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

Smith, Terry; Crossman, Arthur; Borissow, Charles N.; Paterson, Michael; Dix, Alex; Brimacombe, J. S.; Ferguson, Michael A. J.

doi:10.1093/emboj/20.13.3322

Cited by 55 publications

(76 citation statements)

References 52 publications

(96 reference statements)

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“…Several potential inhibitors that exploit differences in the substrate specificity of the trypanosome and mammalian enzymes have been developed (317,325,327,329). Inhibitors of the fatty acid remodeling reactions also have potent trypanocidal activity (81).…”

Section: Biosynthesis Of Gpi Protein Anchorsmentioning

confidence: 99%

Secretory Pathway of Trypanosomatid Parasites

McConville

Mullin

Ilgoutz

et al. 2002

Microbiol Mol Biol Rev

222

191

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The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Trypanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles in the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory transport and protein glycosylation that may be exploited in developing new antiparasite drugs

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Section: Biosynthesis Of Gpi Protein Anchorsmentioning

confidence: 99%

Secretory Pathway of Trypanosomatid Parasites

McConville

Mullin

Ilgoutz

et al. 2002

Microbiol Mol Biol Rev

222

191

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“…3 H]Ac-PI as well as on the natural ␣-anomer (39), but the abilities of downstream enzymes to recognize the ␤-linked analogue are unknown. Here, we show that the addition of either GlcNAc-␤-PI ( Fig.…”

Section: Substrate Specificities Of T Brucei Mt-1 and Downstreammentioning

confidence: 99%

“…3 H]Ac-PI (39). It is by no means clear why the enzymes downstream of the de-N-acetylase should recognize GlcN-PI(Me,C18) so poorly.…”

Section: The Effects Of Lipid Structure On Substrate Recognition By Tmentioning

confidence: 99%

“…Thus, GlcN-(2-Ohexadecyl)-PI was shown to inhibit MT-1, whereas GlcN-(2-Ooctyl)-PI and its N-acetylated version inhibited inositol acylation of Man 1-3 GlcN-PI and prevented the subsequent addition of an ethanolamine phosphate bridge (37). Another series of parasite-specific GPI pathway inhibitors containing L-myo-inositol inhibited T. brucei MT-1 (38), and very recently, two potent suicide substrate inhibitors of the parasite GlcNAc-PI de-N-acetylase have been reported (39). Another example of a species-specific inhibitor is a terpenoid natural product that inhibits yeast and human, but not parasite, GPI biosynthesis (40).…”

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confidence: 99%

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Specificities of Enzymes of Glycosylphosphatidylinositol Biosynthesis in Trypanosoma brucei and HeLa Cells

