RESUMEN
Conjugation of small interfering RNA (siRNA) to tris N-acetylgalactosamine [(GalNAc)3] can enable highly selective, potent, and durable knockdown of targeted proteins in the liver. However, potential knowledge gaps between in vitro experiments, preclinical species, and clinical scenarios remain. A minimal physiologically based pharmacokinetic-pharmacodynamic model for GalNAc-conjugated siRNA (GalNAc-siRNA) was developed using published data for fitusiran (ALN-AT3), an investigational compound targeting liver antithrombin (AT), to delineate putative determinants governing the whole-body-to-cellular pharmacokinetic (PK) and pharmacodynamic (PD) properties of GalNAc-siRNA and facilitate preclinical-to-clinical translation. The model mathematically linked relevant mechanisms: 1) hepatic biodistribution, 2) tris-GalNAc binding to asialoglycoprotein receptors (ASGPRs) on hepatocytes, 3) ASGPR endocytosis and recycling, 4) endosomal transport and escape of siRNA, 5) cytoplasmic RNA-induced silencing complex (RISC) loading, 6) degradation of target mRNA by bound RISC, and 7) knockdown of protein. Physiologic values for 36 out of 48 model parameters were obtained from the literature. Kinetic parameters governing (GalNAc)3-ASGPR binding and internalization were derived from published studies of uptake in hepatocytes. The proposed model well characterized reported pharmacokinetics, RISC dynamics, and knockdown of AT mRNA and protein by ALN-AT3 in mice. The model bridged multiple PK-PD data sets in preclinical species (mice, rat, monkey) and successfully captured reported plasma pharmacokinetics and AT knockdown in a phase I ascending-dose study. Estimates of in vivo potency were similar (â¼2-fold) across species. Subcutaneous absorption and serum AT degradation rate constants scaled across species by body weight with allometric exponents of -0.29 and -0.22. The proposed mechanistic modeling framework characterizes the unique PK-PD properties of GalNAc-siRNA. SIGNIFICANCE STATEMENT: Tris N-acetylgalactosamine (GalNAc)3-conjugated small interfering RNA (siRNA) therapeutics enable liver-targeted gene therapy and precision medicine. Using a translational and systems-based minimal physiologically based pharmacokinetic-pharmacodynamic (mPBPK-PD) modeling approach, putative determinants influencing GalNAc-conjugated siRNA (GalNAc-siRNA) functionality in three preclinical species and humans were investigated. The developed model successfully integrated and characterized relevant published in vitro-derived biomeasures, mechanistic PK-PD profiles in animals, and observed clinical PK-PD responses for an investigational GalNAc-siRNA (fitusiran). This modeling effort delineates the disposition and liver-targeted pharmacodynamics of GalNAc-siRNA.
Asunto(s)
Acetilgalactosamina/farmacocinética , Silenciador del Gen/fisiología , Modelos Biológicos , ARN Interferente Pequeño/farmacocinética , Acetilgalactosamina/genética , Animales , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Haplorrinos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Ratones , ARN Interferente Pequeño/genética , Ratas , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiologíaRESUMEN
We report here the evaluation of a novel in vitro experimental model, prolonged cultured human hepatocytes (PCHC), as an experimental system to evaluate the potency and duration of effects of oligonucleotide therapeutics. A novel observation was made on the redifferentiation of PCHC upon prolonged culturing based on mRNA profiling of characteristic hepatic differentiation marker genes albumin, transferrin, and transthyretin. Consistent with the known de-differentiation of cultured human hepatocytes, decreases in marker gene expression were observed upon culturing of the hepatocytes for 2 days. A novel observation of re-differentiation was observed on day 7 as demonstrated by an increase in expression of the marker genes to levels similar to that observed on the first day of culture. The expression of the differentiation marker genes was highest on day 7, followed by a gradual decrease but remained higher than that on day 2 for up to the longest culture duration evaluated of 41 days. The redifferentiation phenomenon suggests that PCHC may be useful for the evaluation of the duration of effects of oligonucleotide therapeutics on gene expression in human hepatocytes. A proof of concept study was thereby conducted with PCHC with a GalNAc-conjugated siRNA targeting human hypoxanthine phosphoribosyl transferase1 (HPRT1). HPRT1 mRNA expression in siRNA-treated cultures decreased to 21% of that in untreated hepatocytes on day 1, <10% from days 2 to 12, <20% from days 16 to 33, and eventually recovered to 64% by day 41. Our results suggest that PCHC represent a clinically-relevant cost- and time-efficient experimental tool to aid in the evaluation of GalNAc-siRNA silencing activity, providing information on both efficacy and duration of efficacy. PCHC may be applicable in the drug development setting as a species- and cell type-relevant experimental tool to aid the development of oligonucleotide therapeutics.
