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Journal of the American Chemical Society Oct 2018Copper deficiency is implicated in a variety of genetic, neurological, cardiovascular, and metabolic diseases. Current approaches for addressing copper deficiency rely...
Copper deficiency is implicated in a variety of genetic, neurological, cardiovascular, and metabolic diseases. Current approaches for addressing copper deficiency rely on generic copper supplementation, which can potentially lead to detrimental off-target metal accumulation in unwanted tissues and subsequently trigger oxidative stress and damage cascades. Here we present a new modular platform for delivering metal ions in a tissue-specific manner and demonstrate liver-targeted copper supplementation as a proof of concept of this strategy. Specifically, we designed and synthesized an N-acetylgalactosamine-functionalized ionophore, Gal-Cu(gtsm), to serve as a copper-carrying "Trojan Horse" that targets liver-localized asialoglycoprotein receptors (ASGPRs) and releases copper only after being taken up by cells, where the reducing intracellular environment triggers copper release from the ionophore. We utilized a combination of bioluminescence imaging and inductively coupled plasma mass spectrometry assays to establish ASGPR-dependent copper accumulation with this reagent in both liver cell culture and mouse models with minimal toxicity. The modular nature of our synthetic approach presages that this platform can be expanded to deliver a broader range of metals to specific cells, tissues, and organs in a more directed manner to treat metal deficiency in disease.
Topics: Acetylgalactosamine; Animals; Asialoglycoprotein Receptor; Copper; Dietary Supplements; Drug Carriers; Drug Delivery Systems; Ionophores; Liver; Mice
PubMed: 30351140
DOI: 10.1021/jacs.8b08014 -
The Journal of Biological Chemistry Mar 2019Biological functions of nuclear proteins are regulated by post-translational modifications (PTMs) that modulate gene expression and cellular physiology. However, the...
Biological functions of nuclear proteins are regulated by post-translational modifications (PTMs) that modulate gene expression and cellular physiology. However, the role of linked glycosylation (-GalNAc) as a PTM of nuclear proteins in the human cell has not been previously reported. Here, we examined in detail the initiation of GalNAc glycan biosynthesis, representing a novel PTM of nuclear proteins in the nucleus of human cells, with an emphasis on HeLa cells. Using soluble nuclear fractions from purified nuclei, enzymatic assays, fluorescence microscopy, affinity chromatography, MS, and FRET analyses, we identified all factors required for biosynthesis of GalNAc glycans in nuclei: the donor substrate (UDP-GalNAc), nuclear polypeptide GalNAc -transferase activity, and a GalNAc transferase (polypeptide GalNAc-T3). Moreover, we identified GalNAc glycosylated proteins in the nucleus and present solid evidence for GalNAc glycan synthesis in this organelle. The demonstration of GalNAc glycosylation of nuclear proteins in mammalian cells reported here has important implications for cell and chemical biology.
Topics: Acetylgalactosamine; Active Transport, Cell Nucleus; Cell Line, Tumor; Cell Nucleus; Glycosylation; Humans; Lamin Type B; N-Acetylgalactosaminyltransferases; Polysaccharides; Polypeptide N-acetylgalactosaminyltransferase
PubMed: 30591584
DOI: 10.1074/jbc.RA118.005524 -
Liver International : Official Journal... Jan 2022Upregulation of hepatic delta-aminolevulinic acid synthase 1 with accumulation of potentially toxic heme precursors delta-aminolevulinic acid and porphobilinogen is... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND & AIMS
Upregulation of hepatic delta-aminolevulinic acid synthase 1 with accumulation of potentially toxic heme precursors delta-aminolevulinic acid and porphobilinogen is fundamental to the pathogenesis of acute hepatic porphyria.
AIMS
evaluate long-term efficacy and safety of givosiran in acute hepatic porphyria.
METHODS
Interim analysis of ongoing ENVISION study (NCT03338816), after all active patients completed their Month 24 visit. Patients with acute hepatic porphyria (≥12 years) with recurrent attacks received givosiran (2.5 mg/kg monthly) (n = 48) or placebo (n = 46) for 6 months (double-blind period); 93 received givosiran (2.5 mg or 1.25 mg/kg monthly) in the open-label extension (continuous givosiran, n = 47/48; placebo crossover, n = 46/46). Endpoints included annualized attack rate, urinary delta-aminolevulinic acid and porphobilinogen levels, hemin use, daily worst pain, quality of life, and adverse events.
