The role of inositol acylation and inositol deacylation in GPI biosynthesis in Trypanosoma brucei

Güther, Maria Lucia S.; Ferguson, Michael A. J.

doi:10.1016/0169-4758(95)80181-2

Cited by 26 publications

(67 citation statements)

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“…3A). These observations are consistent with previous studies of [ 3 H]Man‐labeling of GPIs in which glycolipid A (or P2), which is cleaved by GPI‐PLC, and glycolipid C (or P3) which is not digested by the enzyme, were described [3,31,33]. Glycolipid C, similar to glycolipid Z (Fig.…”

Section: Resultssupporting

confidence: 92%

“…A second approach was used to test whether T. brucei lost GPIs in response to hypo‐osmotic or mild alkali treatment. Here, T. brucei 427 and RUMP528 were metabolically labeled with d ‐[2,6‐ 3 H]mannose in the presence of tunicamycin, which blocks synthesis of dolichol‐linked glycolipids used for N‐glycosylation of proteins [31]. [ 3 H]Man‐glycolipids were extracted from the cells with organic solvent.…”

Section: Resultsmentioning

confidence: 99%

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Regulated cleavage of intracellular glycosylphosphatidylinositol in a trypanosome

Subramanya

Mensa-Wilmot

2006

The FEBS Journal

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Cell exposure to hypo‐osmolarity and alkalinity triggers a spectrum of responses including activation of phospholipases. Glycosylphosphatidylinositol‐specific phospholipase C (GPI‐PLC) is expressed in Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. We examined possible contributions of GPI‐PLC to the response of T. brucei to hypo‐osmotic or mildly alkaline conditions. GPIs were detected at the endoplasmic reticulum (ER). They were cleaved after exposure of T. brucei to hypo‐osmolarity or mild alkalinity, which also, strikingly, caused translocation of GPI‐PLC from glycosomes (peroxisomes) to the ER. A catalytically inactive Gln81Leu mutant of GPI‐PLC failed to cleave GPIs despite being transported from glycosomes to the ER after hypo‐osmotic or mild alkaline treatment of the parasites. In contrast, a Cys347Ser mutant of the enzyme could not exit glycosomes after treatment of cells expressing the protein with mild base or hypo‐osmotic buffer. We conclude that: (a) GPI‐PLC contributes to loss of GPIs in T. brucei treated with hypo‐osmotic or mildly alkaline buffer; (b) access of GPI‐PLC to its substrate in vivo can be regulated post‐translationally; (c) translocation of GPI‐PLC from glycosomes to the ER is important for in vivo cleavage of GPIs; (d) Cys347 is part of a peptide motif required for post‐translational targeting of GPI‐PLC to the ER. Glycosome‐to‐ER movement of GPI‐PLC reveals a novel pathway for intracellular protein traffic. The physiological significance of GPI digestion in cells exposed to mildly alkalinity or hypo‐osmolarity is discussed.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Regulated cleavage of intracellular glycosylphosphatidylinositol in a trypanosome

Subramanya

Mensa-Wilmot

2006

The FEBS Journal

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“…Firstly, the suggestion that the trypanosomal Dol-P-Man:GlcN-PI α1-4 mannosyltransferase requires a free hydroxyl group at the 2-position of the D-myo-inositol residue for substrate recognition (Güther and Ferguson, 1995;Smith et al, 1996) must be revised. This notion arose from the observation that inositol-acylation of GlcN-PI does not occur until after the first αMan residue is added (Güther and Ferguson, 1995). However, in the light of the successful mannosylation of GlcN-(2-O-Me)PI, this could be reinterpreted in terms of the accessibility of substrates to the inositol-acyltransferase.…”

Section: Discussionmentioning

confidence: 99%

Parasite and mammalian GPI biosynthetic pathways can be distinguished using synthetic substrate analogues

et al. 1997

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Glycosylphosphatidylinositol (GPI) structures are attached to many cell surface glycoproteins in lower and higher eukaryotes. GPI structures are particularly abundant in trypanosomatid parasites where they can be found attached to complex phosphosaccharides, as well as to glycoproteins, and as mature surface glycolipids. The high density of GPI structures at all life-cycle stages of African trypanosomes and Leishmania suggests that the GPI biosynthetic pathway might be a reasonable target for the development of anti-parasite drugs. In this paper we show that synthetic analogues of early GPI intermediates having the 2-hydroxyl group of the D-myo-inositol residue methylated are recognized and mannosylated by the GPI biosynthetic pathways of Trypanosoma brucei and Leishmania major but not by that of human (HeLa) cells. These findings suggest that the discovery and development of specific inhibitors of parasite GPI biosynthesis are attainable goals. Moreover, they demonstrate that inositol acylation is required for mannosylation in the HeLa cell GPI biosynthetic pathway, whereas it is required for ethanolamine phosphate addition in the T.brucei GPI biosynthetic pathway.

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“…Digestion with GPI-specific phospholipase D (GPI-PLD, Glyco) and PI-specific phospholipase C (PI-PLC, Glyco), deamination, base hydrolysis and HF treatment were as described previously (Güther et al, 1994;Güther and Ferguson, 1995;Smith et al, 1996). After deamination, reactions were subjected to butanol/water partitioning and total [ 3 H] cpm in these fractions was determined by scintillation spectrometry using a Beckman LS6000E with formula 989 scintillation fluid (Packard Biosciences).…”

Section: Enzymatic Digests and Chemical Characterization Of Radiolabementioning

confidence: 99%

The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream‐form Trypanosoma brucei is dependent on the de novo synthesis of inositol

Martin

Smith

2006

Molecular Microbiology

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SummaryIn bloodstream-form Trypanosoma brucei (the causative agent of African sleeping sickness) the glycosylphosphatidylinositol (GPI) anchor biosynthetic pathway has been validated genetically and chemically as a drug target. The conundrum that GPI anchors could not be in vivo labelled with [ 3 H]-inositol led us to hypothesize that de novo synthesis was responsible for supplying myo-inositol for phosphatidylinositol (PI) destined for GPI synthesis. The rate-limiting step of the de novo synthesis is the isomerization of glucose 6-phosphate to 1-Dmyo-inositol-3-phosphate, catalysed by a 1-D-myoinositol-3-phosphate synthase (INO1). When grown under non-permissive conditions, a conditional double knockout demonstrated that INO1 is an essential gene in bloodstream-form T. brucei. It also showed that the de novo synthesized myo-inositol is utilized to form PI, which is preferentially used in GPI biosynthesis. We also show for the first time that extracellular myo-inositol can in fact be used in GPI formation although to a limited extent. Despite this, extracellular inositol cannot compensate for the deletion of INO1. Supporting these results, there was no change in PI levels in the conditional double knockout cells grown under non-permissive conditions, showing that perturbation of growth is due to a specific lack of de novo synthesized myo-inositol and not a general inositol-less death. These results suggest that there is a distinction between de novo synthesized myo-inositol and that from the extracellular environment.

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The role of inositol acylation and inositol deacylation in GPI biosynthesis in Trypanosoma brucei

Cited by 26 publications

References 0 publications

Regulated cleavage of intracellular glycosylphosphatidylinositol in a trypanosome

Regulated cleavage of intracellular glycosylphosphatidylinositol in a trypanosome

Parasite and mammalian GPI biosynthetic pathways can be distinguished using synthetic substrate analogues

The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream‐form Trypanosoma brucei is dependent on the de novo synthesis of inositol

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