Asunto(s)
Acetilgalactosamina/biosíntesis , Técnicas de Cultivo de Célula/métodos , Silenciador del Gen/fisiología , Hepatocitos/metabolismo , Hipoxantina Fosforribosiltransferasa/biosíntesis , ARN Interferente Pequeño/administración & dosificación , Acetilgalactosamina/antagonistas & inhibidores , Acetilgalactosamina/genética , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Silenciador del Gen/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Humanos , Hipoxantina Fosforribosiltransferasa/antagonistas & inhibidores , Hipoxantina Fosforribosiltransferasa/genética , ARN Interferente Pequeño/genéticaRESUMEN
RNA interference (RNAi) is an ancient biological mechanism used to defend against external invasion. It theoretically can silence any disease-related genes in a sequence-specific manner, making small interfering RNA (siRNA) a promising therapeutic modality. After a two-decade journey from its discovery, two approvals of siRNA therapeutics, ONPATTRO® (patisiran) and GIVLAARI™ (givosiran), have been achieved by Alnylam Pharmaceuticals. Reviewing the long-term pharmaceutical history of human beings, siRNA therapy currently has set up an extraordinary milestone, as it has already changed and will continue to change the treatment and management of human diseases. It can be administered quarterly, even twice-yearly, to achieve therapeutic effects, which is not the case for small molecules and antibodies. The drug development process was extremely hard, aiming to surmount complex obstacles, such as how to efficiently and safely deliver siRNAs to desired tissues and cells and how to enhance the performance of siRNAs with respect to their activity, stability, specificity and potential off-target effects. In this review, the evolution of siRNA chemical modifications and their biomedical performance are comprehensively reviewed. All clinically explored and commercialized siRNA delivery platforms, including the GalNAc (N-acetylgalactosamine)-siRNA conjugate, and their fundamental design principles are thoroughly discussed. The latest progress in siRNA therapeutic development is also summarized. This review provides a comprehensive view and roadmap for general readers working in the field.
Asunto(s)
Acetilgalactosamina/análogos & derivados , Enfermedades Genéticas Congénitas/terapia , Pirrolidinas/uso terapéutico , ARN Interferente Pequeño/uso terapéutico , Acetilgalactosamina/genética , Acetilgalactosamina/uso terapéutico , Enfermedades Genéticas Congénitas/genética , Terapia Genética/tendencias , Humanos , Interferencia de ARN , ARN Bicatenario/uso terapéutico , ARN Interferente Pequeño/genéticaRESUMEN
Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-acetylgalactosamine ligand to facilitate delivery to hepatocytes via uptake by the asialoglycoprotein receptors. Revusiran, in development for the treatment of hereditary transthyretin-mediated amyloidosis, was discontinued after an imbalance in deaths in the "ENDEAVOUR" phase 3 clinical trial. Nonclinical safety assessments included safety pharmacology, acute and repeat-dose toxicity, genotoxicity, and carcinogenicity. There were no effects on cardiovascular or respiratory function in monkeys after single doses of up to 100 mg/kg. No neurological effects were noted in monkeys in repeat-dose studies up to 300 mg/kg. Revusiran was well tolerated in repeat-dose mouse (weekly doses) and rat and monkey (five daily doses followed by weekly doses) toxicity studies. The no observed adverse effect level (NOAEL) in rats was 30 mg/kg based on reversible microscopic changes in liver that were accompanied by correlating elevations in clinical chemistry at higher doses. Dose-limiting toxicity was absent in monkeys, and the NOAEL was 200 mg/kg. There was no evidence of genotoxicity in vitro or in vivo at limit doses or carcinogenicity in a 2-year study in rats at doses up to 100 mg/kg. Overall, these results demonstrate that revusiran had a favorable nonclinical safety profile.
