conjugates by installing the targeting ligand GalNAc (N-acetylgalactosamine) onto atorvastatin (AT). Compared to the parent drug, these two conjugates, termed G2-AT and G2-K-AT, showed increased hepatic cellular uptake. Moreover, both conjugates were able to release atorvastatin, and consequently showed dramatic inhibition of β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) reductase and increased LDL receptors on cell surface.
Topics: Acetylgalactosamine; Animals; Asialoglycoprotein Receptor; Atorvastatin; Cell Line, Tumor; Hepatocytes; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Ligands; Receptors, LDL; Swine
PubMed: 31005367
DOI: 10.1016/j.bmc.2019.04.019 -
International Journal of Molecular... Jul 2022Galactose and -acetyl-D-galactosamine-inhibitable lectin of have roles in the pathogenicity of intestinal amoebiasis. Igl1, the intermediate subunit lectin-1 of , has...
Galactose and -acetyl-D-galactosamine-inhibitable lectin of have roles in the pathogenicity of intestinal amoebiasis. Igl1, the intermediate subunit lectin-1 of , has been shown to have both hemolytic and cytotoxic activities that reside in the C-terminus of the protein. To identify the amino acid regions responsible for these activities, recombinant proteins were prepared and used in hemolytic and cytotoxic assays. The results revealed that Igl1 has multiple domains with hemolytic and cytotoxic activities and that amino acids 787-846, 968-1028 and 1029-1088 are involved in these activities. The hemolytic activities of the fragments were partly inhibited by mannose, galactose and -acetylgalactosamine, and glucose showed lower or negligible inhibitory effects for the activities. This is the first report of a protozoan protein with hemolytic and cytotoxic activities in multiple domains.
Topics: Acetylgalactosamine; Cytotoxins; Entamoeba histolytica; Galactose; Hemolysis; Humans; Lectins; Protozoan Proteins
PubMed: 35887043
DOI: 10.3390/ijms23147700 -
PLoS Neglected Tropical Diseases Feb 2022Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids (Sia) from sialoglycoconjugates to terminal...
Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids (Sia) from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Enzymes comprise a N-terminal catalytic domain (CD) followed by a lectin-like domain (LD). Most work on trypanosomal TS has been done on enzymatic activities focusing on the CD of TS from Trypanosoma cruzi (causing Chagas disease in Latin America), subspecies of Trypanosoma brucei, (causing human sleeping sickness in Africa) and Trypanosoma congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS)-LD binds to several carbohydrates, such as 1,4-β-mannotriose. In this study we investigated the influence of TconTS3-LD on Sia transfer efficiency of TconTS1a-CD by swapping domains. in silico analysis on structure models of TconTS enzymes revealed the potential of domain swaps between TconTS1a and TconTS3 without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear Sia transfer activity, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolytic release of free Neu5Ac as a side product was suppressed resulting in increased transfer efficiency. Presence of 1,4-β-mannotriose during TS reactions modulates enzyme activities enhancing transfer efficiency possibly due to occupation of the binding site in TconTS1a-LD. Interestingly this effect was in the same range as that observed when swapping TconTS1a-CD and TconTS3-LD. In summary, this study demonstrate the proof-of-principle for swapping CDs and LDs of TconTS and that TconTS3-LD influences enzymatic activity of TconTS1a-CD providing evidence that LDs play pivotal roles in modulating activities and biological functions of TconTS and possibly other TS.
Topics: Acetylgalactosamine; Binding Sites; Catalysis; Galactose; Glycoproteins; Neuraminidase; Oligosaccharides; Protozoan Proteins; Sialic Acids; Trypanosoma congolense
PubMed: 35130274
DOI: 10.1371/journal.pntd.0009585 -
The Journal of Biological Chemistry Jan 1990We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked...
We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types.
