Investigation of the mechanism of transfer of a-tocopherol by the human a-tocopherol transfer protein (H-a-TTP) /

The human a-tocopherol transfer protein (h-a-TTP) is understood to be the entity responsible for the specific retention of a-tocopherol (a-toc) in human tissues over all other forms of vitamin E obtained from the diet. a-Tocopherol is the most biologically active form of vitamin E, and to date has been studied extensively with regard to its antioxidant properties and its role of terminating membrane lipid peroxidation chain reactions. However, information surrounding the distribution of a-tocopherol, specifically its delivery to intracellular membranes by a-TTP, is still unclear and the molecular factors influencing transfer remain elusive. To investigate the mechanism of ligand transfer by the h-a-TTP, a fluorescent analogue of a-toc has been used in the development of a fluorescence resonance energy transfer (FRET) assay. (/?)-2,5,7,8-tetramethyl-2-[9-(7-nitro-benzo[l,2,5]oxdiazol-4-ylamino)-nonyl]- chroman-6-ol (NBD-toc) has allowed for the development of the FRET-based ligand transfer assay. This ligand has been utilized in a series of experiments where changes were made to acceptor lipid membrane concentration and composition, as well as to the ionic strength and viscosity of the buffer medium. Such changes have yielded evidence supporting a collisional mechanism of ligand transfer by a-TTP, and have brought to light a new line of inquiry pertaining to the nature of the forces governing the collisional transfer interaction. Through elucidation of the transfer mechanism type, a deeper understanding of the transfer event and the in vivo fate of a-tocopherol have been obtained. Furthermore, the results presented here allow for a deeper investigation of the forces controlling the collisional protein-membrane interaction and their effect on the transfer of a-toc to membranes. Future investigation in this direction will raise the possibility of a complete understanding of the molecular events surrounding the distribution of a-toc within the cell and to the body's tissues.

Isolation and analysis of a corprinus cinereus DNA fragment showing homology to a fimbrial cDNA from ustilago violacea

Surface proteinaceous fibrils, termed fimbriae, were first identified on gram negative bacteria in the 1940s. Fungal fimbriae, discovered some 25 years later, are found on members of all fungal classes. In the present study, polyclonal antiserum raised against the fimbrial proteins of U. vio/acea were used in order to identify antigenically related proteins from Coprinus cinereus and Schizophy//um commune. Two polypeptides with molecular masses of 37 and 39 kDa from C. cinereus were observed and confirm earlier results. A single previously unidentified 50 kDa polypeptide in S. commune crossreacted with the antiserum. The 50 kDa protein was found to consist of 3 isoforms with isoelectric points ranging from 5.6 to 5.8. A fimbrial cDNA derived from U. vio/acea was used to identify DNA restriction fragments from C. cinereus and S. commune showing homology to the fimbrial transcript of U. vio/acea. Heterologous hybridization with this cDNA was used in order to screen a C. cinereus genomic DNA library. A single clone, A2-3A, with a 14 kbp insert showed strong homology to the pfim3-1 cDNA. The region of homology, a 700 bp Xba I fragment, was subcloned into pUG19. This plasmid was refered to as pXX8. DNA sequence determinations of pXX8 and adjacent fragments from A2-3A suggested that the cloned DNA was a portion of the rONA repeat encoding the small subunit rRNA. DNA sequence analysis of pfim3-1 yielded an incomplete open reading frame. The predicted amino acid sequence codes for a 206 amino acid, 22 kDa polypeptide which contains a domain similar to a transmembrane domain from rat leukocyte antigen, GDS3. As well, an untranslated 576 nucleotide domain showed 81 % homology to pXX8 and 830/0 homology to the 188 rRNA sequence of Ustilago maydis. This sequence was found adjacent to a region of adenine-thymine base pairs presumed to represent the polyadenylation sequence of the fimbrial transcript. The size and extent of homology is sufficient to account for the hybridization of pfim3-1 to rDNA. It is suggested that this domain represents a completely novel regulatory domain within eukaryotes that may enable the observed rapid regeneration of fimbriae in U. violacea.

The effect of lysobisphosphatidic acid (LBPA) on α-tocopherol transfer protein (α-TTP) binding to lipid membranes

The a-tocopherol transfer protein (a-TTP) is responsible for the retention of the atocopherol form of vitamin E in living organisms. The detailed ligand transfer mechanism by a-TTP is still yet to be fully elucidated. To date, studies show that a-TTP transfers a-tocopherol from late endosomes in liver cells to the plasma membrane where it is repackaged into very low density lipoprotein (VLDL) and released into the circulation. Late endosomes have been shown to contain a lipid known as lysobisphosphatidic acid (LBP A) that is unique to this cellular compartment. LBPA plays a role in intracellular trafficking and controlling membrane curvature. Taking these observations into account plus the fact that certain proteins are recruited to membranes based on membrane curvature, the specific aim of this project was to examine the effect of LBP A on a-TTP binding to lipid membranes. To achieve this objective, dual polarization interferometry (DPI) and a vesicle binding assay were employed. Whilst DPI allows protein binding affinity to be measured on a flat lipid surface, the vesicle binding assay determines protein binding affinity to lipid vesicles mimicking curved membranes. DPI analysis revealed that the amount of a-TTP bound to lipid membranes is higher when LBPA is present. Using the vesicle binding assay, a similar result was seen where a greater amount of protein is bound to large unilamellar vesicles (LUV s) containing LBP A. However, the effect of LBP A was attenuated when small unilamellar vesicles (SUVs) were replaced with LUVs. The outcome of this project suggests that aTTP binding to membranes is influenced by membrane curvature, which in turn is induced by the presence of LBP A.