Asunto(s)
Acetilgalactosamina/farmacología , Neuropatías Amiloides Familiares/tratamiento farmacológico , ARN Interferente Pequeño/farmacología , Acetilgalactosamina/química , Acetilgalactosamina/genética , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/patología , Animales , Pruebas de Carcinogenicidad , Modelos Animales de Enfermedad , Haplorrinos , Hepatocitos/efectos de los fármacos , Humanos , Ratones , Pruebas de Mutagenicidad , ARN Interferente Pequeño/genéticaRESUMEN
Small interfering RNAs (siRNAs) conjugated to N-acetylgalactosamine (GalNAc) ligands have been used to treat disease in patients. However, conjugates with other ligands deliver siRNA less efficiently, limiting the development of new targeted therapies. Most approaches to enhancing the potency of such conjugates have concentrated on increasing ligand effectiveness and/or the chemical stability of the siRNA drug. One complementary and unexplored alternative is to increase the number of siRNAs delivered per ligand. An ideal system would be a single chemical entity capable of delivering multiple copies of an oligonucleotide drug and/or several such drugs simultaneously. Here we report that siRNAs can be stably linked together under neutral aqueous conditions to form chemically defined siRNA "multimers," and that these multimers can be delivered in vivo by a GalNAc ligand. Conjugates containing multiple copies of the same siRNA showed enhanced activity per unit of ligand, whereas siRNAs targeting different genes linked to a single ligand facilitated multigene silencing in vivo; this is the first demonstration of silencing several genes simultaneously in vivo using ligand-directed multimeric siRNA. Multimeric oligonucleotides represent a powerful and practical new approach to improve intracellular conjugate delivery.
Asunto(s)
Transporte Biológico/genética , Silenciador del Gen , Terapia Genética/tendencias , ARN Interferente Pequeño/uso terapéutico , Acetilgalactosamina/genética , Acetilgalactosamina/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Ligandos , Oligonucleótidos/genética , Oligonucleótidos/uso terapéutico , ARN Bicatenario , ARN Interferente Pequeño/genéticaRESUMEN
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.
Asunto(s)
Glicoproteínas/genética , Lectinas Tipo C/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Acetilgalactosamina/genética , Acetilgalactosamina/metabolismo , Antígenos de Carbohidratos Asociados a Tumores/genética , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Glicoproteínas/inmunología , Glicosilación , Humanos , Inmunidad Innata/genética , Células Jurkat , Lectinas Tipo C/inmunología , Antígenos Comunes de Leucocito/genética , Leucosialina/genética , Ligandos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologíaRESUMEN
We report rapid, potent reversal of GalNAc-siRNA-mediated RNA interference (RNAi) activity in vivo with short, synthetic, high-affinity oligonucleotides complementary to the siRNA guide strand. We found that 9-mers with five locked nucleic acids (LNAs) have the highest potency across several targets. Our modular, sequence-specific approach, named REVERSIR, may enhance the therapeutic profile of any long-acting GalNAc-siRNA (short interfering RNA) conjugate by enabling control of RNAi pharmacology.
Asunto(s)
Silenciador del Gen , ARN Interferente Pequeño/genética , Acetilgalactosamina/genética , Animales , Secuencia de Bases , Biotecnología , Células Cultivadas , Femenino , Hepatocitos/metabolismo , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos/química , Oligonucleótidos/genética , Interferencia de ARN , ARN Interferente Pequeño/químicaRESUMEN
Capsular polysaccharide A (CPSA) is a four-sugar repeating unit polymer found on the surface of the gut symbiont Bacteroides fragilis that has therapeutic potential in animal models of autoimmune disorders. This therapeutic potential has been credited to its zwitterionic character derived from a positively charged N-acetyl-4-aminogalactosamine (AADGal) and a negatively charged 4,6-O-pyruvylated galactose (PyrGal). In this report, using a fluorescent polyisoprenoid chemical probe, the complete enzymatic assembly of the CPSA tetrasaccharide repeat unit is achieved. The proposed pyruvyltransferase, WcfO; galactopyranose mutase, WcfM; and glycosyltransferases, WcfP and WcfN, encoded by the CPSA biosynthesis gene cluster were heterologously expressed and functionally characterized. Pyruvate modification, catalyzed by WcfO, was found to occur on galactose of the polyisoprenoid-linked disaccharide (AADGal-Gal), and did not occur on galactose linked to uridine diphosphate (UDP) or a set of nitrophenyl-galactose analogues. This pyruvate modification was also found to be required for the incorporation of the next sugar in the pathway N-acetylgalactosamine (GalNAc) by the glycosyltransferase WcfP. The pyruvate acetal modification of a galactose has not been previously explored in the context of a polysaccharide biosynthesis pathway, and this work demonstrates the importance of this modification to repeat unit assembly. Upon production of the polyisoprenoid-linked AADGal-PyrGal-GalNAc, the proteins WcfM and WcfN were found to work in concert to form the final tetrasaccharide, where WcfM formed UDP-galactofuranose (Galf) and WcfN transfers Galf to the AADGal-PyrGal-GalNAc. This work demonstrates the first enzymatic assembly of the tetrasaccharide repeat unit of CPSA in a sequential single pot reaction.