Smith

Crossman

Paterson³

et al. 2002

Journal of Biological Chemistry

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A series of synthetic analogues of D-GlcN␣1-6-D-myoinositol-1-HPO 4 -sn-1,2-dipalmitoylglycerol, consisting of 22 variants of the D-GlcN or lipid components, were tested in trypanosomal and human (HeLa) cell-free systems. The assays measured the abilities of the analogues to act as substrates or inhibitors of the enzymes of glycosylphosphatidylinositol biosynthesis downstream of GlcNAc-phosphatidylinositol (GlcNAc-PI) de-N-acetylase. One compound, 4-deoxy-D-GlcN␣1-6-D-myo-inositol-1-HPO 4 -sn-1,2-dipalmitoylglycerol, proved to be an inhibitor of both the trypanosomal and HeLa pathways, whereas 4-O-methyl-D-GlcN␣1-6-D-myo-inositol-1-HPO 4 -sn-1,2-dipalmitoylglycerol and the 4-epimer, D-GalN-␣1-6-D-myo-inositol-1-HPO 4 -sn-1,2-dipalmitoylglycerol, were neither substrates nor inhibitors. The results with other analogues showed that the 6-OH of the ␣-D-GlcN residue is not required for substrate recognition in the trypanosomal and human pathways, whereas the 3-OH group is essential for both. Parasite-specific recognition of the ␤-linked analogue D-GlcN␤1-6-D-myo-inositol-1-HPO 4 -sn-1,2-dipalmitoylglycerol is striking. This suggests that, like the GlcNAc-PI de-N-acetylase, the trypanosomal glycosylphosphatidylinositol ␣-mannosyltransferases, inositol acyltransferse and ethanolamine phosphate transferase, do not recognize the 2-, 3-, 4-, and 5-OH groups of the D-myo-inositol residue, whereas the human inositol acyltransferase and/or first ␣-mannosyltransferase recognizes one or more of these groups. All of the various lipid analogues tested served as substrates in both the trypanosomal and HeLa cell-free systems, suggesting that a precise lipid structure and stereochemistry are not essential for substrate recognition. However, an analogue containing a single C18:0 alkyl chain in place of sn-1,2-dipalmitoylglycerol proved to be a better substrate in the trypanosomal than in the HeLa cell-free system. These findings should have a bearing on the design of future generations of specific inhibitors of the trypanosomal glycosylphosphatidylinositol biosynthetic pathway.A significant proportion of eukaryotic cell-surface glycoproteins is attached to the plasma membrane by covalent linkage to a glycosylphosphatidylinositol (GPI) 1 anchor. The structure and biosynthesis of GPI membrane anchors and related molecules have been reviewed recently (1-5). The basic GPI core structure attached to protein is composed of NH 2 CH 2 CH 2 PO 4 H-6Man␣1-2Man␣1-6Man␣1-4GlcN␣1-6-D-myo-inositol-1-HPO 4 -lipid (EtN-P-Man 3 GlcN-PI), where the lipid can be diacylglycerol, alkylacylglycerol, or ceramide. This minimal GPI structure may be embellished with additional ethanolamine phosphate groups and/or carbohydrate side chains in a species-and tissue-specific manner (2). Protozoa tend to express significantly higher densities of cell-surface GPI-anchored proteins than do higher eukaryotes. For example, Trypanosoma brucei, the causative agent of African sleeping sickness, expresses a dense cell-surface coat consisting of ϳ5 ϫ 10 6 dimers of a GPI-a...

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“…For example, the yeast and human homologues of PIG-L are inhibited by a naturally occurring terpenoid, whereas the trypanosomal PIG-L is not (10). The Trypanosoma PIG-L enzyme, unlike the human PIG-L, recognizes and catalyzes the deacetylation of GlcNAc-[L]-PI, GlcNAc-␤-PI, and GlcNAc-(2-O-alkyl)PI, a fact that has been exploited for the design of Trypanosoma-specific inhibitors (11). Likewise, the Plasmodium falciparum GlcNAc-PI de-N-acetylase is distinct from the human PIG-L counterpart, leading to the synthesis of Plasmodium-specific inhibitors (12).…”

mentioning

confidence: 99%

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

Ashraf¹,

Yadav²,

Sreejith

et al. 2011

Journal of Biological Chemistry

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PIG-L/GPI12 proteins are endoplasmic reticulum-resident membrane proteins involved in the second step of glycosylphosphatidylinositol anchor biosynthesis in eukaryotes. We show that the Entamoeba histolytica PIG-L protein is optimally active in the acidic pH range. The enzyme has an intrinsic low level of de-N-acetylase activity in the absence of metal and is significantly stimulated by divalent cations. Metal binding induces a large conformational change in the protein that appears to improve catalytic rates while not altering the affinity of the enzyme for its substrate.

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

Cited by 55 publications

References 52 publications

Secretory Pathway of Trypanosomatid Parasites

Secretory Pathway of Trypanosomatid Parasites

Specificities of Enzymes of Glycosylphosphatidylinositol Biosynthesis in Trypanosoma brucei and HeLa Cells

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

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