Purification and Use of Recombinant Glycosylphosphatidylinositol-Phospholipase C

This chapter describes the assay for glycosylphosphatidylinositol–phospholipase C (GPI–PLC), purification of the recombinant enzyme from Escherichia coli extracts and use of the purified enzyme for analyzing GPIs. E. coli cells expressing GPI–PLC and monoclonal antibodies used in purification are also reviewed. The protozoan parasite Trypanosoma brucei is the causative agent of sleeping sickness (African trypanosomiasis). In the human or animal host, T. brucei is completely covered with about 10⁷ identical copies of a variant surface glycoprotein (VSG). The VSG coat protects underlying membrane structures from host immune defense mechanisms. The VSG is tethered to the plasma membrane by a GPI protein anchor. The GPI–PLC releases dimyristoylglycerol from the GPI anchor, converting VSG from an amphipathic membrane form (mfVSG) to a hydrophilic soluble form (sVSG). The cleaved VSG contains GlcN(αl,6)inositol 1,2-cyclic monophosphate linked to the remaining components of the GPI glycan. The assay for GPI-PLC uses [³H]myristate-labeled VSG as a substrate. The enzyme releases ³H-labeled dimyristoylglycerol, which can be extracted into n-butanol and quantitated in a scintillation counter.