Asunto(s)
Bacteroides fragilis/enzimología , Bacteroides fragilis/metabolismo , Vías Biosintéticas , Polisacáridos Bacterianos/metabolismo , Acetilgalactosamina/genética , Acetilgalactosamina/metabolismo , Transferasas de Aldehído-Cetona/genética , Transferasas de Aldehído-Cetona/metabolismo , Animales , Bacteroides fragilis/química , Bacteroides fragilis/genética , Expresión Génica , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Familia de Multigenes , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/genéticaRESUMEN
Human Gb3/CD77 synthase (α1,4-galactosyltransferase) is the only known glycosyltransferase that changes acceptor specificity because of a point mutation. The enzyme, encoded by A4GALT locus, is responsible for biosynthesis of Gal(α1-4)Gal moiety in Gb3 (CD77, Pk antigen) and P1 glycosphingolipids. We showed before that a single nucleotide substitution c.631C > G in the open reading frame of A4GALT, resulting in replacement of glutamine with glutamic acid at position 211 (substitution p. Q211E), broadens the enzyme acceptor specificity, so it can not only attach galactose to another galactose but also to N-acetylgalactosamine. The latter reaction leads to synthesis of NOR antigens, which are glycosphingolipids with terminal Gal(α1-4)GalNAc sequence, never before described in mammals. Because of the apparent importance of position 211 for enzyme activity, we stably transfected the 2102Ep cells with vectors encoding Gb3/CD77 synthase with glutamine substituted by aspartic acid or asparagine, and evaluated the cells by quantitative flow cytometry, high-performance thin-layer chromatography and real-time PCR. We found that cells transfected with vectors encoding Gb3/CD77 synthase with substitutions p. Q211D or p. Q211N did not express Pk, P1 and NOR antigens, suggesting complete loss of enzymatic activity. Thus, amino acid residue at position 211 of Gb3/CD77 synthase is critical for specificity and activity of the enzyme involved in formation of Pk, P1 and NOR antigens. Altogether, this approach affords a new insight into the mechanism of action of the human Gb3/CD77 synthase.
