RESUMEN
Recombinant glycosyltransferases are potential biocatalysts for the construction of a compound library of oligosaccharides, glycosphingolipids, glycopeptides, and various artificial glycoconjugates on the basis of combined chemical and enzymatic synthetic procedures. The structurally defined glycan-related compound library is a key resource both in the basic studies of their functional roles in various biological processes and in the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that the immobilization of extremely unstable membrane-bound glycosyltransferases on some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile separation of products and recycling use of enzymes possible. Until now, however, it seems that no standardized protocol preventing a significant loss of enzyme activity is available due to the lack of a general method of site-selective anchoring between glycosyltransferases and scaffold materials through a stable covalent bond. Here we communicate a versatile and efficient method for the immobilization of recombinant glycosyltransferases onto commercially available solid supports by means of transpeptidase reaction by Staphylococcus aureus sortase A. This protocol allowed for the first time highly specific conjugation at the designated C-terminal signal peptide moiety of recombinant human beta1,4-galactosyltransferase or recombinant Helicobacter pylori alpha1,3-fucosyltransferase with simple aliphatic amino groups displayed on the surface of solid materials. Site-specifically immobilized enzymes exhibited the desired sugar transfer activity, an improved stability, and a practical reusability required for rapid and large-scale synthesis of glycoconjugates. Considering that most mammalian enzymes responsible for the posttranslational modifications, including the protein kinase family, as well as glycosyltransferases are unstable and highly oriented membrane proteins, the merit of our strategy based on "site-specific" transpeptidation is evident because the reaction proceeds only at an engineered C-terminus without any conformational influence around the active sites of both enzymes as well as heptad repeats of rHFucT required to maintain native secondary and quaternary structures during the dimerization on cell surfaces.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Enzimas Inmovilizadas/metabolismo , Glicosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/enzimología , Aminas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Enzimas Inmovilizadas/química , Fucosiltransferasas/química , Fucosiltransferasas/metabolismo , Glicosiltransferasas/química , Helicobacter pylori/enzimología , Humanos , Antígeno Lewis X/biosíntesis , Antígeno Lewis X/química , Proteínas de la Membrana/química , Modelos Moleculares , N-Acetil-Lactosamina Sintasa/química , N-Acetil-Lactosamina Sintasa/metabolismo , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes/química , Sefarosa/química , Sefarosa/metabolismo , Especificidad por SustratoRESUMEN
An efficient protocol for the construction of MUC1-related glycopeptide analogues having complex O-glycan and N-glycan chains was established by integrating chemical and enzymatic approaches on the functional polymer platforms. We demonstrated the feasibility of sortase A-mediated ligation between two glycopeptide segments by tagging with signal peptides, LPKTGLR and GG, at each C- or N-terminal position. Structural analysis of the macromolecular N,O-glycopeptides was performed by means of ESI-TOFMS (MS/MS) equipped with an electron-captured dissociation device. Immunological assay using MUC1 glycopeptides synthesized in this study revealed that N-glycosylation near the antigenic O-glycosylated PDTR motif did not disturb the interaction between the anti-MUC1 monoclonal antibody and this crucial O-glycopeptide moiety. NMR study indicated that the N-terminal immunodominant region [Ala-Pro-Asp-Thr(O-glycan)-Arg] forms an inverse gamma-turn-like structure, while the C-terminal region composed of N-glycopeptide and linker SrtA-peptide was proved to be an independently random structure. These results indicate that the bulky O- and N-glycan chains can function independently as disease-relevant epitopes and ligands for carbohydrate-binding proteins, when both are combined by an artificial intervening peptide having a possible effect of separating N- and C-terminal regions. The present strategy will greatly facilitate rapid synthesis of multiply functionalized complex neoglycopeptides as new types of convenient tools or models for the investigation of thhe structure-function relationship of various glycoproteins and development of novel class glycopeptide-based biopharmaceuticals, drug delivery systems, and biomedical materials.
