RESUMO
The human erythrocyte glucose transporter is a fully integrated membrane glycoprotein having only one N-linked carbohydrate chain on the extracellular part of the molecule. Several authors have suggested the involvement of the carbohydrate moiety in glucose transport, but not definitive results have been published to date. Using transport glycoproteins reconstituted in proteoliposomes, kinetic studies of zero-trans influx were performed before and after N-glycanase treatment of the proteoliposomes: this enzymatic treatment results in a 50% decrease of the Vmax. The orientation of transport glycoproteins in the lipid bilayer of liposomes was investigated and it appears that about half of the reconstituted transporter molecules are oriented properly. Finally, it could be concluded that the release of the carbohydrate moiety from the transport glycoproteins leads to the loss of their transport activity.
Assuntos
Glicemia/metabolismo , Eritrócitos/metabolismo , Proteínas de Transporte de Monossacarídeos/sangue , Amidoidrolases/metabolismo , Transporte Biológico , Glicosilação , Humanos , Cinética , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Proteínas de Transporte de Monossacarídeos/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Relação Estrutura-AtividadeRESUMO
A series of double-tailed hydrocarbon and/or fluorocarbon glycolipids derived from galactose and glucose have been prepared. These compounds were obtained upon opening a lactono- and maltonolactone moiety by the amino group of either a glycine, glycylglycine or lysine residue. The carboxyl terminus of the glycyl and glycylglycine conjugates was further reacted with the appropriate double-tailed amine. In the case of lysine, the lactonamide conjugate was functionalized with a hydrocarbon and/or fluorocarbon fatty amine and acid, respectively. The ability of such glycolipids to disperse in water, the morphology of self-assemblies formed and the stability of the supramolecular structure obtained were shown to depend on the presence or absence and on the nature of the aminoacid spacer. Most of the compounds described were shown by conventional techniques (TEM, Cryo-TEM, LLS, etc.) to produce stable vesicular systems.
Assuntos
Materiais Biocompatíveis/química , Fluorocarbonos/análise , Glicolipídeos/química , Hidrocarbonetos/análise , Animais , Varredura Diferencial de Calorimetria , Sequência de Carboidratos , Células Cultivadas , Técnica de Fratura por Congelamento , Galactose , Glucose , Lipossomos , Camundongos , Microscopia Eletrônica , Dados de Sequência Molecular , Tensoativos , TermodinâmicaRESUMO
Natural-abundance 13C nuclear magnetic resonance (13C-n.m.r.) was used to study the effect of monoglycosylation on the structure and dynamics of a pentapeptide related to the N-terminus of glycophorin AM. The results of this study indicate that a single point of glycosylation, on the pentapeptide, can significantly affect its structure. Moreover, glycosylation of this pentapeptide also affects its dynamic motion in solution. This study further defines the role that the carbohydrate residue plays in determining the structure about the N-terminus of glycophorin AM.
Assuntos
Glicoforinas , Oligopeptídeos/síntese química , Sialoglicoproteínas , Sequência de Carboidratos , Glicopeptídeos , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Espectroscopia de Ressonância MagnéticaRESUMO
The N-terminus of glycophorin A, the main transmembrane erythrocyte glycoprotein responsible for the MN blood-group specificity, has been modelled. As the minimum size of the protein recognised by the antiglycophorin A antibodies is the N-terminal glycopentapeptide, attention was focused on the TN and T antigenic determinants of this size in order to determine wether differences in 3D structure exist and how a specific response with different antibodies is induced.