Asunto(s)
Galactosiltransferasas , Glicoesfingolípidos/biosíntesis , Mutación Missense , Acetilgalactosamina/genética , Acetilgalactosamina/metabolismo , Sustitución de Aminoácidos , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Línea Celular Tumoral , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Glicoesfingolípidos/genética , Humanos , Especificidad por SustratoRESUMEN
Polysaccharides are essential and immunologically relevant components of bacterial cell walls. These biomolecules can be found covalently attached to lipids (e.g., O-polysaccharide (PS) contains undecaprenyl and lipopolysaccharide (LPS) contains lipid A) or noncovalently associated with cell wells (e.g., capsular PS (CPS)). Although extensive genetic studies have indicated that the Wzy-dependent biosynthetic pathway is primarily responsible for producing such polysaccharides, in vitro biochemical studies are needed to determine, for example, which gene product is responsible for catalyzing each step in the pathway, and to reveal molecular details about the Wzx translocase, Wzy polymerase and O-PS chain-length determinant. Many of these biochemical studies require access to a structurally well-defined PS repeating unit undecaprenyl pyrophosphate (RU-PP-Und), the key building block in this pathway. We describe herein the chemoenzymatic synthesis of Escherichia coli (serotype O157) RU-PP-Und. This involves (i) chemical synthesis of precursor N-acetyl-D-galactosamine (GalNAc)-PP-Und (2 weeks) and (ii) enzymatic extension of the precursor to produce RU-PP-Und (2 weeks). Undecaprenyl phosphate and peracetylated GalNAc-1-phosphate are prepared from commercially available undecaprenol and peracetylated GalNAc. The chemical coupling of these two products, followed by structural confirmation (mass spectrometry and NMR) and deprotection, generates GalNAc-PP-Und. This compound is then sequentially modified by enzymes in the E. coli serotype O157 (E. coli O157) O-PS biosynthetic pathway. Three glycosyltransferases (GTs) are involved (WbdN, WbdO and WbdP) and they transfer glucose (Glc), L-fucose (L-Fuc) and N-acetylperosamine (PerNAc) onto GalNAc-PP-Und to form the intact RU-PP-Und in a stepwise manner. Final compounds and intermediates are confirmed by mass spectrometry. The procedure can be adapted to the synthesis of analogs with different PS or lipid moieties.
Asunto(s)
Vías Biosintéticas , Escherichia coli/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Polisacáridos Bacterianos/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/genética , Acetilgalactosamina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Familia de Multigenes , Fosfatos de Poliisoprenilo/química , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/genéticaRESUMEN
Enzymatic addition of GalNAc to isotopically labeled IFNα2a produced in Escherichia coli yielded the O-linked glycoprotein GalNAcα-[(13)C,(15)N]IFNα2a. The three-dimensional structure of GalNAcα-IFNα2a has been determined in solution by NMR spectroscopy at high resolution. Proton-nitrogen heteronuclear Overhauser enhancement measurements revealed that the addition of a single monosaccharide unit at Thr-106 significantly slowed motions of the glycosylation loop on the nanosecond time scale. Subsequent addition of a Gal unit produced Gal(ß1,3)GalNAcα-[(13)C,(15)N]IFNα2a. This extension resulted in a further decrease in the dynamics of this loop. The methodology used here allowed the first such description of the structure and dynamics of an O-glycoprotein and opens the way to the study of this class of proteins.
Asunto(s)
Acetilgalactosamina/química , Interferón-alfa/metabolismo , Polisacáridos/química , Treonina/química , Acetilgalactosamina/genética , Biología Computacional/métodos , Disulfuros/química , Escherichia coli/metabolismo , Glicoproteínas/química , Glicosilación , Humanos , Interferón alfa-2 , Interferones/química , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Conformación Molecular , Péptidos/química , Conformación Proteica , Proteínas Recombinantes/metabolismoRESUMEN
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.
Asunto(s)
Acetilgalactosamina/metabolismo , Regulón/fisiología , Shewanella/metabolismo , Acetilgalactosamina/genética , Transporte Biológico Activo/fisiología , Hidrolasas de Éster Carboxílico/biosíntesis , Hidrolasas de Éster Carboxílico/genética , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Shewanella/genéticaRESUMEN
O-Linked glycosylation is a functionally and structurally diverse type of protein modification present in many tissues and across many species. α-Dystroglycan (α-DG), a protein linked to the extracellular matrix, whose glycosylation status is associated with human muscular dystrophies, displays two predominant types of O-glycosylation, O-linked mannose (O-Man) and O-linked N-acetylgalactosamine (O-GalNAc), in its highly conserved mucin-like domain. The O-Man is installed by an enzyme complex present in the endoplasmic reticulum. O-GalNAc modifications are initiated subsequently in the Golgi apparatus by the UDP-GalNAc polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) enzymes. How the presence and position of O-Man influences the action of the ppGalNAc-Ts on α-DG and the distribution of the two forms of glycosylation in this domain is not known. Here, we investigated the interplay between O-Man and the addition of O-GalNAc by examining the activity of the ppGalNAc-Ts on peptides and O-Man-containing glycopeptides mimicking those found in native α-DG. These synthetic glycopeptides emulate intermediate structures, not otherwise readily available from natural sources. Through enzymatic and mass spectrometric methods, we demonstrate that the presence and specific location of O-Man can impact either the regional exclusion or the site of O-GalNAc addition on α-DG, elucidating the factors contributing to the glycosylation patterns observed in vivo. These results provide evidence that one form of glycosylation can influence another form of glycosylation in α-DG and suggest that in the absence of proper O-mannosylation, as is associated with certain forms of muscular dystrophy, aberrant O-GalNAc modifications may occur and could play a role in disease presentation.
