RESUMEN
The chemical synthesis of human interleukin-2 (IL-2) , having a core 1 sugar, by a ligation method is reported. Although IL-2 is a globular glycoprotein, its C-terminal region, in particular (99-133), is extremely insoluble when synthesized by solid-phase method. To overcome this problem, the side-chain carboxylic acid of the Glu residues was protected by a picolyl ester, thus reversing its polarity from negative to positive. This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28-65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL-2 having full biological activity.
Asunto(s)
Glicoproteínas/química , Interleucina-2/análogos & derivados , Péptidos/química , Humanos , Interleucina-2/síntesis química , Interleucina-2/químicaRESUMEN
Human glycodelin consists of 162 amino acid residues and two N-linked glycans at Asn(28) and Asn(63) . In this study, we synthesized it by a fully convergent strategy using native chemical ligation (NCL) in N to C direction. The four peptide segments corresponding to 1-31, 32-65, 66-105 and 106-162 sequences were synthesized by 9-fluorenylmethoxycarbonyl based solid-phase peptide synthesis. At the C-terminus of the second segment, N-ethyl-S-acetamidomethyl-cysteine was attached as a post-ligation thioesterification device. The N-terminal two segments were condensed by the homocysteine-mediated NCL at Leu-Met site, and the product was methylated to convert homocysteine to methionine. After deprotection of acetamidomethyl group on the N-ethylcysteine residue, the peptide was thioesterified by N-alkylcysteine-assisted method. The product was then ligated with the C-terminal half, which was obtained by the NCL of third and fourth segments, to give the full-length glycodelin.
Asunto(s)
Glicoproteínas/síntesis química , Secuencia de Aminoácidos , Aminoácidos , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Cisteína/química , Esterificación , Fluorenos , Glicodelina , Glicoproteínas/aislamiento & purificación , Humanos , Técnicas de Síntesis en Fase SólidaRESUMEN
A novel GlcNAc-Asn unit carrying trifluoroacetic acid (TFA)-sensitive O-protecting groups was prepared. The unit was used for the solid-phase peptide synthesis (SPPS) of the N-acetylglucosaminylated emmprin (35-69) thioester via one-step deprotection by TFA combined with the N-alkylcysteine thioesterification method. This segment was used for the synthesis of the first Ig domain (22-104) of emmprin carrying GlcNAc by one-pot ligation with other segments using the thioester method. Finally, the sugar chain was elongated by transglycosylation using glycosynthase to give the Ig domain carrying the disialo- and asialo-complex-type sugar chain.
Asunto(s)
Glucosamina/química , Glucosamina/síntesis química , Glicoproteínas/biosíntesis , Glicósido Hidrolasas/metabolismo , Péptidos/química , Péptidos/síntesis química , Glucosamina/análogos & derivados , Glicoproteínas/química , Glicósido Hidrolasas/química , Glicosilación , Estructura MolecularRESUMEN
The complex-type N-linked octasaccharide oxazoline having LacNAc as the nonreducing end sugar was efficiently synthesized using the benzyl-protected LacNAc, mannose, and ß-mannosyl GlcNAc units as key building blocks. To achieve a highly ß-selective glycosylation with the LacNAc unit, the N-trichloroacetyl group was used for the protection of the amino group in the LacNAc unit. After complete assembly of these units and deprotection, the obtained free sugar was successfully derivatized into the corresponding sugar oxazoline. On the other hand, the N-acetylglucosaminylated saposin C, a hydrophobic lipid-binding protein, was chemically synthesized by the native chemical ligation reaction. On the basis of the previous results related to the synthesis of the nonglycosylated saposin C, the O-acyl isopeptide structure was introduced to the N-terminal peptide thioester carrying GlcNAc to improve its solubility toward aqueous organic solvents. The ligation reaction efficiently proceeded with the simultaneous O- to N-acyl shift at the O-acyl isopeptide moiety. After the removal of the cysteine-protecting group and folding, saposin C carrying GlcNAc was successfully obtained. The synthetic sugar oxazoline was then transferred to this glycoprotein using the mutant of endo-ß-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) (glycosynthase), and the saposin C carrying the complex-type nonasaccharide was successfully obtained.
