Biosynthesis of Glycosyl-Phosphatidylinositol Protein Anchors in African Trypanosomes

A simple purification of procyclic acidic repetitive protein and demonstration of a sialylated glycosyl-phosphatidylinositol membrane anchor

Biochemical Journal ◽

10.1042/bj2910051 ◽

1993 ◽

Vol 291 (1) ◽

pp. 51-55 ◽

Cited By ~ 81

Author(s):

M A Ferguson ◽

P Murray ◽

H Rutherford ◽

M J McConville

Keyword(s):

Hydrophobic Interaction Chromatography ◽

Glycosylation Site ◽

Surface Glycoprotein ◽

Membrane Anchor ◽

Cell Surface Glycoprotein ◽

African Trypanosomes ◽

Glycosyl Phosphatidylinositol ◽

Repeat Domain ◽

Rapid Purification ◽

Repetitive Protein

The procyclic acidic repetitive protein is the major cell-surface glycoprotein of the insect-dwelling procyclic forms of the Trypanosoma brucei species of African trypanosomes. The glycoprotein contains an acidic Glu-Pro repeat domain, a glycosyl-phosphatidylinositol membrane anchor and a putative asparagine glycosylation site. In this paper we describe a rapid purification scheme for this glycoprotein, using solvent extraction and hydrophobic interaction chromatography, and a partial characterization of the glycosylphosphatidylinositol membrane anchor. The carbohydrate composition of the anchor is extremely unusual; it contains on average nine GlcNAc, nine Gal, and five sialic acid residues. This is the first description of such a heavily substituted and negatively charged anchor. A comparison between the trypanosome procyclic surface and the Leishmania promastigote surface is also presented.

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Developmentally regulated, phospholipase C-mediated release of the major surface glycoprotein of amastigotes of Trypanosoma cruzi.

Journal of Experimental Medicine ◽

10.1084/jem.167.2.300 ◽

1988 ◽

Vol 167 (2) ◽

pp. 300-314 ◽

Cited By ~ 64

Author(s):

N W Andrews ◽

E S Robbins ◽

V Ley ◽

K S Hong ◽

V Nussenzweig

Keyword(s):

Trypanosoma Cruzi ◽

Phospholipase C ◽

Myristic Acid ◽

Surface Glycoprotein ◽

Developmentally Regulated ◽

African Trypanosomes ◽

Glycosyl Phosphatidylinositol ◽

Major Surface Glycoprotein ◽

Major Stage ◽

Major Surface

The surface of amastigotes of Trypanosoma cruzi is covered by Ssp-4, a major stage-specific glycoprotein. Ssp-4 is anchored to the cell membrane by GPI. It can be metabolically labeled with [3H]myristic acid, and is converted into a hydrophilic form by treatment with the glycan-specific phospholipase C of T. brucei, or after lysis of the parasites in non-ionic detergents. The hydrophilic form of Ssp-4 is recognized by antibodies to the cross-reactive determinant of the variant surface glycoprotein of African trypanosomes. Ssp-4 is progressively shed during the intra- or extracellular development of amastigotes preceding their transformation into epi- and trypomastigotes. We show here that T. cruzi contains a phospholipase C and that most shed Ssp-4 is hydrophilic, does not contain myristic acid, and reacts with anti-CRD. These observations provide strong evidence that phospholipase C mediates the release of this glycosyl-phosphatidylinositol-anchored protein under physiological conditions, as the parasite undergoes differentiation.

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Glycosyl Phosphatidylinositol Myristoylation in African Trypanosomes

Journal of Biological Chemistry ◽

10.1074/jbc.275.19.14147 ◽

2000 ◽

Vol 275 (19) ◽

pp. 14147-14154 ◽

Cited By ~ 19

Author(s):

Yasu S. Morita ◽

Alvaro Acosta-Serrano ◽

Laurence U. Buxbaum ◽

Paul T. Englund

Keyword(s):

African Trypanosomes ◽

Glycosyl Phosphatidylinositol

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Effects of phenothiazine neuroleptic drugs on the microtubular–membrane complex in bloodstream forms of Trypanosoma brucei

Parasitology ◽

10.1017/s0031182000066002 ◽

1995 ◽

Vol 111 (4) ◽

pp. 493-504 ◽

Cited By ~ 5

Author(s):