Asunto(s)
Acetilgalactosamina/metabolismo , Distroglicanos/metabolismo , Manosa/metabolismo , Complejos Multienzimáticos/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Acetilgalactosamina/genética , Animales , Línea Celular , Distroglicanos/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Glicosilación , Humanos , Manosa/genética , Ratones , Complejos Multienzimáticos/genética , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , N-Acetilgalactosaminiltransferasas/genética , Uridina Difosfato N-Acetilgalactosamina/genéticaRESUMEN
BACKGROUND: The alpha1,3-galactosyl epitope (alpha1-3Gal epitope), a major xenotransplant antigen, is synthesized by alpha1,3-galactosyltransferase (alpha1-3Gal transferase), which is evolutionarily related to the histo-blood group A/B transferases. STUDY DESIGN AND METHODS: We constructed structural chimeras between the human type A and murine alpha1-3Gal transferases and examined their activity and specificity. RESULTS: In many instances, a total loss of transferase activity was observed. Certain areas could be exchanged, with a potential diminishing of activity. With a few constructs, changes in acceptor substrate specificity were suspected. Unexpectedly, a functional conversion from A to B transferase activity was observed after replacing the short sequence of human A transferase with the corresponding sequence from murine alpha1-3Gal transferase. CONCLUSION: Because these two paralogous enzymes differ in 16 positions of the 38 amino acid residues in the replaced region, our finding may suggest that despite separate evolution and diversified acceptors, these glycosyltransferases still share the three-dimensional domain structure that is responsible for their sugar specificity, arguing against the functional requirement of a strong purifying selection playing a role in the evolution of the ABO family of genes.
Asunto(s)
Evolución Molecular , Galactosiltransferasas/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Acetilgalactosamina/genética , Acetilgalactosamina/metabolismo , Animales , Galactosiltransferasas/genética , Células HeLa , Humanos , Ratones , N-Acetilgalactosaminiltransferasas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. Vesicle trafficking events, such as phagocytosis and delivery of plasma membrane proteins, have been implicated in pathogenicity. Rab GTPases are proteins whose primary function is to regulate vesicle trafficking; therefore, understanding the function of Rabs in this organism may provide insight into virulence. E. histolytica possesses a number of unique Rabs that exhibit limited homology to host Rabs. In this study we examined the function of one such Rab, EhRabA, by characterizing a mutant overexpressing a constitutively GTP-bound version of the protein. Overexpression of mutant EhRabA resulted in decreased adhesion to and phagocytosis of human red blood cells and in the appearance of large tubular organelles that could be stained with endoplasmic reticulum (ER)-specific but not Golgi complex-specific antibodies. Consistent with the adhesion defect, two subunits of a cell surface adhesin, the galactose/N-acetylgalactosamine lectin, were mislocalized to the novel organelle. A cysteine protease, EhCP2, was also localized to the ER-like compartment in the mutant; however, the localization of two additional cell surface proteins, Igl and SREHP, remained unchanged in the mutant. The phenotype of the mutant could be recapitulated by treatment with brefeldin A, a cellular toxin that disrupts ER-to-Golgi apparatus vesicle traffic. This suggests that EhRabA influences vesicle trafficking pathways that are also sensitive to brefeldin A. Together, the data indicate that EhRabA directly or indirectly influences the morphology of secretory organelles and regulates trafficking of a subset of secretory proteins in E. histolytica.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Retículo Endoplásmico/enzimología , Entamoeba histolytica/enzimología , Galectinas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/genética , Acetilgalactosamina/metabolismo , Animales , Brefeldino A/farmacología , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/genética , Compartimento Celular/genética , Diferenciación Celular/fisiología , Retículo Endoplásmico/genética , Retículo Endoplásmico/ultraestructura , Entamoeba histolytica/genética , Entamoeba histolytica/ultraestructura , Galectinas/genética , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutación/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas/genética , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
