RESUMO
The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is one of the few glycosylated proteins found in Escherichia coli. Glycosylation is mediated by a specific heptosyltransferase encoded by the aah gene, but little is known about the role of this modification and the mechanism involved. In this study, we identified several peptides of AIDA-I modified by the addition of heptoses by use of mass spectrometry and N-terminal sequencing of proteolytic fragments of AIDA-I. One threonine and 15 serine residues were identified as bearing heptoses, thus demonstrating for the first time that AIDA-I is O-glycosylated. We observed that unglycosylated AIDA-I is expressed in smaller amounts than its glycosylated counterpart and shows extensive signs of degradation upon heat extraction. We also observed that unglycosylated AIDA-I is more sensitive to proteases and induces important extracytoplasmic stress. Lastly, as was previously shown, we noted that glycosylation is required for AIDA-I to mediate adhesion to cultured epithelial cells, but purified mature AIDA-I fused to GST was found to bind in vitro to cells whether or not it was glycosylated. Taken together, our results suggest that glycosylation is required to ensure a normal conformation of AIDA-I and may be only indirectly necessary for its cell-binding function.
Assuntos
Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/metabolismo , Aderência Bacteriana/fisiologia , Escherichia coli/química , Escherichia coli/metabolismo , Conformação Proteica , Processamento de Proteína Pós-Traducional , Linhagem Celular , Células Epiteliais/microbiologia , Glicosilação , Heptoses/metabolismo , Humanos , Espectrometria de Massas , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Análise de Sequência de Proteína , Serina/química , Treonina/químicaRESUMO
A 7beta-O-glycosylated docetaxel analogue was semi-synthesized from 9-dihydro-13-acetylbaccatin III, the most abundant taxane isolated from the needles of Taxus canadensis. It was shown to be more bioactive than paclitaxel according to the tubulin assay. It had a reduced potency in the MCF7 cell line cytotoxicity assay compared to paclitaxel, but it demonstrated better activity against the drug resistant cell line MCF7-ADR. In addition, the presence of one sugar moiety on C-7 doubled the water solubility versus that of paclitaxel.
Assuntos
Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/farmacologia , Paclitaxel/análogos & derivados , Paclitaxel/síntese química , Paclitaxel/farmacologia , Taxoides , Animais , Antibióticos Antineoplásicos/farmacologia , Química Encefálica/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Bovinos , Docetaxel , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Glicosilação , Humanos , Técnicas In Vitro , Indicadores e Reagentes , Tubulina (Proteína)/biossíntese , Células Tumorais CultivadasRESUMO
Reduction of 5 alpha-hydroxy-7 beta,9 alpha,10 beta-triacetoxy-4(20), 11(12)-taxadien-13-one 1 with activated zinc in glacial acetic acid led to rearranged products, including compounds with double bonds at C3-C4, C10-C11 or with an epoxide at C11-C12. Molecular modeling studies suggested that addition of a side chain at C-20 or C-5 of the taxanes with a C3-C4 double bond might lead to bioactivity. Semi-syntheses and results of bioactivities are discussed.