Asunto(s)
Acetilglucosaminidasa/química , Acetilglucosaminidasa/metabolismo , Carbohidratos/química , Glicopéptidos/química , Glicoproteínas/química , Glicoproteínas/síntesis química , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/química , Saposinas/química , Saposinas/síntesis química , Proteínas Portadoras , Glicopéptidos/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Interacciones Hidrofóbicas e Hidrofílicas , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Datos de Secuencia Molecular , Saposinas/metabolismoRESUMEN
Pyrrolysine-tRNA(Pyl) complex is produced by pyrrolysyl-tRNA synthetase (PylRS). In this study, we investigated the substrate specificity of Desulfitobacterium hafnience PylRS. PylRS incorporated various L-lysine derivatives into tRNA(Pyl) in vitro. In addition, the PylRS/tRNA(Pyl) pair introduced these lysine derivatives into the recombinant protein by the Escherichia coli expression system, indicating that this PylRS/tRNA(Pyl) pair can be used in protein engineering technology.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Desulfitobacterium/enzimología , Lisina/análogos & derivados , Aminoacil-ARNt Sintetasas/genética , Escherichia coli/genética , Lisina/química , Lisina/metabolismo , Unión Proteica , Especificidad por SustratoRESUMEN
BACKGROUND: Emmprin, a glycoprotein containing two Ig domains, is enriched on tumor cell surfaces and stimulates matrix metalloproteinase (MMP) production by adjacent stromal cells. Its first Ig domain (ECI) contains the biologically active site. The dependence of emmprin activity on N-glycosylation is controversial. We investigated whether synthetic ECI with the shortest sugar is functionally active. METHODS: The whole ECI peptides carrying sugar chains, a chitobiose unit or N-linked core pentasaccharide, were synthesized by the thioester method and added to fibroblasts to examine whether they stimulate MMP-2 production. RESULTS: ECI carrying a chitobiose unit, ECI-(GlcNAc) 2, but not ECI without a chitobiose unit or the chitobiose unit alone, dose-dependently stimulated MMP-2 production by fibroblasts. ECI with longer chitobiose units, ECI-[(Man)3(GlcNAc)2], also stimulated MMP-2 production, but the extent of its stimulation was lower than that of ECI-(GlcNAc)2. CONCLUSIONS: Our results indicate that ECI can mimic emmprin activity when substituted with chitobiose, the disaccharide with which N-glycosylation starts.
Asunto(s)
Basigina/farmacología , Disacáridos/química , Fibroblastos/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/biosíntesis , Fragmentos de Péptidos/farmacología , Procesamiento Proteico-Postraduccional , Acetilglucosamina/química , Adenocarcinoma/patología , Adenocarcinoma/secundario , Adulto , Basigina/química , Secuencia de Carbohidratos , Células Cultivadas , Neoplasias Cerebelosas/patología , Neoplasias Cerebelosas/secundario , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Fascitis/patología , Fibroblastos/enzimología , Glicosilación , Humanos , Neoplasias Pulmonares , Mananos/química , Metaloproteinasa 2 de la Matriz/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Estructura Terciaria de Proteína , Sarcoma/patología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The biantennary complex-type N-glycans bearing LacNAc and LacdiNAc as the nonreducing end motif were synthesized in a protected form suitable to use in the Fmoc solid-phase peptide synthesis studies. Two approaches for the nonasaccharide synthesis were examined by taking advantage of the highly ß-selective glycosylation with GlcNTCA (N-phenyl)trifluoroacetimidate. An earlier approach, which involved the reaction of the trisaccharide donor (Gal-GlcNTCA-Man) and trisaccharide acceptor (Man-GlcNPhth(2)-N(3)), produced a mixture of nonasaccharide isomers. On the other hand, mannosylation of the trisaccharide acceptor (Man-GlcNPhth(2)-N(3)) stereoselectively afforded the known pentasaccharide (Man(3)-GlcNPhth(2)-N(3)), which was reacted with the disaccharyl glycosyl donor (Gal-GlcNTCA or GalNTCA-GlcNTCA) to produce the desired nonasaccharide as a single stereoisomer. Selective dephthaloylation followed by N-acetylation furnished the GlcNAc(2) functionality. The resulting nonasaccharyl azides were condensed with Fmoc-Asp(OPfp)-OBu(t) or Fmoc-Asp(OPfp)-OPac in the presence of Ph(CH(3))(2)P and HOOBt. Finally, the Zn reduction and cleavage of the tert-butyl ester or Zn reduction alone produced the targeted nonasaccharyl Asn building blocks.
Asunto(s)
Glicopéptidos/síntesis química , Polisacáridos/síntesis química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicopéptidos/química , Datos de Secuencia Molecular , Polisacáridos/química , EstereoisomerismoRESUMEN
In the so-called thioester method for the condensation of peptide segments, protecting groups for amino and thiol groups are required for chemoselective ligation. In this study, we developed a novel thiol protecting group, N-methyl-phenacyloxycarbamidomethyl (Pocam). We used it for protection of cysteine side chains, and synthesized Pocam-containing peptides and peptide thioesters. These were condensed by the thioester method. After the condensation reaction, Pocam groups were cleaved by Zn/AcOH treatment. At the same time, the azido group, which was used for the protection of lysine side chains, was also converted to an amino group, demonstrating that this protecting group strategy simplified the deprotecting reaction after the peptide condensation reaction to only one step.
