Differences between the trypanosomal and human GlcNAc-PI de-N-acetylases of glycosylphosphatidylinositol membrane anchor biosynthesis

De-N-acetylation of N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) is the second step of glycosylphosphatidylinositol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent processing of glucosaminyl-phosphatidylinositol (GlcN-PI) that leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum, In this article, we used a direct de-N-acetylase assay, based on the release of [C-14]acetate from synthetic GlcN[C-14]Ac-PI and analogues thereof, and an indirect assay, based on the mannosylation of GlcNAc-PI analogues, to study the substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells. The HeLa enzyme was found to be more fastidious than the trypanosomal enzyme such that, unlike the trypanosomal enzyme, it was unable to act on a GlcNAc-PI analogue containing 2-O-octyl-D-myo-inositol or on the GlcNAc-PI diastereoisomer containing L-myo-inositol (GlcNAc-P(L)I). These results suggest that selective inhibition of the trypanosomal de-N-acetylase may be possible and that this enzyme should be considered as a possible therapeutic target. The lack of strict stereospecificity of the trypanosomal de-N-acetylase for the D-myo-inositol component was also seen for the trypanosomal GPI alpha-mannosyltransferases when GlcNAc-P(L)I was added to the trypanosome cell-free system, but not when GlcN-P(L)I was used. In an attempt to rationalize these data, we modeled the structure and dynamics of D-GlcNAc alpha 1-6D-myo-inositol-1-HPO4-(sn)-3-glycerol and its diastereoisomer D-GlcNAc alpha 1-6L-myo-inositol-1-HPO4-(sn)-3-glycerol. These studies indicate that the latter compound visits two energy minima, one of which resembles the low-energy conformer of former compound. Thus, it is conceivable that the trypanosomal de-N-acetylase acts on GlcNAc-P(L)I when it occupies a GlcNAc-PI-like conformation and that GlcN-P(L)I emerging from the de-N-acetylase may be channeled to the alpha-mannosyltransferases in this conformation.