CEL-I is a C-type lectin isolated from the Holothuroidea Cucumaria echinata. This lectin shows very high N-acetylgalactosamine-binding specificity. We constructed an artificial gene encoding recombinant CEL-I (rCEL-I) using a combination of synthetic oligonucleotides, and expressed it in Escherichia coli cells. Since the recombinant protein was obtained as inclusion bodies, the latter were solubilized using urea and 2-mercaptoethanol, and the protein was refolded during the purification and dialysis steps. The purified rCEL-I showed comparable hemagglutinating activity to that of native CEL-I at relatively high Ca(2+)-concentrations, whereas it was weaker at lower Ca(2+)-concentrations due to decreased Ca(2+)-binding affinity. rCEL-I exhibited similar carbohydrate-binding specificity to native CEL-I, including strong GalNAc-binding specificity, as examined by hemagglutination inhibition assay. Comparison of the far UV-CD spectra of recombinant and native CEL-I revealed that the two proteins undergo a similar conformational change upon binding of Ca(2+). Single crystals of rCEL-I were also obtained under the same conditions as those used for the native protein, suggesting that they have similar tertiary structures. Although native CEL-I exhibited strong cytotoxicity toward cultured cells, rCEL-I showed low cytotoxicity. These results indicate that rCEL-I has a tertiary structure and carbohydrate-binding specificity similar to those of native CEL-I. Howeger, there is a subtle difference in the properties between the two proteins probably due to the additional methionine residue at the N-terminus of rCEL-I.
Asunto(s)
Acetilgalactosamina/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Sintéticos , Lectinas Tipo C/biosíntesis , Lectinas Tipo C/química , Acetilgalactosamina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cristalización , Relación Dosis-Respuesta a Droga , Células HeLa , Hemaglutininas/biosíntesis , Hemaglutininas/química , Hemaglutininas/genética , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/fisiología , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/toxicidad , Difracción de Rayos XRESUMEN
The cyst wall of Giardia intestinalis contains proteins and a novel N-acetylgalactosamine (GalNAc) polysaccharide, which is its major constituent. GalNAc is not present in growing trophozoites, but is synthesized during encystment via an inducible pathway of enzymes that produce UDP-GalNAc from fructose 6-phosphate. This report focuses on the regulation of these enzymes and thus the genes for glucosamine 6-phosphate N-acetyltransferase (GNA), phosphoacetylglucosamine mutase (AGM), UDP-N-acetylglucosamine pyrophosphorylase (UAP), and UDP-N-acetylglucosamine 4-epimerase (UAE) were cloned and expressed in Escherichia coli. Each of these expressed enzymes had the predicted activity and was used to generate antibodies. Northern and Western blot analyses demonstrated that both the mRNA and protein levels for all of these enzymes increase during encystment. Nuclear run-on assays of these and the previously analyzed glucosamine 6-phosphate deaminase (GNP; glucosamine 6-P isomerase) showed that all of the genes responsible for UDP-GalNAc synthesis during encystment are induced at the transcription level.
Asunto(s)
Giardia lamblia/enzimología , Giardia lamblia/crecimiento & desarrollo , Polisacáridos/biosíntesis , Acetilgalactosamina/genética , Acetilgalactosamina/aislamiento & purificación , Acetilgalactosamina/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/aislamiento & purificación , Animales , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/aislamiento & purificación , Clonación Molecular , Enzimas/clasificación , Enzimas/genética , Regulación de la Expresión Génica , Giardia lamblia/genética , Giardia lamblia/fisiología , Glucosamina 6-Fosfato N-Acetiltransferasa , Humanos , Datos de Secuencia Molecular , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/aislamiento & purificación , Fosfotransferasas (Fosfomutasas)/genética , Fosfotransferasas (Fosfomutasas)/aislamiento & purificación , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Transcripción GenéticaRESUMEN
Sambucus nigra agglutinin I (SNA-I) is a type 2 ribosome-inactivating protein. Site-directed mutagenesis was used to mimic the conversion of the highly active B-chain of fruit-specific SNA (SNA-If) into the completely inactive B-chain of the closely related and naturally occurring loss-of-activity mutant called S. nigra agglutinin lectin-related protein. In the first mutant SNA-If-M1 the high-affinity site 2 of SNA-If was disrupted by replacing the presumed critical residue Asp231 with Glu231. In the double mutant SNA-If-M2, site 1 of SNA-If-M1 was also disrupted by substituting the presumed critical residue Asn48 with Ser48. The parent type 2 ribosome-inactivating protein and both mutants were expressed in Nicotiana tabacum Samsun NN and the recombinant proteins were purified and analysed. Recombinant SNA-If agglutinated rabbit erythrocytes equally well as SNA-If, but both mutants were completely inactive in this test. Binding assays to immobilized galactose and fetuin revealed that the mutation Asp231-->Glu231 reduces the affinity of the B-chain for galactose and fetuin by more than 50%. Furthermore, the introduction of the second mutation Asn48-->Ser48 reduces the binding activity to less than 20% of the original activity.