Asunto(s)
Química Farmacéutica/métodos , Cisteína/química , Indicadores y Reactivos/síntesis química , Lisina/química , Péptidos/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos , Animales , Azidas/química , Cromatografía de Fase Inversa , Humanos , Péptidos/análisis , Zinc/químicaRESUMEN
The synthesis of Fmoc-aminoacyl-N-ethyl-S-triphenylmethylcysteine, an N- to S-acyl migratory device for the preparation of peptide thioesters by Fmoc-SPPS (solid-phase peptide synthesis) is described. Condensation of Fmoc-aminoacyl pentafluorophenyl ester and N-ethyl-S-triphenylmethylcysteine was efficiently performed in the presence of HOOBt (3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine) in DMF. A small amount of diastereomer yielded during the reaction was easily separated by HPLC purification and the highly pure devices were obtained for most of the proteinogenic amino acids.
Asunto(s)
Técnicas de Química Sintética/métodos , Cisteína/análogos & derivados , Péptidos/síntesis química , Compuestos de Sulfhidrilo/síntesis química , Cisteína/síntesis química , Cisteína/química , Estructura Molecular , Péptidos/química , Estereoisomerismo , Compuestos de Sulfhidrilo/químicaRESUMEN
Androgenic gland hormone (AGH) of the woodlouse, Armadillidium vulgare, is a heterodimeric glycopeptide. In this study, we synthesized AGH with a homogeneous N-linked glycan using the expressed protein ligation method. Unexpectedly, disulfide bridge arrangement of a semisynthetic peptide differed from that of a recombinant peptide prepared in a baculovirus expression system, and the semisynthetic peptide showed no biological activity in vivo. To confirm that the loss of biological activity resulted from disulfide bond isomerization, AGH with a GlcNAc moiety was chemically synthesized by the selective disulfide formation. This synthetic AGH showed biological activity in vivo. These results indicate that the native conformation of AGH is not the most thermodynamically stable form, and correct disulfide linkages are important for conferring AGH activity.
Asunto(s)
Hormonas Gonadales/química , Hormonas Gonadales/síntesis química , Animales , Crustáceos , Electroforesis en Gel de Poliacrilamida , Hormonas Gonadales/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Estructura Molecular , TermodinámicaRESUMEN
The thioester method is a peptide condensation reaction, which requires the protection of Lys side chains for chemoselective ligation. We recently found that the azido group could be used as an amino protecting group in the peptide condensation by the thioester method. In this study, we synthesized the glycosylated mouse pro-opiomelanocortin (1-74) by the thioester method. The N-terminal peptide thioester segment, whose Lys side chain was protected by an azido group, was prepared using a 9-fluorenylmethoxycarbonyl (Fmoc) strategy and an N-alkylcysteine (NAC)-assisted thioesterification reaction. The C-terminal azido-glycopeptide segment carrying N- and O-linked glycans was also prepared by the Fmoc chemistry and condensed with the N-terminal segment by the silver ion-free thioester method. These results showed that our azido-based strategy was fully compatible with the NAC-assisted method and glycoprotein synthesis.
Asunto(s)
Glicopéptidos/química , Proopiomelanocortina/síntesis química , Secuencia de Aminoácidos , Animales , Azidas/síntesis química , Azidas/química , Glicopéptidos/síntesis química , Ratones , Datos de Secuencia Molecular , Proopiomelanocortina/química , Compuestos de Sulfhidrilo/síntesis química , Compuestos de Sulfhidrilo/químicaRESUMEN
A 32-year-old woman visited another hospital complaining of productive cough and fever at the beginning of February 2006. Her symptoms improved after the administration of antibiotics, while infiltration shadows on chest radiographs remained unchanged. Bronchoscopic examination revealed stenosis of the left upper division bronchus, while lung biopsy was negative. She was referred to Saga University Hospital for further examination. Bronchoscopy on August 14th showed severe stenosis of the left upper division and lingular bronchi. Her illness was diagnosed as sarcoidosis on the basis of non-caseating granulomas seen in biopsy specimens from the bronchial wall and the periphery of the left upper division bronchus. Pulmonary function test revealed a marked decrease of vital capacity, while the FEV1.0/FVC ratio was 81%. Arrhythmia on electrocardiogram and marked right ventricular enlargement on cardiography were noted. We diagnosed cardiac sarcoidosis on the basis of gallium scintigraphy, thalium scintigraphy and cardiac MRI findings. We report the rare presentation of sarcoidosis with stenosis of proximal airways and marked dilatation of the right ventricle.
