Dolichol Research Articles

Candida albicans; exploring glycosylation pathway in the search of targets for antimicrobial agents and yeast to hyphae transition.

Microbial cell wall is mostly synthesized by the glycosylated proteins with the distinct enzymatic activity. In this review we have concentrated on the description of the certain steps of glycosylation and their effect on the cell wall integrity and yeast to hyphae transition, the process enhancing the pathogenic properties of C.albicans. The glycoproteins play an invaluable role in C. albicans virulence and they modulate adhesive, invasive, morphogenetic and immune stimulating properties of the pathogen as well as its susceptibility to the antifungal agents. Therefore, understanding of C. albicans glycobiology might let us expand the arsenal in the war against fungal enemies. The early stages of N-, O-glycans and GPI-anchor synthesis requires dolichol - the lipid carrier of sugar residues. Diminished supply of dolichol causes series of defects in C. albicans cells, among which aberrant protein glycosylation is the most evident. Furthermore, the relations between the cell wall composition and integrity, resistance to some antifungal and cell wall disturbing agents and dolichol dependent glycosylation are observed. Moreover relevance of these reactions for the morphological differentiation of C.albicans is described.

Bacterial Lipid II Analogs: Novel In Vitro Substrates for Mammalian Oligosaccharyl Diphosphodolichol Diphosphatase (DLODP) Activities

Mammalian protein N-glycosylation requires the transfer of an oligosaccharide containing 2 residues of N-acetylglucosamine, 9 residues of mannose and 3 residues of glucose (Glc3Man9 GlcNAc2) from Glc3Man9GlcNAc2-diphospho (PP)-dolichol (DLO) onto proteins in the endoplasmic reticulum (ER). Under some pathophysiological conditions, DLO biosynthesis is perturbed, and truncated DLO is hydrolyzed to yield oligosaccharyl phosphates (OSP) via unidentified mechanisms. DLO diphosphatase activity (DLODP) was described in vitro, but its characterization is hampered by a lack of convenient non-radioactive substrates. Our objective was to develop a fluorescence-based assay for DLO hydrolysis. Using a vancomycin-based solid-phase extraction procedure coupled with thin layer chromatography (TLC) and mass spectrometry, we demonstrate that mouse liver membrane extracts hydrolyze fluorescent bacterial lipid II (LII: GlcNAc-MurNAc(dansyl-pentapeptide)-PP-undecaprenol) to yield GlcNAc-MurNAc(dansyl-pentapeptide)-P (GM5P). GM5P production by solubilized liver microsomal proteins shows similar biochemical characteristics to those reported for human hepatocellular carcinoma HepG2 cell DLODP activity. To conclude, we show, for the first time, hydrolysis of lipid II by a eukaryotic enzyme. As LII and DLO are hydrolyzed by the same, or closely related, enzymes, fluorescent lipid II analogs are convenient non-radioactive substrates for investigating DLODP and DLODP-like activities.

Molecular Dynamics Simulations of Lipid-Linked Oligosaccharide in Membrane Bilayers

Lipid-linked oligosaccharides (LLO) are intermediates in glycosylation in prokaryotes and eukaryotes. An LLO is composed of a lipid molecule joined by diphosphate to a sugar chain. However, the structure, dynamics, and orientation (with respect to bilayers) of LLO within biological membranes have not been explored previously. Using the recent CHARMM carbohydrate and general force fields, we have constructed two types of LLO: one based on the lipid molecule dolichol (DOL), which is found ubiquitously in eukaryotes, and one based on undecaprenol (UND), which is found in bacteria. We have simulated each LLO type in four different pure bilayer types with different hydrophobic thicknesses and saturations: DLPC (dilauroylphosphatidylcholine), DMPC (dimyristoylphosphatidylcholine), DOPC (dioleoylphosphatidylcholine), and SAPC (stearoylarachidonylcholine). The simulation results of each system will be discussed in terms of density distribution of each component along the membrane normal, RMSD and RMSF of oligosaccharides, oligosaccharides’ conformations, DOL and UND conformations, orientations of oligosaccharides with respect to the bilayer normal, and interactions of oligosaccharide with the bilayer.

Effects of hexagonal phase induction by dolichol on phospholipid membrane permeability and morphology

The effect of H 11-phase induction by dolichol (DOL) (C 80–105, 0–2 wt%) within the bilayers of multilamellar liposomes (MLV, DOPE:DOPC 2:1 or 3:1 w/w) on their permeability, lipid mixing and morphology was determined. Low-angle X-ray diffraction patterns were consistent with mixtures of bilaycr and H 11 phases, the latter increasing with increasing DOL or DOPE content and temperature. Efflux rate constants for 6-carboxyfluorescein (6-CF) from 2:1 DOPE:DOPC vesicles depended on temperature and DOPE/DOL content, increasing as much as 200-fold over DOL-free controls at 2% w/w DOL. Fluorescence resonance energy transfer assays detected lipid mixing with unlabeled target MLV. It was appreciable only when target MLV contained DOPE and increased with DOL content. Confocal scanning fluorescence microscopy was applied to study the morphological structure of fully-hydrated samples and field scanning electron microscopy the ultrastructure of cryo-stabilized samples. 3:1 DOPE/DOPC MLV, stable at pH 9.5, underwent rapid morphological changes at pH 7.4. Within minutes filaments formed and large areas of membrane surface became studded with 10–15 nm bumps and 5 nm holes, resembling in size and shape unilamellarly covered intcrlamellar micellar intermediates and interlamellar attachments (ILA) previously associated with H 11-phase transitions. The filaments, seen in MLV with and without DOL, may represent extensions of IMI into coaxial assemblages of rod micellar intermediates (RMI). These phenomena may have implications for liposomal delivery of therapeutic peptides/proteins if they can be made to trigger the convective release of liposomal contents via controlled formation of ILA between adjacent lamellae of MLV.

Phosphorylation and mannosylation of dolichol (C55) in microsomes from hepatocytes preincubated with dolichol

Phosphorylation and mannosylation of dolichol (C55) in microsomes from hepatocytes preincubated with dolichol