Assuntos
Glicoforinas/química , Sequência de Aminoácidos , Configuração de Carboidratos , Epitopos/química , Glicoforinas/imunologia , Humanos , Imunoquímica , Sistema do Grupo Sanguíneo MNSs/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Conformação ProteicaRESUMO
13C-N.m.r. spectral data as well as spin-lattice relaxation times (T1 values) are presented for the core glycopeptides beta-D-Gal-(1----3)-alpha-D-GalNAc----Ser, Thr. The binding of Gd3+ to these model compounds containing N-terminal blocking groups and esterified carboxyl groups indicates that the disaccharide contains a rather weak, but unique, binding-site in the vicinity of C-2 of alpha-D-GalNAc (possibly involving N-2', the acetamido carbonyl group, O-3' and/or possibly the glycosidic oxygen atom (O-3)).
Assuntos
Gadolínio/metabolismo , Glicopeptídeos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Rotação Ocular , Ligação ProteicaRESUMO
The reaction of L-serine methyl ester hydrochloride (1) with paraformaldehyde (2) in dichloromethane in the presence of triethylamine afforded a novel compound: [lS,2S,6S,7S]-1,6-diaza-4,9-dioxa-2,7-dimethoxycarbonylbicyclo[4.4.1]undecane (4) as a 2:3 adduct of 1 with 2. 1H and 13C NMR spectroscopy were unable to discriminate between two possible symmetrical structures. The latter was unambiguously proved by X-ray crystallography. The crystal structure established: (i) the existence of two identical seven-membered rings each containing a N-C-O grouping; (ii) the existence of a long C-O-C-N-C-N-C-O-C sequence in which each nitrogen belongs simultaneously to a N-C-O (oxazolidine) and to a N-C-N (aminal) motifs; (iii) the existence of a chair-like conformation for both seven-membered rings; (iv) the antiperiplanar geometry of pN-C-O and consequently the manifestation of a strong anomeric effect in both N-C-O groupings, whereas anomeric effect was virtually absent in the N-C-N sequence, as corroborated by bond distances and bond angles. Chemical shifts, coupling constants and NOE effects confirm that the conformational features of 4 are preserved in solution.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Ácidos Dicarboxílicos/química , Formaldeído/química , Serina/química , Cristalografia por Raios X , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Serina/análogos & derivados , Soluções , EstereoisomerismoRESUMO
Tris(hydroxymethyl)aminomethane (Tris) can react with benzaldehyde (1:2 molar ratio) to produce cis-2,8-diphenyl-5-hydroxymethyl-1-aza-3,7-dioxabicyclo[3.3.0]octa ne, the structure of which has been confirmed by nuclear magnetic resonance spectroscopy and X-ray crystallography. The crystal structure showed that both oxazolidine rings A and B are puckered in opposite directions. Ring A exists in an E3 envelope form with 0-3 noticeably down (0.65 A) the plane of the remaining atoms, whereas ring B adopts the 7E envelope conformation with the 0-7 atom displaced up from the mean reference plane by 0.70 A. Comparison of bond angles and bond distances showed that both oxazolidine rings A and B exhibit cross endo-anomeric effects resulting from electron delocalization over the bond sequence O-3-C-2-N-1-C-8-O-7.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Oxazóis/química , Configuração de Carboidratos , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Estereoisomerismo , TermodinâmicaRESUMO
Natural-abundance, 13C-n.m.r. spectroscopy was used to study the mode of binding of Gd3+ to mono-O-glycosylated L-serine and tripeptides variously composed of Gly and L-Thr. When the amino and carboxyl groups of the amino acid are not blocked, strong interaction of Gd3+ with them is observed; this is also readily apparent with some related, nonglycosylated peptides. When the amino and carboxyl groups of the amino acid are blocked, noticeable interaction of Gd3+ with the glycosidic oxygen atom (O-3) and O-2' for the glycopeptide containing alpha-D-Galp, and with O-3 and N-2' for the glycopeptide containing alpha-D-GalpNAc, is observed. Weak interactions are also possible with O-4' and O-6' of the glycosyl groups. Although the amino acids were protected, these metal ion-carbohydrate interactions may still be mediated, to some extent, by the acetyl protecting the amino group and by the ester group on the amino acid.