RESULTS
Patients receiving continuous givosiran had sustained annualized attack rate reduction (median 1.0 in double-blind period, 0.0 in open-label extension); in placebo crossover patients, median annualized attack rate decreased from 10.7 to 1.4. Median annualized days of hemin use were 0.0 (double-blind period) and 0.0 (open-label extension) for continuous givosiran patients and reduced from 14.98 to 0.71 for placebo crossover patients. Long-term givosiran led to sustained lowering of delta-aminolevulinic acid and porphobilinogen and improvements in daily worst pain and quality of life. Safety findings were consistent with the double-blind period.
CONCLUSIONS
Long-term givosiran has an acceptable safety profile and significantly benefits acute hepatic porphyria patients with recurrent attacks by reducing attack frequency, hemin use, and severity of daily worst pain while improving quality of life.
Topics: Acetylgalactosamine; Humans; Porphyria, Acute Intermittent; Porphyrias, Hepatic; Pyrrolidines; Quality of Life
PubMed: 34717041
DOI: 10.1111/liv.15090 -
Nucleic Acids Research May 2020In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully...
Chimeric siRNAs with chemically modified pentofuranose and hexopyranose nucleotides: altritol-nucleotide (ANA) containing GalNAc-siRNA conjugates: in vitro and in vivo RNAi activity and resistance to 5'-exonuclease.
In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2'-deoxy-2'-fluoro and 2'-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro. Although ANA residues were detrimental at the 5' end of the antisense strand, the siRNAs with ANA at position 6 or 7 in the seed region had activity comparable to the parent. The siRNA with ANA at position 7 in the seed region was active in a mouse model. An Oligonucleotide with ANA at the 5' end was more stable in the presence of 5'-exonuclease than an oligonucleotide of the same sequence and chemical composition without the ANA modification. Modeling studies provide insight into the origins of regiospecific changes in potency of siRNAs and the increased protection against 5'-exonuclease degradation afforded by the ANA modification.
Topics: Acetylgalactosamine; Animals; COS Cells; Carbohydrates; Chlorocebus aethiops; Exoribonucleases; Hepatocytes; Mice; Nucleic Acid Conformation; Prealbumin; RNA Interference; RNA, Small Interfering; Ribonucleotides; Sugar Alcohols
PubMed: 32170309
DOI: 10.1093/nar/gkaa125 -
Bioconjugate Chemistry Jun 2013Dynamic alterations in cell surface glycosylation occur in numerous biological processes that involve cell-cell communication and cell migration. We report here imaging...
Dynamic alterations in cell surface glycosylation occur in numerous biological processes that involve cell-cell communication and cell migration. We report here imaging of cell surface glycosylation in live mice using double click chemistry. Cell surface glycans were metabolically labeled using peracetylated azido-labeled N-acetylgalactosamine and then reacted, in the first click reaction, with either a cyclooctyne, in a Huisgen [3 + 2] cycloaddition, or with a Staudinger phosphine, via Staudinger ligation. The second click reaction was a [4 + 2] inverse electron demand Diels-Alder reaction between a trans-cyclooctene and a tetrazine, where the latter reagent had been fluorescently labeled with a far-red fluorophore. After administration of the fluorescent tetrazine, the bifunctional cyclooctyne-cyclooctene produced significant azido sugar-dependent fluorescence labeling of tumor, kidney, liver, spleen, and small intestine in vivo, where the kidney and tumor could be imaged noninvasively in the live mouse.
Topics: Acetylgalactosamine; Animals; Azides; Click Chemistry; Cyclization; Cyclooctanes; Female; Glycosylation; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Structure; Neoplasms; Polysaccharides; Tumor Cells, Cultured
PubMed: 23642228
DOI: 10.1021/bc300621n -
Journal of Proteome Research Mar 2019C-type lectins are a diverse group of proteins involved in many human physiological and pathological processes. Most C-type lectins are glycan-binding proteins, some of...
