Substrate specificity of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol membrane anchor biosynthesis in African trypanosomes and human cells

Abstract

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 mannosylation of glucosaminyl-phosphatidylinositol (GlcN-PI) which leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. The substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells were studied with respect to the N-acyl groups (R) that could be removed from a series of GlcNR-PI substrates, where R = acetyl (Ac), propionyl (Pr), butyryl (Bu), isobutyryl (iBu), pentanoyl (Pen) or hexanoyl (Hex). The data show that the trypanosomal and HeLa enzymes had similar specificities and that the turnover of GlcNR-PIs by the trypanosomal enzyme was in the order GlcNAc-PI > GlcNPr-PI GlcNBu - PI ≈ GlcNiBu - PI ≈ GlcNPen - PI GlcNHex - PI. The trypanosome and HeLa de-N-acetylases were unable to de-N-acetylate mannosylated GlcNAc-PI intermediates, which explains why de-N-acetylation must precede mannosylation in the GPI biosynthetic pathway.

Probing Enzymes Late in the Trypanosomal Glycosylphosphatidylinositol Biosynthetic Pathway with Synthetic Glycosylphosphatidylinositol Analogues

“…The order of the glycolipids in the table reflects their relative migration on hptlc (highest migrating/most hydrophobic first) and for clarity are grouped according to their lipid moiety (Dolichol, PI or IPC 18 ). ND ϭ not determined.…”

“…The production of endogenous radiolabeled GPI intermediates was prevented by inclusion of N-ethylmaleimide, which inhibits the UDP-GlcNAc:PI ␣1-6 GlcNAc transferase but not the downstream enzymes of GPI biosynthesis (28), and the production of dolichol cycle intermediates was prevented by the addition of tunicamycin. As a positive control, the production of radiolabeled GPI intermediates was stimulated by the addition of 10 M of synthetic GlcNAc-PI or GlcNAc-IPC 18 (22−24). The radiolabeled glycolipids were separated by hptlc and visualized by fluorography ( Figure 3, panel a), and their identities were determined by chemical and enzymatic treatments ( Table 1).…”

Mammalian PIG-L and its yeast homologue Gpi12p are N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential in glycosylphosphatidylinositol biosynthesis

“…They share 77 % and 24 % amino acid identity with rat PIG-L protein respectively. The yeast homologue, termed GPI12, was essential for growth, suggesting that it is the only gene that encodes GlcNAc-PI de-N-acetylase in S. cere isiae and that de-N-acetylation of GlcNAc-PI is an essential step in GPI biosynthesis in yeast, as it is in mammals [34] and trypanosomes [12,35].…”

“…6-fold better substrate than GlcN-PI for the later mannosyltransferase [35]. Because the de-N-acetylation of GlcNAc-PI must precede mannosylation in the GPI biosynthetic pathway [12], this suggests a degree of substrate channelling via the GlcNAc-PI de-N-acetylase to the mannosyltransferase, indicating complex formation between the two enzymes. The identification of a trypanosomal homologue of GlcNAc-PI de-N-acetylase might lead to the identification of the subsequent GlcN-PI mannosyltransferase.…”

“…The substrate specificity of de-N-acetylase was studied by using various GlcNAc-PI analogues [11][12][13]. However, this enzyme was so unstable in the presence of the detergent that purification was impossible [11].…”