Topics: Acetylgalactosamine; Borohydrides; Carbohydrate Conformation; Chromatography, Affinity; Galactose; Glucosamine; Humans; Hydrogen-Ion Concentration; Immunosorbent Techniques; Mannose; N-Acetylneuraminic Acid; Oligosaccharides; Receptors, Transferrin; Sialic Acids; Tumor Cells, Cultured
PubMed: 2403553
DOI: No ID Found -
Journal of Biomolecular Techniques : JBT Sep 2017-Linked glycosylation is a common post-translational modification that can alter the overall structure, polarity, and function of proteins. Reverse-phase (RP)...
-Linked glycosylation is a common post-translational modification that can alter the overall structure, polarity, and function of proteins. Reverse-phase (RP) chromatography is the most common chromatographic approach to analyze -glycosylated peptides and their unmodified counterparts, even though this approach often does not provide adequate separation of these two species. Hydrophilic interaction liquid chromatography (HILIC) can be a solution to this problem, as the polar glycan interacts with the polar stationary phase and potentially offers the ability to resolve the peptide from its modified form(s). In this paper, HILIC is used to separate peptides with --acetylgalactosamine (-GalNAc), --acetylglucosamine (-GlcNAc), and -fucose additions from their native forms, and coefficients representing the extent of hydrophilicity were derived using linear regression analysis as a means to predict the retention times of peptides with these modifications.
Topics: Acetylgalactosamine; Acetylglucosamine; Amino Acid Sequence; Chromatography, Liquid; Chromatography, Reverse-Phase; Fucose; Glycopeptides; Glycosylation; Hydrophobic and Hydrophilic Interactions; Polysaccharides; Protein Processing, Post-Translational
PubMed: 28785176
DOI: 10.7171/jbt.17-2803-003 -
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 -
PLoS Neglected Tropical Diseases Aug 2023In the human host, the protozoan parasite Entamoeba histolytica is adapted to a non-invasive lifestyle in the colon as well as to an invasive lifestyle in the...
In the human host, the protozoan parasite Entamoeba histolytica is adapted to a non-invasive lifestyle in the colon as well as to an invasive lifestyle in the mesenterial blood vessels and the liver. This means to cope with bacteria and human cells as well as various metabolic challenges. Galactose and N-acetylgalactosamine (GalNAc) are sugars of great importance for the amoebae, they attach to the host mucus and enterocytes via their well-studied Gal/GalNAc specific lectin, they carry galactose residues in their surface glycans, and they cleave GalNAc from host mucins. The enzyme UDP-glucose 4-epimerase (GalE) works as a bridge between the galactose and glucose worlds, it can help to generate glucose for glycolysis from phagocytosis products containing galactose as well as providing UDP-galactose necessary for the biosynthesis of galactose-containing surface components. E. histolytica contains a single galE gene. We recombinantly expressed the enzyme in Escherichia coli and used a spectrophotometric assay to determine its temperature and pH dependency (37°C, pH 8.5), its kinetics for UDP-glucose (Km = 31.82 μM, Vmax = 4.31 U/mg) and substrate spectrum. As observed via RP-HPLC, the enzyme acts on UDP-Glc/Gal as well as UDP-GlcNAc/GalNAc. Previously, Trypanosoma brucei GalE and the bloodstream form of the parasite were shown to be susceptible to the three compounds ebselen, a selenoorganic drug with antioxidant properties, diethylstilbestrol, a mimic of oestrogen with anti-inflammatory properties, and ethacrynic acid, a loop diuretic used to treat oedema. In this study, the three compounds had cytotoxic activity against E. histolytica, but only ebselen inhibited the recombinant GalE with an IC50 of 1.79 μM (UDP-Gal) and 1.2 μM (UDP-GalNAc), suggesting that the two other compounds are active against other targets in the parasite. The importance of the ability of GalE to interconvert UDP-GalNAc and UDP-GlcNAc may be that the trophozoites can generate precursors for their own cyst wall from the sugar subunits cleaved from host mucins. This finding advances our understanding of the biochemical interactions of E. histolytica in its colonic environment.
