The ability of glycosphingolipids (GSLs) to organize into microdomains, i.e.'rafts', in biomembranes is proposed to be a key feature in the lateral organization of many lipid-anchored signaling proteins. The processes by which GSL-enriched domains are formed and maintained are not well defined and may involve specific proteins that can bind and transfer GSLs between membrane surfaces. Our long- range goal is to understand how the structural features of GSLs govern their mixing behavior with other membrane lipids and the impact of GSL organization on the functional regulation of soluble lipid transfer proteins that selectively accelerate glycolipid transfer between membranes (GLTPs). The objective of this application is to directly assess GLTP interaction witty model membranes of differing composition and to determine how GLTP functions mechanistically during the GSL transfer process. The central hypothesis is that the physical environment produced by lipid compositional changes and the resulting changes in lateral organizational state of GSL 'substrates' regulates GLTP translocation on and off the membrane, thereby controlling GLTP activity. The hypothesis has been formulated on the basis of strong preliminary data produced by the applicants. The rationale for the research is that, once it is known how GLTP transfers GSLs between membrane surfaces and how the physical environment and GSL organizational state in the membrane modulates GLTP activity, then this protein can be used in innovative ways to manipulate GSL composition in cell surfaces by introducing new GSL antigens into the cell surface to immunotherapeutically target diseased cells. We are uniquely prepared to undertake the proposed research, because we recently cloned and expressed fully-active GLTP; to our knowledge, this has been accomplished by no one else. The central hypothesis will be tested and the objectives of this application accomplished by pursuing three specific aims:1) Determine how GSL-lipid packing interactions within and across the bilayer regulate GLTP translocation on and off membranes; 2) Ascertain GLTP's functional relationship with two related GLTP-like proteins and identify essential structural features in GLTP by site-directed mutagenesis; and 3) Identify the structural features of GSLs that modulate their mixing interactions with phospholipids and sterols, and define the physical nature of the lamellar environment that is produced by GSL-lipid interactions within and across the bilayer. The proposed work is innovative because it capitalizes on the first-ever cloning and expression of GLTP and the approaches will provide novel insights into 'raft' and caveolar lipid interactions. It is our expectation that the resultant approach will define membrane physiochemical features that regulate the function of GLTP and related GLTP-like proteins. The outcomes will be significant because using and controlling proteins with the ability to alter the glycosphingolipid composition of cell membranes and their microdomains is likely to be of therapeutic value due to the established roles of GSLs in oncogenesis and infectious diseases (HIV, N. gonorrhea, cholera, rotavirus, H. pylori).