Mechanism of tocopherol transfer by human α-tocopherol transfer protein (α-hTTP)

Vitamin E is a well known fat soluble chain breaking antioxidant. It is a general tenn used to describe a family of eight stereoisomers of tocopherols. Selective retention of a-tocopherol in the human circulation system is regulated by the a -Tocopherol Transfer Protein (a-TIP). Using a fluorescently labelled a-tocopherol (NBD-a-Toc) synthesized in our laboratory, a fluorescence resonance energy transfer (FRET) assay was developed to monitor the kinetics of ligand transfer by a-hTTP in lipid vesicles. Preliminary results implied that NBD-a-Toe simply diffused from 6-His-a-hTTP to acceptor membranes since the kinetics of transfer were not responsive to a variety of conditions tested. After a series of trouble shooting experiments, we identified a minor contaminant, E coli. outer membrane porin F (OmpF) that co-purified with 6-His-a-hTTP from the metal affinity column as the source of the problem. In order to completely avoid OmpF contamination, a GST -a-hTTP fusion protein was purified from a glutathione agarose column followed by an on-column thrombin digestion to remove the GST tag. We then demonstrated that a-hTTP utilizes a collisional mechanism to deliver its ligand. Furthennore, a higher rate of a-tocopherol transfer to small unilamellar vesicles (SUV s) versus large unilamellar vesicles (LUV s) indicated that transfer is sensitive to membrane curvature. These findings suggest that ahTTP mediated a-Toc transfer is dominated by the hydrophobic nature of a-hTTP and the packing density of phospholipid head groups within acceptor membranes. Based on the calculated free energy change (dG) when a protein is transferred from water to the lipid bilayer, a model was generated to predict the orientation of a-hTTP when it interacts with lipid membranes. Guided by this model, several hydrophobic residues expected to penetrate deeply into the bilayer hydrophobic core, were mutated to either aspartate or alanine. Utilizing dual polarization interferometry and size exclusion vesicle binding assays, we identified the key residues for membrane binding to be F 165, F 169 and 1202. In addition, the rates of ligand transfer of the u-TTP mutants were directly correlated to their membrane binding capabilities, indicating that membrane binding was likely the rate limiting step in u-TTP mediated transfer of u-Toc. The propensity of u-TTP for highly curved membrane provides a connection to its colocalization with u-Toc in late endosomes.

Role of Phosphorylation of the Oxysterol Binding Protein (OSBP) in (PI(4)P) and Sterol-Containing Membrane Recognition and Binding

Studies have demonstrated that the oxysterol binding protein (OSBP) acts as a phosphatidylinositol phosphate (PIP)-sterol exchanger at membrane contact sites (MCS) of the endoplasmic reticulum (ER) and Golgi. OSBP is known to pick up phosphatidylinositol-4-phosphate (PI(4)P) from the ER, transfer it to the trans-Golgi in exchange for a cholesterol molecule that is then transferred from the trans-Golgi to the ER. Upon further examination of this pathway by Ridgway et al. (1), it appeared that phosphorylation of OSBP played a role in the localization of OSBP. The dephosphorylation state of OSBP was linked to Golgi localization and the depletion of cholesterol at the ER. To mimic the phosphorylated state of OSBP, the mutant OSBP-S5E was designed by Ridgway et al. (1). The lipid and sterol recognition by wt-OSBP and its phosphomimic mutant OSBP-S5E were investigated using immobilized lipid bilayers and dual polarization interferometry (DPI). DPI is a technique in which the protein binding affinity to immobilized lipid bilayers is measured and the binding behavior is examined through real time. Lipid bilayers containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of PI(4)Ps or sterols (cholesterol or 25-hydroxycholesterol) were immobilized on a silicon nitride chip. It was determined that wt-OSBP binds differently to PI(4)P-containing bilayers compared to OSBP-S5E. The binding behavior suggested that wt-OSBP extracts PI(4)P and the change in the binding behavior, in the case of OSBP-S5E, suggested that the phosphorylation of OSBP may prevent the recognition and/or extraction of PI(4)P. In the presence of sterols, the overall binding behavior of OSBP, regardless of phosphorylation state, was fairly similar. The maximum specific bound mass of OSBP to sterols did not differ as the concentration of sterols increased. However, comparing the maximum specific bound mass of OSBP to cholesterol with oxysterol (25-hydroxycholesterol), OSBP displayed nearly a 2-fold increase in bound mass. With the absence of the wt-OSBP-PI(4)P binding behavior, it can be speculated that the sterols were not extracted. In addition, the binding behavior of OSBP was further tested using a fluorescence based binding assay. Using 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD cholesterol), wt-OSBP a one site binding dissociation constant Kd, of 15 ± 1.4 nM was determined. OSBP-S5E did not bind to 22-NBD cholesterol and Kd value was not obtained.