Asunto(s)
Acetilgalactosamina/metabolismo , Metabolismo de los Hidratos de Carbono , Lectinas/metabolismo , Ribosomas/metabolismo , Sambucus nigra/metabolismo , Acetilgalactosamina/genética , Secuencia de Aminoácidos , Lectinas/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Lectinas de Plantas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N-acetyl-D-galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6-P isomerase (GPI), the first inducible enzyme unique to this pathway appears, oxygen uptake rates double from non-encysting levels, and metronidazole (MTZ) inhibits oxygen uptake. Within 12 h, GPI and its activity are detectable and OU decreases 50% from non-encysting levels; glucose's stimulation and MTZ's inhibition of oxygen uptake cease. In contrast, aspartate uptake remained constant throughout the 40 h monitored. Two genes, gpi 1 and 2 encode for GPI, but only gpi1 is expressed during encystment. Glucosamine 6-P (GlcN6P), the synthetic product of GPI, activates UDP-N-acetylglucosamine (UDP-GlcNAc) pyrophosphorylase, a downstream enzyme, 3 to 5-fold in the direction of UDP-GlcNAc synthesis. UDP-GlcNAc is epimerized to UDP-GalNAc and UDP-GalNAc is polymerized by "cyst wall synthase" (beta 1 --> 3 GalNAc transferase) into a highly insoluble beta 1,3-linked homopolymer. This GalNAc polysaccharide, the major component of cyst wall filaments, forms, in conjunction with polypeptides, the outer cyst wall of Giardia.
Asunto(s)
Acetilgalactosamina/metabolismo , Isomerasas Aldosa-Cetosa/genética , Regulación Enzimológica de la Expresión Génica , Giardia lamblia/crecimiento & desarrollo , Polisacáridos/metabolismo , Acetilgalactosamina/genética , Isomerasas Aldosa-Cetosa/metabolismo , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Genes Protozoarios , Giardia lamblia/genética , Giardia lamblia/metabolismo , Giardiasis/parasitología , Humanos , Datos de Secuencia MolecularRESUMEN
Using differential mRNA display to uncover potential mediators associated with chronic rejection, we identified a cDNA fragment induced in Lewis to F344 rat cardiac allografts with arteriosclerosis but not Lewis syngrafts. The full-length cDNA (1.4 kb) isolated from a rat cardiac allograft cDNA library was 99% identical to galactose/N-acetylgalactosamine (Gal/GalNAc) macrophage lectin, a cell-surface receptor. This cDNA hybridized in Northern analysis with total RNA from eight cardiac allografts but not with host hearts, syngrafts, or other organs. There was a significant allograft-specific increase in transcript levels measured by reverse transcriptase PCR at days 7, 14, 28, and 75 in comparison with paired F344 host hearts (subject to same circulation but histologically normal), day-0 hearts, and syngrafts (P < 0.008, n = 4 at each time). Transcript levels in cardiac allografts were higher than those in paired host spleens (a major source of inflammatory cells) (P < 0.0001), indicating the localized nature of Gal/GalNAc lectin induction. By in situ hybridization and immunostaining, Gal/GalNAc lectin expression localized to a subset of inflammatory cells in cardiac allografts. These findings link Gal/GalNAc macrophage lectin to the chronic rejection process, as a possible mediator of macrophage infiltration.