C-type lectins are a diverse group of proteins involved in many human physiological and pathological processes. Most C-type lectins are glycan-binding proteins, some of which are pivotal for innate immune responses against pathogens. Other C-type lectins, such as the macrophage galactose-type lectin (MGL), have been shown to induce immunosuppressive responses upon the recognition of aberrant glycosylation on cancer cells. MGL is known to recognize terminal N-acetylgalactosamine (GalNAc), such as the Tn antigen, which is commonly found on malignant cells. Even though this glycan specificity of MGL is well described, there is a lack of understanding of the actual glycoproteins that bind MGL. We present a glycoproteomic workflow for the identification of MGL-binding proteins, which we applied to study MGL ligands on the human Jurkat leukemia cell line. In addition to the known MGL ligands and Tn antigen-carrying proteins CD43 and CD45 on these cells, we have identified a set of novel cell-surface ligands for MGL. Importantly, for several of these, O-glycosylation has hitherto not been described. Altogether, our data provide new insight into the identification and structure of novel MGL ligands that presumably act as modulatory molecules in cancer immune responses.
Topics: Acetylgalactosamine; Antigens, Tumor-Associated, Carbohydrate; Carrier Proteins; Glycoproteins; Glycosylation; Humans; Immunity, Innate; Jurkat Cells; Lectins, C-Type; Leukocyte Common Antigens; Leukosialin; Ligands; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 30582698
DOI: 10.1021/acs.jproteome.8b00796 -
Structural analysis of the Rhizoctonia solani agglutinin reveals a domain-swapping dimeric assembly.The FEBS Journal Apr 2013Rhizoctonia solani agglutinin (RSA) is a 15.5-kDa lectin accumulated in the mycelium and sclerotia of the soil born plant pathogenic fungus R. solani. Although it is...
UNLABELLED
Rhizoctonia solani agglutinin (RSA) is a 15.5-kDa lectin accumulated in the mycelium and sclerotia of the soil born plant pathogenic fungus R. solani. Although it is considered to serve as a storage protein and is implicated in fungal insecticidal activity, its physiological role remains unclear as a result of a lack of any structure/function relationship information. Glycan arrays showed that RSA displays high selectivity towards terminal nonreducing N-acetylgalactosamine residues. We determined the amino acid sequence of RSA and also determined the crystal structures of the free form and the RSA-N-acetylgalactosamine complex at 1.6 and 2.2 Å resolution, respectively. RSA is a homodimer comprised of two monomers adopting the β-trefoil fold. Each monomer accommodates two different carbohydrate-binding sites in an asymmetric way. Despite RSA topology similarities with R-type lectins, the two-monomer assembly involves an N-terminal swap, thus creating a dimer association novel to R-type lectins. Structural characterization of the two carbohydrate-binding sites offers insights on the structural determinants of the RSA carbohydrate specificity.
DATABASE
Structural data have been deposited in the Protein Data Bank database under accession numbers 4G9M and 4G9N.
STRUCTURED DIGITAL ABSTRACT
RSA and RSA bind by x-ray crystallography (View interaction).
Topics: Acetylgalactosamine; Agglutinins; Amino Acid Sequence; Base Sequence; Cloning, Molecular; Dimerization; Molecular Sequence Data; Protein Structure, Tertiary; Rhizoctonia
PubMed: 23402398
DOI: 10.1111/febs.12190 -
Microbes and Infection May 2000The human pathogenic protozoan Entamoeba histolytica is a motile cell polarized into a front pseudopod and a rear uroid. The amoebic Gal/GalNAc surface lectin is a major... (Review)
Review
The human pathogenic protozoan Entamoeba histolytica is a motile cell polarized into a front pseudopod and a rear uroid. The amoebic Gal/GalNAc surface lectin is a major adhesion molecule composed of an immunodominant 170-kDa heavy subunit, mostly extracellular except for a short cytoplasmic tail, and of an extracellular light subunit. The binding of multivalent ligands triggers lectin capping and recruitment to the uroid. The properties of the Gal/GalNAc lectin and its role in amoeba adhesion and uroid polarization are reviewed in the context of the molecular mechanisms underlying cell polarization and locomotion.
Topics: Acetylgalactosamine; Animals; Cell Adhesion; Cell Movement; Cell Polarity; Entamoeba histolytica; Entamoebiasis; Galactose; Humans; Lectins; Protozoan Proteins
PubMed: 10884615
DOI: 10.1016/s1286-4579(00)00361-0 -
Journal of Biochemistry Jul 2003N-Acetylgalactosamine 4-sulfotransferase (GalNAc4ST) transfers sulfate to position 4 of nonreducing terminal GalNAc residues. We previously cloned human GalNAc4ST-1...
Mouse N-acetylgalactosamine 4-sulfotransferases-1 and -2. Molecular cloning, expression, chromosomal mapping and detection of their activity with GalNAcbeta1-4GlcNAcbeta1-octyl.
