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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 -
International Journal of Molecular... Jul 2021In light of recent research, there is increasing evidence showing that extracellular semen components have a significant impact on the immune reaction of the female...
In light of recent research, there is increasing evidence showing that extracellular semen components have a significant impact on the immune reaction of the female partner, leading to the tolerogenic response enabling the embryo development and implantation as well as further progress of healthy pregnancy. Seminal plasma glycoproteins are rich in the unique immunomodulatory glycoepitopes that may serve as ligands for endogenous lectins that decorate the surface of immune cells. Such interaction may be involved in modulation of the maternal immune response. Among immunomodulatory glycans, Lewis type antigens have been of interest for at least two decades, while the importance of T/Tn antigens and related structures is still far from understanding. In the current work, we applied two plant lectins capable of distinguishing glycoepitopes with terminal GalNAc and Gal to identify glycoproteins that are their efficient carriers. By means of lectin blotting and lectin affinity chromatography followed by LC-MS, we identified lactotransferrin, prolactin inducible protein as well as fibronectin and semenogelins 1 and 2 as lectin-reactive. Net-O-glycosylation analysis results indicated that the latter three may actually carry T and/or Tn antigens, while in the case of prolactin inducible protein and lactotransferrin LacdiNAc and lactosamine glycoepitopes were more probable. STRING bioinformatics analysis linked the identified glycoproteins in the close network, indicating their involvement in immune (partially innate) processes. Overall, our research revealed potential seminal plasma ligands for endogenous Gal/GalNAc specific lectins with a possible role in modulation of maternal immune response during fertilization.
Topics: Acetylgalactosamine; Female; Fertilization; Galactose; Glycoproteins; Humans; Semen; Seminal Plasma Proteins
PubMed: 34298952
DOI: 10.3390/ijms22147331 -
Journal of Microbiology and... Aug 2022When , a glucose-specific phosphotransferase system (PTS) component, is deleted in , growth can be severely poor because of the lack of efficient glucose transport. We...
When , a glucose-specific phosphotransferase system (PTS) component, is deleted in , growth can be severely poor because of the lack of efficient glucose transport. We discovered a new PTS transport system that could transport glucose through the growth-coupled experimental evolution of -deficient C strain under anaerobic conditions. Genome sequencing revealed mutations in , which encodes a repressor of -acetylgalactosamine (Aga) PTS expression in evolved progeny strains. RT-qPCR analysis showed that the expression of Aga PTS gene increased because of the loss-of-function of . We confirmed the efficient Aga PTS-mediated glucose uptake by genetic complementation and anaerobic fermentation. We discussed the discovery of new glucose transporter in terms of different genetic backgrounds of strains, and the relationship between the pattern of mixed-acids fermentation and glucose transport rate.
Topics: Acetylgalactosamine; Agar; Escherichia coli; Escherichia coli Proteins; Glucose; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphotransferases
PubMed: 35791075
DOI: 10.4014/jmb.2205.05059 -
Organic & Biomolecular Chemistry Nov 2018The study of complex multivalent carbohydrate-protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. In this...
The study of complex multivalent carbohydrate-protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. In this study, we demonstrate that bio-layer interferometry is an excellent complementary method to standard techniques such as SPR and ITC. Using tetra- and hexadecavalent GalNAc glycoconjugates and Helix pomatia agglutinin (HPA) as a model lectin, we were able to measure reliable kinetic and thermodynamic parameters of multivalent interactions going from the micro to the nanomolar range.
Topics: Acetylgalactosamine; Animals; Glycoconjugates; Helix, Snails; Interferometry; Kinetics; Lectins; Thermodynamics
PubMed: 30264842
DOI: 10.1039/c8ob01664j -
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 -
The Journal of Biological Chemistry Apr 2001We have identified and characterized an N-acetylgalactosamine-4-O-sulfotransferase designated GalNAc-4-ST2 (GenBank(TM) accession number ) based on its homology to HNK-1...
Molecular cloning and expression of an N-acetylgalactosamine-4-O-sulfotransferase that transfers sulfate to terminal and non-terminal beta 1,4-linked N-acetylgalactosamine.
We have identified and characterized an N-acetylgalactosamine-4-O-sulfotransferase designated GalNAc-4-ST2 (GenBank(TM) accession number ) based on its homology to HNK-1 sulfotransferase (HNK-1 ST). The cDNA predicts an open reading frame encoding a type II membrane protein of 443 amino acids with a 12-amino acid cytoplasmic domain, a 23-amino acid transmembrane domain, and a 408-amino acid luminal domain containing four potential N-linked glycosylation sites. GalNAc-4-ST2 displays a high degree of amino acid sequence identity with GalNAc-4-ST1 (46%), HNK-1 ST (23%), chondroitin 4-O-sulfotransferase-1 (C4ST-1) (27%), and chondroitin 4-O-sulfotransferase-2 (C4ST-2) (24%). GalNAc-4-ST2 transfers sulfate to the C-4 hydroxyl of terminal beta1,4-linked GalNAc in the sequence GalNAc-beta1,4GlcNAcbeta-R found on N-linked oligosaccharides and nonterminal beta1,4-linked GalNAc in chondroitin and dermatan. The translated region of GalNAc-4-ST2 is encoded by five exons located on human chromosome 18q11.2. Northern blot analysis reveals a 2.1-kilobase transcript. GalNAc-4-ST2 message is most highly expressed in trachea and to a lesser extent in heart, liver, pancreas, salivary gland, and testis. The I.M.A.G.E. cDNA clone 49547 contains a putative GalNAc-4-ST2 splice form with an open reading frame encoding a protein of 358 amino acids that lacks the transmembrane domain and the stem region. This form of GalNAc-4-ST2 is not retained by transfected cells and is active against chondroitin but not terminal beta1,4-linked GalNAc. Thus, as with GalNAc-4-ST1, sequences N-terminal to the catalytic domain contribute to the specificity of GalNAc-4-ST2 toward terminal beta1,4-linked GalNAc.