A. M. Page ◽

J. R. Lagnado

Keyword(s):

Trypanosoma Brucei ◽

Structural Integrity ◽

Membrane Skeleton ◽

Host Immune System ◽

Surface Coat ◽

Neuroleptic Drugs ◽

African Trypanosomes ◽

Glycosyl Phosphatidylinositol ◽

Membrane Complex

SUMMARYAfrican trypanosomes are parasitic protozoa causing sleeping sickness in humans and related diseases in domestic animals against which no entirely satisfactory forms of chemotherapy are yet available. It was previously shown that related species of trypanosomes, as well as procyclic (insect) forms of Trypanosoma brucei are extremely sensitive to the action of phenothiazine neuroleptic drugs in vitro. In this work, we have carried out a more detailed investigation of the effects of thioridazine, one of the most potent neuroleptic phenothiazine drugs known, on the morphology of the infective bloodstream forms of T. brucei, with particular reference to the parasite's prominent pellicular membrane complex. Our data show that this drug induces rapid changes in cell shape that appear to involve some reorganization of the microtubular membrane skeleton, but does not affect the structural integrity of the microtubular complex. Another early consequence of drug action involved damage to nuclear and cytoplasmic membranes and the appearance of tubular arrays of coated membrane within the flagellar pocket. It was also revealed that the drug induces a rapid release of the variant-specific glycoprotein (VSG) which makes up the surface coat protecting bloodstream forms of the parasite against the host immune system. Our evidence suggests that this release of VSG involves cleavage of the protein's glycosyl-phosphatidylinositol (GPI) membrane anchor by endogenous GPI-specific phospholipase C, probably as a consequence of minor damage to the parasite plasma membrane induced by the drug.

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Myristate exchange. A second glycosyl phosphatidylinositol myristoylation reaction in African trypanosomes.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)43799-4 ◽

1994 ◽

Vol 269 (48) ◽

pp. 30212-30220

Author(s):

L U Buxbaum ◽

J Raper ◽

F R Opperdoes ◽

P T Englund

Keyword(s):

African Trypanosomes ◽

Glycosyl Phosphatidylinositol

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Glycosyl phosphatidylinositol myristoylation in African trypanosomes

Molecular and Biochemical Parasitology ◽

10.1016/s0166-6851(96)02820-4 ◽

1997 ◽

Vol 85 (1) ◽

pp. 1-7 ◽

Cited By ~ 11

Author(s):

Karl A Werbovetz ◽

Paul T Englund

Keyword(s):

African Trypanosomes ◽

Glycosyl Phosphatidylinositol

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Glycosyl-phosphatidylinositol molecules of the parasite and the host

Parasitology ◽

10.1017/s0031182000075715 ◽

1994 ◽

Vol 108 (S1) ◽

pp. S45-S54 ◽

Cited By ~ 47

Author(s):

M. A. J. Ferguson ◽

J. S. Brimacombe ◽

S. Cottaz ◽

R. A. Field ◽

L. S. Güther ◽

...

Keyword(s):

Mammalian Cells ◽

Chemotherapeutic Agents ◽

Surface Glycoprotein ◽

Molecular Architecture ◽

Protozoan Parasites ◽

Gpi Anchor ◽

African Trypanosomes ◽

Glycosyl Phosphatidylinositol ◽

Protein Membrane ◽

Parasite Survival

SUMMARYThe glycosyl-phosphatidylinositol (GPI) protein-membrane anchors are ubiquitous among the eukaryotes. However, while mammalian cells typically express in the order of 100 thousand copies of GPI-anchor per cell, the parasitic protozoa, particularly the kinetoplastids, express up to 10–20 million copies of GPI-anchor and/or GPI-related glycolipids per cell. Thus GPI-family members dominate the cell surface molecular architecture of these organisms. In several cases, GPI-anchored proteins, such as the variant surface glycoprotein (VSG) of the African trypanosomes, or GPI-related glycolipids, such as the lipophosphoglycan (LPG) of the Leishmania, are known to be essential for parasite survival and infectivity. The highly elevated levels and specialised nature of GPI metabolism in the kinetoplastid parasites suggest that the GPI biosynthetic pathways might be good targets for the development of chemotherapeutic agents. This article introduces the range of GPI structures found in protozoan parasites, and their mammalian hosts, and discusses some aspects of GPI biosynthesis.

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