Topics: Humans; UDPglucose 4-Epimerase; Entamoeba histolytica; Galactose; Glucose; Acetylgalactosamine; Cysts; Uridine Diphosphate
PubMed: 37616327
DOI: 10.1371/journal.pntd.0011574 -
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 Aug 2009To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature...
To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature for 1) the spontaneous methanolysis of ATP and 2) reactions catalyzed by kinases to which different mechanisms of action have been ascribed. For each of these enzymes, the minor effects of changing viscosity indicate that k(cat)/K(m) is governed by the central chemical events in the enzyme-substrate complex rather than by enzyme-substrate encounter. Individual Arrhenius plots, obtained at intervals between pH 4.8 and 11.0, yielded Delta H(#) and T Delta S(#) for the nonenzymatic methanolysis of ATP(2-), ATP(3-), and ATP(4-) in the absence of Mg(2+). The addition of Mg(2+) led to partly compensating changes in Delta H(#) and T Delta S(#), accelerating the nonenzymatic methanolysis of ATP 11-fold at pH 7 and 25 degrees C. The rate enhancements produced by yeast hexokinase, homoserine kinase, and N-acetylgalactosamine kinase (obtained by comparison of their k(cat)/K(m) values in the presence of saturating phosphoryl acceptor with the second order rate constant for methanolysis of MgATP) ranged between 10(12)- and 10(14)-fold. Their nominal affinities for the altered substrates in the transition state were 2.1 x 10(-16) m for N-acetylgalactosamine kinase, 7.4 x 10(-17) m for homoserine kinase, and 6.4 x 10(-18) m for hexokinase. Compared with nonenzymatic phosphoryl transfer, all three kinases were found to produce major reductions in the entropy of activation, in accord with the likelihood that substrate juxtaposition and desolvation play prominent roles in their catalytic action.
Topics: Acetylgalactosamine; Adenosine Triphosphate; Calorimetry; Glucose; Hexokinase; Homoserine; Humans; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Methanococcus; Methanol; Phosphotransferases (Alcohol Group Acceptor); Thermodynamics; Viscosity
PubMed: 19531469
DOI: 10.1074/jbc.M109.017806 -
Drug Design, Development and Therapy 2022Small interfering ribonucleic acids [siRNAs] are short ribonucleic acid (RNA) fragments cleaved from double-stranded RNA molecules that target and bind to specific... (Review)
Review
Small interfering ribonucleic acids [siRNAs] are short ribonucleic acid (RNA) fragments cleaved from double-stranded RNA molecules that target and bind to specific sequences on messenger RNA (mRNA), leading to their destruction. Therefore, the siRNA down-regulates the formation of selected mRNAs and their protein products. Givosiran is one such siRNA that uses this mechanism to treat acute hepatic porphyrias. Acute hepatic porphyrias are a group of rare, inherited metabolic disorders, characterized by acute potentially life-threatening attacks as well as chronic symptoms with a negative impact on quality of life. It has four types, each associated with distinct enzyme defects in the heme biosynthesis pathway in the liver. By targeting the expression of hepatic 5-aminolevulinic acid [ALA] synthase-1 [ALAS1], givosiran can down-regulate levels of toxic metabolites, leading to biochemical and clinical improvement. Givosiran selectively targets hepatocytes due to its linkage to -acetylgalactosamine (GalNac) leading to its selective uptake via asialoglycoprotein receptors (ASGPR). We provide an up-to-date literature review regarding givosiran in the context of a clinical overview of the porphyrias, an overview of siRNAs for therapy of human disorders, the design and development of givosiran, key clinical trial results of givosiran for prevention of acute porphyric attacks, emerging concerns regarding chronic use of givosiran, and the overall management of acute hepatic porphyrias. These insights are important not only for the management of acute hepatic porphyrias but also for the emerging field of siRNAs and their role in novel therapies for various diseases.
Topics: Acetylgalactosamine; Adult; Humans; Porphobilinogen Synthase; Porphyrias, Hepatic; Pyrrolidines; Quality of Life; RNA, Messenger; RNA, Small Interfering
PubMed: 35734365
DOI: 10.2147/DDDT.S281631