Assuntos
Gadolínio , Glicopeptídeos , Glicopeptídeos/síntese química , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Relação Estrutura-AtividadeAssuntos
Aminoácidos/análise , Carboidratos/análise , Glicoproteínas/análise , Oligossacarídeos/análise , Configuração de Carboidratos , Sequência de Carboidratos , Isótopos de Carbono , Fenômenos Químicos , Química , Glicopeptídeos/análise , Glicosídeos/análise , Espectroscopia de Ressonância Magnética/métodosRESUMO
The human Ss blood group antigens are located on glycophorin B, a minor human erythrocyte membrane glycoprotein. The structural difference in Ss antigens is determined by a Met/Thr polymorphism at position 29. This report describes the first synthesis of the two peptides carrying the Ss specificities, SS: Asn-Gly-Glu-Met-Gly-Gln-Leu-Val and ss: Asn-Gly-Glu-Thr-Gly-Gln-Leu-Val.
Assuntos
Antígenos de Grupos Sanguíneos , Glicoforinas , Oligopeptídeos , Peptídeos/síntese química , Sialoglicoproteínas , Sequência de Aminoácidos , Humanos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Rotação Ocular , Relação Estrutura-AtividadeRESUMO
Three 2-acetamido-2-deoxy-alpha-D-galactopyranoses attached to Ser2, Thr3 and Thr4 of the amino-terminal portion of glycophorin AM are responsible for the so-called TN blood group specificity. The corresponding glycopeptide H2N-Ser-Ser*-Thr*-Gly-OH obtained by a stepwise peptide coupling strategy was submitted to a detailed high-field nuclear magnetic resonance (n.m.r.) analysis. 13C-n.m.r. spectrum confirms the validity of previous assignments made on M sialo and asialoglycopeptides obtained by specific degradation of human glycophorin AM. In addition, the 400 MHz 1H-n.m.r. spectrum allowed most of the proton resonances to be assigned. A careful examination of the chemical shifts and coupling constants revealed some interesting features of the conformational properties of the GalNAc-Ser and GalNAc-Thr linkage as well as of the rotational isomerism of Thr and Ser side-chains. The data give conclusive evidence that high-field n.m.r. spectroscopy can be successfully used to gain structural and dynamic information on rather sophisticated glycopeptides.
Assuntos
Antígenos de Grupos Sanguíneos , Glicopeptídeos/síntese química , Glicoforinas , Sialoglicoproteínas , Sequência de Carboidratos , Humanos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Serina , TreoninaRESUMO
Three 2-acetamido-2-deoxy-alpha-D-galactopyranose residues attached to Ser2, Thr3 and Thr4 of the amino-terminal portion of glycophorin AM are responsible for the so-called TN blood group specificity. In a continuation of earlier work, this report describes the first chemical synthesis of the triglycosylated pentapeptide H2N-Ser1-Ser2*-Thr3*-Thr4*-Gly5-OH, in which (*) represents the 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residue. This compound constitutes the G1-G5 sequence of the amino-terminal portion of human glycophorin AM, the main erythrocyte membrane glycoprotein. The above compound was obtained by a stepwise peptide coupling strategy alternatively using aminoacids and adequately protected and/or activated O-glycosyl-aminoacids. Since the desired sequence possesses both unglycosylated and glycosylated serine this route was preferred to that in which the glycosylation is carried out on the preformed pentapeptide H2N-Ser-Ser-Thr-Thr-Gly-OH. Carbohydrate residues were introduced into the sequence as 2-azido-2-deoxy-alpha-D-galactopyranosyl-L-serine and L-threonine derivatives. The azido functions were further converted into the corresponding acetamido groups by treatment of the final triglyco-pentapeptide with sodium borohydride in the presence of nickel chloride followed by acetylation.
