ABSTRACT
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.
Subject(s)
Adhesins, Escherichia coli/chemistry , Adhesins, Escherichia coli/metabolism , Bacterial Adhesion/physiology , Escherichia coli/chemistry , Escherichia coli/metabolism , Protein Conformation , Protein Processing, Post-Translational , Cell Line , Epithelial Cells/microbiology , Glycosylation , Heptoses/metabolism , Humans , Mass Spectrometry , Peptide Hydrolases/metabolism , Peptides/chemistry , Peptides/isolation & purification , Peptides/metabolism , Sequence Analysis, Protein , Serine/chemistry , Threonine/chemistryABSTRACT
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.
Subject(s)
Antineoplastic Agents, Phytogenic/chemical synthesis , Antineoplastic Agents, Phytogenic/pharmacology , Paclitaxel/analogs & derivatives , Paclitaxel/chemical synthesis , Paclitaxel/pharmacology , Taxoids , Animals , Antibiotics, Antineoplastic/pharmacology , Brain Chemistry/drug effects , Breast Neoplasms/drug therapy , Cattle , Docetaxel , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Female , Glycosylation , Humans , In Vitro Techniques , Indicators and Reagents , Tubulin/biosynthesis , Tumor Cells, CulturedABSTRACT
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.