Topics: Acetylgalactosamine; Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA, Complementary; Humans; Molecular Sequence Data; Sequence Alignment; Sequence Analysis; Sulfates; Sulfotransferases; Transfection
PubMed: 11139592
DOI: 10.1074/jbc.M011560200 -
Nucleic Acids Research Jan 2021Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In...
Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial-both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2'-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.
Topics: 3T3-L1 Cells; Acetylgalactosamine; Animals; Asialoglycoprotein Receptor; Cesium; HEK293 Cells; HeLa Cells; Humans; Kidney; Liver; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron; Myocardium; Oligonucleotides, Antisense; Phosphorothioate Oligonucleotides; Pseudopodia; RNA, Long Noncoding; Spectrometry, Mass, Secondary Ion; Subcellular Fractions; Sulfur; Sulfur Isotopes; Tissue Distribution
PubMed: 33275144
DOI: 10.1093/nar/gkaa1112 -
Biochemistry May 2021The human macrophage galactose lectin (MGL) is an endocytic type II transmembrane receptor expressed on immature monocyte-derived dendritic cells and activated...
The human macrophage galactose lectin (MGL) is an endocytic type II transmembrane receptor expressed on immature monocyte-derived dendritic cells and activated macrophages and plays a role in modulating the immune system in response to infections and cancer. MGL contains an extracellular calcium-dependent (C-type) carbohydrate recognition domain (CRD) that specifically binds terminal acetylgalactosamine glycan residues such as the Tn and sialyl-Tn antigens found on tumor cells, as well as other and glycans displayed on certain viruses and parasites. Even though the glycan specificity of MGL is known and several binding glycoproteins have been identified, the molecular basis for substrate recognition has remained elusive due to the lack of high-resolution structures. Here we present crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactions with the natural ligand, GalNAc, the cancer-associated Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like the asialoglycoprotein receptor, additional calcium atoms are present and contribute to stabilization of the MGL CRD fold. The structure provides the molecular basis for preferential binding of acetylgalactosamine over galactose and prompted the re-evaluation of the binding modes previously proposed in solution. Saturation transfer difference nuclear magnetic resonance data acquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in solution. Models of MGL1 and MGL2, the mouse homologues of MGL, explain how these proteins might recognize Lewis and GalNAc, respectively.
Topics: Acetylgalactosamine; Animals; Antigens, Tumor-Associated, Carbohydrate; Crystallography, X-Ray; Humans; Lectins, C-Type; Ligands; Mice; Protein Binding; Protein Domains
PubMed: 33724805
DOI: 10.1021/acs.biochem.1c00009 -
ACS Chemical Biology Dec 2009The nematode Caenorhabditis elegans is an excellent model organism for studies of glycan dynamics, a goal that requires tools for imaging glycans in vivo. Here we...
The nematode Caenorhabditis elegans is an excellent model organism for studies of glycan dynamics, a goal that requires tools for imaging glycans in vivo. Here we applied the bioorthogonal chemical reporter technique for the molecular imaging of mucin-type O-glycans in live C. elegans. We treated worms with azidosugar variants of N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and N-acetylmannosamine (ManNAc), resulting in the metabolic labeling of their cell-surface glycans with azides. Subsequently, the worms were reacted via copper-free click reaction with fluorophore-conjugated difluorinated cyclooctyne (DIFO) reagents. We identified prominent localization of mucins in the pharynx of all four larval stages, in the adult hermaphrodite pharynx, vulva and anus, and in the tail of the adult male. Using a multicolor, time-resolved imaging strategy, we found that the distribution and dynamics of the glycans varied anatomically and with respect to developmental stage.
Topics: Acetylgalactosamine; Acetylglucosamine; Anal Canal; Animals; Caenorhabditis elegans; Female; Fluorescent Dyes; Hexosamines; Larva; Male; Mucins; Pharynx; Polysaccharides; Tail; Vulva
PubMed: 19954190
DOI: 10.1021/cb900254y -
Methods in Enzymology 2019The majority of proteins excreted by human cells and borne at the cell surface are modified with carbohydrates. Glycoproteins mediate a wide range of processes and adopt...
The majority of proteins excreted by human cells and borne at the cell surface are modified with carbohydrates. Glycoproteins mediate a wide range of processes and adopt fundamental roles in many diseases. The carbohydrates covalently attached to proteins during maturation in the cell directly impact protein structure and function as integral and indispensable components. However, the ability to study the structure of glycoproteins to high resolution was historically limited by technical barriers including a limited availability of appropriate recombinant protein expression platforms, limited methods to generate compositional homogeneity, and difficulties analyzing glycoprotein composition. Furthermore, glycoproteins and in particular the glycan moieties themselves often exhibit a high degree of conformational heterogeneity. Solution NMR spectroscopy is a powerful tool to study biological macromolecules that is capable of characterizing mobile elements of molecules with atomic-level resolution. Methods to express glycoproteins, incorporate stable isotope labels, and analyze glycoproteins have recently opened new avenues to prepare and investigate glycoproteins. These methods are accessible to many laboratories with experience expressing and purifying proteins from prokaryotic expression hosts.
Topics: Acetylgalactosamine; Carbohydrate Sequence; Cell Line; Chromatography, Affinity; Gene Expression; Glycoproteins; Glycosylation; HEK293 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Immunoglobulin Fc Fragments; Immunoglobulin G; Isotope Labeling; Nuclear Magnetic Resonance, Biomolecular; Serine; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Threonine
PubMed: 30611426
DOI: 10.1016/bs.mie.2018.08.021