Assuntos
Antígenos de Grupos Sanguíneos , Glicopeptídeos/síntese química , Glicoforinas , Sialoglicoproteínas , Sequência de Aminoácidos , Sequência de Carboidratos , Humanos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Rotação OcularRESUMO
The Mg antigen is a well known rare mutation of the MN blood group system. The amino-terminal pentapeptide related to human glycophorin AMg, Leu-Ser-Thr-Asn-Glu, as well as pentapeptides representing the peptide backbone of glycophorin AM, AN and AMc and other analogs, were synthesized to serve both as glycosyl transferase acceptors and as artificial antigens. These compounds were obtained by a stepwise peptide coupling strategy in solution.
Assuntos
Glicoforinas/imunologia , Sistema do Grupo Sanguíneo MNSs , Oligopeptídeos/síntese química , Sialoglicoproteínas/imunologia , Sequência de Aminoácidos , Epitopos , HumanosRESUMO
In this paper, we report the solid-phase synthesis of peptides containing O-phosphonoserine using BOP as coupling reagent. Commercially available Fmoc amino-acids linked to p-alkoxybenzyl resin were used in the first step and Alloc amino acids in the following. Alloc group was removed by catalytic hydrostannolytic cleavage. Acid-labile side-chain protecting groups (including phosphate residue) were used. Thus, both removal of side-chain protecting groups and cleavage of the phosphopeptide from the resin were achieved in one step by treatment with TFA. Alloc serine was phosphorylated by the phosphoramidite method. This strategy enables the preparation of peptides with selectively phosphorylated residue and overcomes problems due to repetitive treatments with TFA and final cleavage with HF.
Assuntos
Fluorenos , Peptídeos/síntese química , Fosfosserina , Aminoácidos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Métodos , Compostos Organofosforados , Peptídeos/química , Fosforilação , Ácido TrifluoracéticoRESUMO
Several phosphoserine, phosphothreonine and phosphotyrosine synthons suitable for the stepwise synthesis of phosphopeptides were prepared. Treatment of methylthiomethyl (MTM) esters of either Z-, Boc-, Allocserine and threonine with phosphochloridate in pyridine followed by MgBr2 cleavage of MTM in diethyl ether afforded the title compounds in good yield. Thiophosphoserine and phosphotyrosine synthons were also obtained by the phosphoramidite method using di-(2,2,2-trichloroethyl)-N,N-diisopropylphosphoramidite and MCPBA as oxidizing reagent. Trichloroethyl proved valuable as phosphate protecting group especially in phosphotyrosine derivatives owing to its stability in acidic conditions. These synthons were involved in the liquid-phase synthesis of several phospho and/or thiophosphopeptides related to either src-protein kinase or rat liver pyruvate kinase.
Assuntos
Organotiofosfatos/síntese química , Fosfopeptídeos/síntese química , Fosfosserina/química , Fosfotreonina/química , Proteínas Tirosina Quinases/síntese química , Tirosina/análogos & derivados , Sequência de Aminoácidos , Proteína Tirosina Quinase CSK , Dados de Sequência Molecular , Fosforilação , Fosfotirosina , Tirosina/química , Quinases da Família srcRESUMO
One- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy (at 11.7 Tesla) was used to gain some structural and spectral information about glycophorin AM, glycophorin AM tryptic glycopeptide, a related pentapeptide, and two related monoglycosylated pentapeptides. The protein spectral information suggests that the highly glycosylated N-terminus of glycophorin does not seem to possess a unique tertiary structure. Furthermore, the spectral information provided by the carbohydrate residues also indicates that there is no strong carbohydrate-protein interaction resulting in a unique tertiary structure. This result does not preclude any unique protein-carbohydrate interactions. For the small monoglycosylated pentapeptide containing alpha-D-GalNAc attached to Thr, a unique NOESY cross-peak was observed between the anomeric proton and the beta-proton of Thr. A cross-peak between the beta-proton of Ser and the anomeric proton was not observed for a related monoglycosylated pentapeptide containing alpha-D-GalNAc O-linked to Ser.