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

Abstract

Glycosylphosphatidylinositol (GPI) is used as a membrane anchor by many eukaryotic cell-surface proteins. The second step of GPI biosynthesis is de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI). We have previously cloned the rat PIG-L gene by expression cloning that complemented a mutant Chinese hamster ovary cell line defective in this step. Here we show that recombinant rat PIG-L protein purified from Escherichia coli as a complex with GroEL has GlcNAc-PI de-N-acetylase activity in vitro. The activity was not enhanced by GTP, which is known to enhance the de-N-acetylase activity of mammalian cell microsomes. As with other de-N-acetylases that act on the GlcNAc moiety, metal ions, in particular Mn2+ and Ni2+, enhanced the enzyme activity of PIG-L. The Saccharomyces cerevisiae YMR281W open reading frame encodes a protein (termed Gpi12p) with 24% amino acid identity with rat PIG-L. On transfection into mammalian PIG-L-deficient cells, this gene, GPI12, restored the cell-surface expression of GPI-anchored proteins and GlcNAc-PI de-N-acetylase activity. The disruption of the gene caused lethality in S. cerevisiae. These results indicate that GlcNAc-PI de-N-acetylase is conserved between mammals and yeasts and that the de-N-acetylation step is also indispensable in yeasts.

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…Once formed, GlcNAc-PI is de-N-acetylated at the cytoplasmic face of the ER membrane by Gpi12 (Vidugiriene and Menon 1993;Watanabe et al 1999). GlcN-PI is the precursor likely to be translocated to the lumenal side of the ER membrane.…”

N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase from Entamoeba histolytica

“…This is followed by a second essential step, the de-Nacetylation of GlcNAc-PI to yield glucosamine-PI (GlcN-PI), a step catalyzed by PIG-L/GPI12 (7,8). PIG-L proteins have been identified in almost all eukaryotic organisms.…”

The Role of Inositol Acylation and Inositol Deacylation in the Toxoplasma gondii Glycosylphosphatidylinositol Biosynthetic Pathway

“…In all cases, GPI biosynthesis involves the initial addition of GlcNAc from UDP-GlcNAc to phosphatidylinositol (GPIno) to form GlcNAc-PI, which is then de-N-acetylated to GlcN-PI (43)(44). De-N-acetylation is a prerequisite for mannosylation of GlcN-PI to form later GPI intermediates (45)(46). These early events occur on the cytoplasmic face of the endoplasmic reticulum, whereas the subsequent steps of mannosylation by three separate Dol-P-Man-dependent mannosyltransferases (23) occurs on the luminal face (10), this followed by the transfer of ethanolamine phosphate to the third mannose from phosphatidylethanolamine (48).…”