Identification and Analysis of Glycoinositol Phospholipid Anchors in Membrane Proteins

This chapter highlights the criteria (identification and analysis) to demonstrate the presence of a glycoinositol phospholipid in a protein of interest. Some of the glycoinositol phospholipid anchors identified include trypanosome variant surface glycoproteins (VSG), decay accelerating factor (DAF), Thy-1, and G₂ acetylcholinesterase (AChE). All appear to reside on the extracellular face of the cell plasma membrane. In most cases, the identification has been based on their susceptibility to release from the cell surface by purified bacterial phosphatidylinositol- specific phospholipase C (PIPLC). Cleavage of anchored proteins with exogenous PIPLC has been achieved in a variety of culture media or isotonic buffers. The loss of anchored Thy-1 antigen from the cells is monitored by flow cytometry. In all anchored proteins, the inositol phospholipid is the only nonionic detergent-binding domain. Cleavage of the anchor by PIPLC removes the hydrophobic diacylglycerol or alkylacylglycerol and generates a hydrophilic protein that retains the glycan portion of the anchor. Two procedures most widely employed to demonstrate the loss of detergent-binding capacity following incubation with PIPLC include phase partitioning with Triton X-114 and nondenaturing poly acrylamide gel electrophoresis (PAGE).