N-Acetylgalactosamine 4-sulfotransferase (GalNAc4ST) transfers sulfate to position 4 of nonreducing terminal GalNAc residues. We previously cloned human GalNAc4ST-1 cDNA. In this paper, we report the cloning, characterization and chromosomal mapping of mouse GalNAc4ST-1 and GalNAc4ST-2. Mouse GalNAc4ST-1 and GalNAc4ST-2 contain single open reading frames that predict type II transmembrane proteins composed of 417 and 413 amino acid residues, respectively. The amino acid sequence identity between the two isoforms is 49%. When the cDNA was transfected to COS-7 cells, sulfotransferase activities toward carbonic anhydrase VI and GalNAcbeta1-4GlcNAcbeta1-octyl were overexpressed, but the sulfotransferase activity toward chondroitin showed no increase over the control level. Northern blot analysis showed that the 2.4 kb messages of GalNAc4ST-1 and GalNAc4ST-2 were strongly expressed in the kidney, where both of the human isoforms were hardly expressed. Reverse transcription-PCR analysis showed that, unlike human GalNAc4ST-1, the expression of mouse GalNAc4ST-1 in the pituitary gland was only marginal, while that of GalNAc4ST-2 in the pituitary gland was as high as that in the kidney. These results suggest that the functions of the two GalNAc4ST isoforms may differ between human and mouse. By fluorescence in situ hybridization, the GalNAc4ST-1 and GalNAc4ST-2 genes were localized to mouse chromosome 7B3 distal-B5 proximal and chromosome 18A2 distal-B1 proximal, respectively.
Topics: Acetylgalactosamine; Acetylglucosamine; Amino Acid Sequence; Amino Acids; Animals; Base Sequence; COS Cells; Chlorocebus aethiops; Chromosome Mapping; Chromosomes; Cloning, Molecular; DNA, Complementary; Disaccharides; Humans; Isoenzymes; Mice; Molecular Sequence Data; Recombinant Proteins; Sulfates; Sulfotransferases; Tissue Distribution
PubMed: 12944377
DOI: 10.1093/jb/mvg118 -
Toxicological Sciences : An Official... Sep 2022SLN360 is a liver-targeted N-acetyl galactosamine (GalNAc)-conjugated small interfering RNA (siRNA) with a promising profile for addressing lipoprotein (a)-related...
SLN360 is a liver-targeted N-acetyl galactosamine (GalNAc)-conjugated small interfering RNA (siRNA) with a promising profile for addressing lipoprotein (a)-related cardiovascular risk. Here, we describe the findings from key preclinical safety studies. In vitro, SLN360 specifically reduced LPA expression in primary human hepatocytes with no relevant off-target effects. In rats, 10 mg/kg subcutaneous SLN360 was distributed specifically to the liver and kidney (peak 126 or 246 mg/g tissue at 6 h, respectively), with <1% of peak liver levels observed in all other tested organs. In vitro, no genotoxicity and no effect on human Ether-a-go-go Related Gene currents or proinflammatory cytokine production was observed, whereas in vivo, no SLN360-specific antibodies were detected in rabbit serum. In rat and nonhuman primate 29-day toxicology studies, SLN360 was well tolerated at all doses. In both species, known GalNAc-conjugated siRNA-induced microscopic changes were observed in the kidney and liver, with small increases in alanine aminotransferase and alkaline phosphatase observed in the high dose rats. Findings were in line with previously described siRNA-GalNAc platform-related effects and all observations were reversible and considered nonadverse. In cynomolgus monkeys, liver LPA messenger RNA and serum lipoprotein (a) were significantly reduced at day 30 and after an 8-week recovery period. No dose-related changes in safety assessment endpoints were noted. No SLN360-induced cytokine production, complement activation, or micronucleus formation was observed in vivo. The toxicological profile of SLN360 presented here is restricted to known GalNAc siRNA effects and no other toxicity associated with SLN360 has been noted. The preclinical profile of SLN360 confirmed suitability for entry into clinical studies.
Topics: Acetylgalactosamine; Alanine Transaminase; Alkaline Phosphatase; Animals; Cardiovascular Diseases; Cytokines; Ethers; Humans; Lipoprotein(a); Macaca fascicularis; RNA, Messenger; RNA, Small Interfering; Rabbits; Rats
PubMed: 35737426
DOI: 10.1093/toxsci/kfac067