Asunto(s)
Glicoproteínas/química , Mucina-1/química , Polisacáridos/química , Secuencia de Aminoácidos , Aminoaciltransferasas/química , Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas/química , Unión Competitiva/inmunología , Biocatálisis , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Cisteína Endopeptidasas/química , Glicoproteínas/biosíntesis , Glicoproteínas/síntesis química , Glicoproteínas/inmunología , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Estructura Molecular , Mucina-1/biosíntesis , Mucina-1/inmunología , Polisacáridos/biosíntesis , Polisacáridos/síntesis química , Polisacáridos/inmunología , Staphylococcus aureus/enzimología , Espectrometría de Masas en TándemAsunto(s)
Acetilglucosamina/análogos & derivados , Pared Celular/química , Pared Celular/metabolismo , Lactobacillus plantarum/química , Lactobacillus plantarum/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Ácido Láctico/biosíntesis , Ácido Láctico/química , Estructura MolecularRESUMEN
The general and efficient method for the site-directed glycosylation of proteins is a key step in order to understand the biological importance of the carbohydrate chains of proteins and to control functional roles of the engineered glycoproteins in terms of the development of improved glycoprotein therapeutics. We have developed a novel method for site-directed glycosylation of proteins based on chemoselective blotting of common reducing sugars by genetically encoded proteins. The oxylamino-functionalized L-homoserine residues, 2-amino-4-O-(N-methylaminooxy) butanoic acid and 2-amino-4-aminooxy butanoic acid, were efficiently incorporated into proteins by using the four-base codon/anticodon pair strategy in Escherichia coli in vitro translation. Direct and chemoselective coupling between unmodified simple sugars and N-methylaminooxy group displayed on the engineered streptavidin allowed for the combinatorial synthesis of novel glycoprotein mimetics.
Asunto(s)
Aminoácidos/química , Carbohidratos/química , Glicoproteínas/química , Imitación Molecular , Ingeniería de Proteínas , Western Blotting , Secuencia de Carbohidratos , Cromatografía de Afinidad , Cromatografía en Capa Delgada , Electroforesis en Gel de Poliacrilamida , Glicosilación , Espectrometría de Masas , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
Mono-, di-, and trisialyloligosaccharides were introduced to mutant insulins through enzymatic reactions. Sugar chains were sialylated by alpha2,6-sialyltransferase (alpha2,6-SiaT) via an accessible glutamine residue at the N-terminus of the B-chain attached by transglutaminase (TGase). Sia2,6-di-LacNAc-Ins(B-F1Q) and Sia2,6-tri-LacNAc-Ins(B-F1Q), displaying two and three sialyl-N-acetyllactosamines, respectively, were administered to hyperglycemic mice. Both branched glycoinsulins showed prolonged glucose-lowering effects compared to native or lactose-carrying insulins, showing that sialic acid is important in obtaining a prolonged effect. Sia2,6-tri-LacNAc-Ins(B-F1Q), in particular, induced a significant delay in the recovery of glucose levels.
Asunto(s)
Glicoproteínas/síntesis química , Glicoproteínas/farmacología , Insulina/análogos & derivados , Oligosacáridos/síntesis química , Oligosacáridos/farmacología , Células 3T3-L1 , Secuencia de Aminoácidos , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Glutamina/química , Glutamina/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Humanos , Insulina/síntesis química , Insulina/metabolismo , Insulina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis , Oligosacáridos/química , Conformación Proteica , Receptor de Insulina/metabolismoRESUMEN
4-Fluorinated UDP-MurNAc pentapeptide, 2, has been synthesized. In our previous study, UDP-MurNAc pentapeptide analogue 1 was found to be incorporated into the bacterial cell wall through biosynthesis. Compound 2 showed growth-inhibition activity against Gram-positive bacteria when it was added to growth media at 0.01 mg/mL. [structure--see text]
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Antibacterianos/química , División Celular/efectos de los fármacos , Pared Celular/química , Pared Celular/metabolismo , Estructura Molecular , Oligopéptidos/químicaRESUMEN
Two non-natural fluorinated 2-N-acetamidosugar nucleotides, uridine 5'-diphosphate (UDP) 2-acetamido-2,4-dideoxy-4-fluoro-alpha-D-glucopyranose (UDP-4-FGlcNAc) 1 and its galacto isomer (UDP-4-FGalNAc) 2, were enzymatically constructed by treating chemically synthesized fluorinated 2-N-acetamidosugar 1-phosphates as the donor with UDP 2-acetamido-2-deoxy-alpha-D-glucopyranose pyrophosphorylase in the presence of uridine 5'-triphosphate (UTP).