Asunto(s)
Enfermedades Bronquiales/patología , Sarcoidosis/patología , Adulto , Dilatación Patológica , Femenino , Ventrículos Cardíacos/patología , HumanosRESUMEN
A unique O-glycan structure, Xylalpha1-3Xylalpha1-3Glcbeta1-O-Ser is found on the consensus sequence C-X-S-X-P-C (X denotes any amino acid) in epidermal growth factor (EGF)-like domains of plasma proteins such as clotting factor VII and IX. One of the enzymes involved in the biosynthesis of this trisaccharide, UDP-d-xylose:beta-d-glucoside 1,3-d-xylosyltransferase has been identified in HepG2 cells (Omichi, K., Aoki, K., Minamida, S., and Hase, S. Eur. J. Biochem. 245, 143-146 [1997]). Here, we report that this enzyme activity can be detected in bovine liver and that the enzyme has been purified from the microsomal fraction. The enzyme was purified 6200-fold in terms of specific activity and ran as a single band on native-PAGE and isoelectric focusing gel electrophoresis. The best acceptor substrate of those tested was the EGF-like domain of bovine factor IX carrying beta-glucoside at Ser53. The Km value for this substrate was 34 muM. Comparison of initial velocity with various acceptor substrates shows that this xylosyltransferase recognizes not only the glucose moiety to which xylose is transferred but also the tertiary structure of the EGF-like domain. With regard to the donor substrate, the enzyme does not recognize UDP-d-glucose but does recognize UDP-d-xylose.
Asunto(s)
Oligosacáridos/metabolismo , Pentosiltransferasa/química , Pentosiltransferasa/aislamiento & purificación , Animales , Bovinos , Factor de Crecimiento Epidérmico , Hígado/enzimología , Pentosiltransferasa/metabolismo , Estructura Terciaria de Proteína , Especificidad por Sustrato , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato Xilosa/metabolismoRESUMEN
A peptide carrying a mercaptomethylated proline derivative at the C-terminus was prepared by solid-phase peptide synthesis (SPPS) and converted to the thioester of 3-mercaptopropionic acid (MPA) by aqueous MPA under microwave irradiation conditions. This post-SPPS thioesterification reaction was successfully applied to the synthesis of a glycopeptide thioester composed of 25 amino acid (AA) residues, which was then used for the preparation of a 61-AA glycopeptide by the thioester condensation method.
Asunto(s)
Ésteres/química , Microondas , Péptidos/síntesis química , Ácido 3-Mercaptopropiónico/química , Cromatografía Líquida de Alta Presión , Glicosilación , Oxidación-Reducción , Péptidos/química , Compuestos de SulfhidriloRESUMEN
[structure: see text] An O-glycosylated peptide carrying two core 2 sialopentasaccharides of different glycoform was synthesized as a model of the heterogeneous surface of mucin glycoprotein. Solid-phase synthesis and the subsequent enzymatic sialylation gave two segments, which were coupled by selective thioester activation to produce the title compound.
Asunto(s)
Glicoproteínas/química , Glicoproteínas/síntesis química , Fragmentos de Péptidos/síntesis química , Sialoglicoproteínas/síntesis química , Antígenos CD/química , Catálisis , Glicoproteínas/análisis , Humanos , Indicadores y Reactivos , Leucosialina , Imitación Molecular , Estructura Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Sialoglicoproteínas/química , Sialiltransferasas/metabolismoRESUMEN
A method to immobilize glycan-linked amino acids with protected α-amino groups, which are key intermediates to produce the desired neoglycoprotein, to a Biacore sensor chip was developed and its utility for interaction analyses was demonstrated. Two types of diN-acetyllactosamine (diLacNAc)-containing glycans, a core 2 hexasaccharide involving linear diLacNAc that is O-linked to N-(9-fluorenyl)methoxycarbonyl (Fmoc)-Thr and a biantennary diLacNAc that is N-linked to Fmoc-Asn, were used as ligands. For immobilization, the free carboxyl groups of the amino acid residues were activated with EDC/NHS, then reacted with the ethylenediamine-derivatized carboxymethyldextran sensor chip to obtain the desired ligand concentrations. Interactions of the ligands with five plant lectins were analyzed by surface plasmon resonance, and the bindings were compared. The resonance unit of each lectin was corrected by subtracting that of the reference cell on which the Fmoc-Thr-core 1 or Fmoc-Asn was immobilized as a ligand. The carbohydrate specificities of interactions were verified by preincubating lectins with their respective inhibitory sugar before injection. By steady state analysis, the Lycopersicon esculentum lectin showed a 27-fold higher affinity to linear diLacNAc than to biantennary diLacNAc, while Datura stramonium and Solanum tuberosum lectins both showed low Ka,apps of 10(6)M(-1) for these two ligands. In contrast, Ricinus communis agglutinin-120 showed a 3.2-fold higher Ka,app to biantennary LacNAc than to linear diLacNAc. A lectin purified from Pleurocybella porrigens mushroom interacted at the high affinity of 10(8)M(-1) with both linear and biantennary diLacNAcs, which identified it as a unique probe. This method provides a useful and sensitive system to analyze interactions by simulating the glycans on the cell surface.