Publications

• Expression cloning of PIG-L, a candidate N-acetylglucosaminyl-phosphatidylinositol deacetylase.

  Nakamura N., Inoue N., Watanabe R., Takahashi M., Takeda J., Stevens V.L., Kinoshita T.

  Many eukaryotic cell surface proteins are bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Several genes involved in GPI anchor biosynthesis have been cloned using complementation of mutant mammalian cell lines and yeasts that are defective in its biosynthesis pathway. Ho ... >> More

  Many eukaryotic cell surface proteins are bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Several genes involved in GPI anchor biosynthesis have been cloned using complementation of mutant mammalian cell lines and yeasts that are defective in its biosynthesis pathway. However, the gene involved in the second step of this pathway, in which N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) is N-deacetylated to form glucosaminyl (GlcN)-PI, has not been cloned. In this study, we established a GPI anchor-deficient mutant of Chinese hamster ovary (CHO) cells defective in the second step. Complementation analysis with the known GPI anchor mutant cells demonstrated that it belonged to the same complementation group as the CHO cell mutant G9PLAP.85. Using the new mutant, we cloned a rat gene termed PIG-L (for phosphatidylinositol glycan class L) that is involved in this step. PIG-L encodes a 252-amino acid, endoplasmic reticulum membrane protein, most of which is in the cytoplasmic side. This orientation of PIG-L protein is consistent with the notion that the second step of GPI anchor biosynthesis occurs on the cytoplasmic side of the endoplasmic reticulum. << Less

  J. Biol. Chem. 272:15834-15840(1997) [PubMed] [EuropePMC]

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

  Watanabe R., Ohishi K., Maeda Y., Nakamura N., Kinoshita T.

  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 complem ... >> More

  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. << Less

  Biochem. J. 339:185-192(1999) [PubMed] [EuropePMC]

• Specificity of GlcNAc-PI de-N-acetylase of GPI biosynthesis and synthesis of parasite-specific suicide substrate inhibitors.

  Smith T.K., Crossman A., Borissow C.N., Paterson M.J., Dix A., Brimacombe J.S., Ferguson M.A.

  The substrate specificities of Trypanosoma brucei and human (HeLa) GlcNAc-PI de-N-acetylases were determined using 24 substrate analogues. The results show the following. (i) The de-N-acetylases show little specificity for the lipid moiety of GlcNAc-PI. (ii) The 3'-OH group of the GlcNAc residue i ... >> More

  The substrate specificities of Trypanosoma brucei and human (HeLa) GlcNAc-PI de-N-acetylases were determined using 24 substrate analogues. The results show the following. (i) The de-N-acetylases show little specificity for the lipid moiety of GlcNAc-PI. (ii) The 3'-OH group of the GlcNAc residue is essential for substrate recognition whereas the 6'-OH group is dispensable and the 4'-OH, while not required for recognition, cannot be epimerized or substituted. (iii) The parasite enzyme can act on analogues containing betaGlcNAc or aromatic N-acyl groups, whereas the human enzyme cannot. (iv) Three GlcNR-PI analogues are de-N-acetylase inhibitors, one of which is a suicide inhibitor. (v) The suicide inhibitor most likely forms a carbamate or thiocarbamate ester to an active site hydroxy-amino acid or Cys or residue such that inhibition is reversed by certain nucleophiles. These and previous results were used to design two potent (IC50 = 8 nM) parasite-specific suicide substrate inhibitors. These are potential lead compounds for the development of anti-protozoan parasite drugs. << Less

  EMBO J 20:3322-3332(2001) [PubMed] [EuropePMC]

• Biosynthesis of the glycosyl phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein. Origin of the non-acetylated glucosamine.

  Doering T.L., Masterson W.J., Englund P.T., Hart G.W.

  Non-acetylated glucosamine is an unusual structural feature shared by all glycosyl phosphatidylinositol (GPI) lipids, including a variety of membrane anchors, the leishmanial lipophosphoglycan, and a mediator of insulin action. We proposed previously a pathway for biosynthesis of glycolipid A, the ... >> More

  Non-acetylated glucosamine is an unusual structural feature shared by all glycosyl phosphatidylinositol (GPI) lipids, including a variety of membrane anchors, the leishmanial lipophosphoglycan, and a mediator of insulin action. We proposed previously a pathway for biosynthesis of glycolipid A, the precursor of the GPI membrane anchor of the trypanosome variant surface glycoprotein (Masterson, W. J., Doering, T. L., Hart, G. W., and Englund, P. T. (1989) Cell 56, 793-800). In this paper we characterize in more detail the initial steps of GPI assembly. The first and committed step in the pathway is the transfer of GlcNAc, from UDP-GlcNAc, to endogenous phosphatidylinositol to form N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). The GlcNAc-PI is then efficiently deacetylated to form glucosaminyl phosphatidylinositol (GlcN-PI), the substrate for subsequent reactions en route to glycolipid A. << Less

  J Biol Chem 264:11168-11173(1989) [PubMed] [EuropePMC]