Our focus will remain on the structure and function of glycoinositol phospholipids (GPIs) which include glycolipids based on a GlcN-(1 -> 6)- inositol phospholipid core.
The first aim i s to identify, isolate and characterize free glycolipids with GPI structures from mammalian cells. Three approaches to their identification will be pursued: a) biosynthetic labeling of intact cells with GPI components like [3H]glucosamine or [3H]inositol; b) exogenous radiomethylation of glucosamine-containing lipids in cell extracts; and c) in vitro labeling of microsomal lipids with UDP-[3H]GlcNAc or GDP-[3H]mannose. GPIs will be identified by their susceptibility to several enzymatic and chemical fragmentation agents including bacterial phosphatidylinositol-specific phospholipase C (PIPLC); trypanosome GPI-specific PLC (GPI-PLC); serum GPI-specific PLD (GPI-PLD); and nitrous acid deamination. The glycan cleavage products will be isolated and analyzed by ion exchange chromatography to identify characteristic GPI components and to deduce GPI structural variants.
The second aim i s to compare the actions of glycans derived from GPIs with those of insulin on intact cells and cell extracts to test the hypothesis that a glycan product formed by PIPLC cleavage of a GPI mediates many of the metabolic effects of insulin. We will continue to pursue this glycan among the GPIs isolated in aim 1. In an alternative approach, the major glycan phosphate derived from the human erythrocyte (Ehu) acetylcholinesterase (AChE) GPI anchor will be compared to insulin in several intact cell and in vitro assays of metabolic enzymes. Preliminary observations that show similar effects with this glycan and insulin will be extended to examine the effects of other glycan fragments derived from the Ehu AChE GPI anchor. In the third aim we will identify and characterize GPI-specific phospholipases. The phospholipid cofactor requirements of PIPLC for optimal activity will be determined as a model for other phospholipases. We will initiate protocols to obtain x-ray crystallographic structural information about the PIPLC active site. The phospholipid requirements for trypanosome GPI-PLC and serum GPI-PLD also will be studied and applied to a search for other endogenous GPI phospholipases in mammalian cells, including those activated by insulin. We propose the chemical synthesis of a simple GPI in the fourth aim. We will undertake the synthesis of radiolabeled GlcN-(1 -> 6)-inositol phospholipids in which the glycerolipid contains short C7 - C10 acyl or alkyl groups. These short lipid groups will insure rapid diffusion into the cell membrane. These GPIs may be useful as 1) substrates for identifying endogenous GPI-specific phospholipases; 2) precursors for glycan extension either in vivo or in vitro; 3) substrates for identifying GPI inositol acylating and/or deacylating enzymes; and 4) labeled ligands to search for receptors on the cell surface.
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