Asunto(s)
Acetilgalactosamina/síntesis química , Acetilglucosamina/síntesis química , Nucleotidiltransferasas/metabolismo , Uridina Difosfato/síntesis química , Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Conformación de Carbohidratos , Escherichia coli/enzimología , Uridina Difosfato/análogos & derivados , Uridina Difosfato/metabolismo , Uridina Trifosfato/químicaAsunto(s)
Insulina/síntesis química , Ácidos Siálicos/química , Secuencia de Aminoácidos , Animales , Glucemia/efectos de los fármacos , Secuencia de Carbohidratos , Diseño de Fármacos , Humanos , Insulina/química , Insulina/farmacología , Masculino , Ratones , Conformación Molecular , Datos de Secuencia Molecular , Estreptozocina/farmacologíaRESUMEN
UDP-MurNAc-pentapeptide derivative bacterial cell-wall precursors were synthesized as effective tools for surface display on living bacteria. Lactobacilli were incubated in the ketone-modified precursor-containing medium, and the ketone moiety was displayed on the bacterial surface through cell-wall biosynthesis. Oligomannose was coupled with the ketone moiety on the bacterial surface via a aminooxyl linker, thereby displaying this oligosaccharide on the surface of the bacteria. The increase in the adhesion of the sugar-displaying bacteria onto a concanavalin A-attached film compared to that of native bacteria was confirmed by microscopic observation and surface plasmon resonance measurement. The incorporation of the artificial cell-wall precursors was enhanced when incubated with fosfomycin, an inhibitor of cell-wall precursor biosynthesis.
Asunto(s)
Adhesión Bacteriana , Pared Celular/metabolismo , Lactobacillus/metabolismo , Oligosacáridos/biosíntesis , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/metabolismo , Antibacterianos/farmacología , Concanavalina A/farmacología , Fosfomicina/farmacología , Estructura Molecular , Resonancia por Plasmón de Superficie , Uridina Difosfato Ácido N-Acetilmurámico/químicaAsunto(s)
Antivirales/síntesis química , Diseño de Fármacos , Glicopéptidos/síntesis química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Péptidos Cíclicos/síntesis química , Antivirales/química , Antivirales/farmacología , Unión Competitiva , Glicopéptidos/química , Glicopéptidos/farmacología , Pruebas de Hemaglutinación , Ligandos , Modelos Moleculares , Estructura Molecular , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Conformación ProteicaRESUMEN
Cell wall precursors that have been modified at their peptide moiety were incorporated into the living bacterial cell wall. Using chemically synthesized bacterial cell wall precursors, a variety of compounds could be attached to the bacterial surface. Escherichia coli took the modified precursors into the cell wall after EDTA treatment, whereas lactobacilli took the compounds more effectively without EDTA treatment. Microscopic observation showed that the incorporated ketone moiety retained its reactivity. On the basis of this strategy, any compound can be displayed on the bacterial surface. This strategy for bacterial cell surface engineering will open the door for new technologies and therapies utilizing bacteria.
Asunto(s)
Bacterias/metabolismo , Carbohidratos/biosíntesis , Pared Celular/metabolismo , Conformación de Carbohidratos , Carbohidratos/química , Microscopía Fluorescente , Espectrometría de Fluorescencia , Vancomicina/metabolismoRESUMEN
A continuous fluorescence coupled enzyme assay was developed to study the acceptor specificity of the glycosyltransferase MurG toward different lipid I analogues with various substituents replacing the undecaprenyl moiety. It was found that most lipid I analogues are accepted as substrates and, amongst these, the saturated C14 analogue exhibits the best activity. This substrate was used to evaluate the inhibition activity of such antibiotics as moenomycin, vancomycin, and two chlorobiphenyl vancomycin derivatives. A vancomycin derivative with a chlorobiphenyl moiety on the aglycon section was identified as a potent inhibitor of MurG.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Monosacáridos/metabolismo , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/metabolismo , Oligopéptidos/metabolismo , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Bambermicinas/química , Bambermicinas/farmacología , Pared Celular/enzimología , Pared Celular/metabolismo , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Concentración 50 Inhibidora , Metabolismo de los Lípidos , Lípidos/química , Monosacáridos/química , Monosacáridos/farmacología , Oligopéptidos/química , Oligopéptidos/farmacología , Especificidad por Sustrato , Vancomicina/análogos & derivados , Vancomicina/farmacologíaRESUMEN
The cell walls of living bacteria were chemically modified by adding cell-wall precursors. As the precursors to be incorporated into the cell wall, UDP-MurNAc pentapeptide, lipid I, and lipid II derivatives were synthesized. The aimed compounds were attached to the amine residue of lysine at the pentapeptide moiety. Fluorescein-attached UDP-MurNAc pentapeptide was efficiently incorporated into both Gram-positive and Gram-negative bacteria. In the case of Gram-negative bacteria, such as Escherichia coli, the permeability of the outer membrane (lipopolysaccharide layer) was enhanced by EDTA treatment before the incorporation. For Gram-positive bacteria, UDP-MurNAc derivatives were incorporated in the cell wall without EDTA treatment due to the lack of the lipopolysaccharide layer. Furthermore, instead of dyes, a ketone group was attached to the UDP-MurNAc pentapeptide. The ketone group was also delivered to the bacterial cell wall of lactic acid bacteria, giving a platform to attach large molecules on the surface.