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

Abstract

De-N-acetylation of N-acetylglucosaminyl-phosphatidylino-sitol (GlcNAc-PI) is the second step of glycosylphosphatidylino-sitol (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 [14C]acetate from synthetic GlcN[14C]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 thatselective 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-manno-syltransferases 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-GlcNAcalpha1-6d- myo -inositol-1-HPO4-( sn )-3-glycerol and its diastereoisomer d-GlcNAcalpha1-6l- 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-likeconformation and that GlcN-P(l)I emerging from the de-N-acetylase may be channeled to the alpha-mannosyltransferases in this conformation.

Cloning of Trypanosoma brucei and Leishmania major Genes Encoding the GlcNAc-Phosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis That Is Essential to the African Sleeping Sickness Parasite

“…The TbGPI12 gene has been shown here to be a single-copy gene that is essential for the disease-causing bloodstream form of T. brucei, thus validating this particular enzyme as a potential drug target for the development of therapeutic agents against African sleeping sickness and, possibly, against related parasitic diseases, such as the leishmaniases and Chagas' disease. The genetic validation of GlcNAc-PI de-N-acetylase as an antitrypanosomal target is particularly significant because there has been considerable biochemical characterization of this enzyme (32)(33)(34)(35)(36)(37)(38). Attractive features of this potential drug FIG.…”

“…Tetracycline was added back to one of the cultures after 6 days (C, arrow), leading to rapid recovery of the culture. target include: (i) relatively low identity and similarity (36 and 54%) between the T. brucei and human enzyme peptide sequences; (ii) significant differences between the substrate specificities of the T. brucei and human enzymes (37,38); and (iii) the recent synthesis of two potent (IC 50 , 8 nM) parasite-specific suicide substrate inhibitors of the enzyme (38).…”

“…The GlcNAc-PI de-N-acetylases from protozoan and mammalian sources are similar with regard to their specificities for the acyl (R) group removed from GlcNR-PI substrates (36), but differ with regard to their specificity for the myo-inositol residue. Thus, the trypanosomal enzyme can de-N-acetylate GlcNAc-PI containing either D-or L-myo-inositol and ␣-or ␤-D-GlcNAc, whereas the human (HeLa) enzyme strictly requires ␣-D-GlcNAc (1-6)D-myo-inositol (37,38). These differences, and the ability of the trypanosomal enzyme to tolerate a C8 O-alkyl substituent on C2 of the D-myo-inositol residue, were recently exploited in the design and synthesis of two parasite-specific GlcNAc-PI de-N-acetylase suicide substrate inhibitors (38).…”

Genes for Glycosylphosphatidylinositol Toxin Biosynthesis inPlasmodium falciparum

“…First, GPI biosynthetic pathways are suitable targets for drug development. It was shown that a block in GPI synthesis by disruption of the PIG-B gene makes blood stages of T. brucei nonviable (48), and specific inactivators have been proposed as possible targets for chemotherapy against sleeping sickness (59,61). While GPI synthesis is also important in mammals, especially for embryogenesis (71), mammalian cells in culture have been shown to survive well in culture without it (46).…”

Biosynthesis of glycosylphosphatidylinositols of Plasmodium falciparum in a cell-free incubation system: inositol acylation is needed for mannosylation of glycosylphosphatidylinositols

“…The first steps in the biosynthesis of GPIs lead to the formation of a glucosaminylinositol-phosphate-lipid intermediate by the transfer of Nacetylglucosamine (GlcNAc) from UDP-GlcNAc to an inositol phosphate lipid and the immediate de-N-acetylation of the GlcNAc. By using different chemically synthesized GlcNAcPtdIns analogues in a de-N-acetylase assay, differences in substrate specificities have been established between the trypanosomal enzyme and a mammalian enzyme [9]. In mammalian cells and yeast, intermediates of GPI biosynthesis receive a palmitoyl residue on the inositol ring before the attachment of the first mannose residue of the evolutionarily conserved trimannosyl core glycan, whereas in trypanosomes the first mannosylation step must precede palmitoylation of the inositol ring [10,11].…”