Developmental regulation of a novel repetitive protein of Trypanosoma brucei.

Mowatt, Michael R.; Clayton, Christine

doi:10.1128/mcb.7.8.2838

Cited by 129 publications

(133 citation statements)

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“…4B, lanes 1 and 2). T. brucei procyclic cells can express two isoforms of procyclins, EP procyclins bearing tandem repeat units of glutamic acid and proline (EP) and GPEET procyclins bearing internal pentapeptide (GPEET) repeats (7,(35)(36)(37). It is known that ratios of these two kinds of procyclins are different among strains and even among clones in the same strain, and can vary with culture length (27,34).…”

Section: Tbgpi10 Is Not Essential But Important For Growth Of Procyclmentioning

confidence: 99%

“…brucei has two distinct proliferative stages, a bloodstream stage living free in mammalian blood and an insect stage (or procyclic form) living in the midguts of tsetse flies. The cell surface of both stages of this unicellular parasite is covered by a large amount of glycosylphosphatidylinositol (GPI)-anchored proteins (3, 4): 10 7 variant surface glycoproteins per cell for the bloodstream form of the parasite and 3 ϫ 10 6 to 6 ϫ 10 6 procyclins (or procyclic acidic repetitive proteins) per cell of the procyclic form of the parasite (4-6), corresponding to 10% and 1-3%, respectively, of total proteins in these parasite stages (7,8). T. brucei evades the host's immune response by expressing structurally different forms of variant surface glycoproteins (4).…”

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Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei

Nagamune

Nozaki

Maeda

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

167

162

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Trypanosoma brucei, the protozoan parasite responsible for sleeping sickness, evades the immune response of mammalian hosts and digestion in the gut of the insect vector by means of its coat proteins tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. To evaluate the importance of GPI for parasite survival, we cloned and disrupted a trypanosomal gene, TbGPI10, involved in biosynthesis of GPI. TbGPI10 encodes a protein of 558 amino acids having 25% and 23% sequence identity to human PIG-B and Saccharomyces cerevisiae Gpi10p, respectively. TbGPI10 restored biosynthesis of GPI in a mouse mutant cell line defective in mouse Pig-b gene. TbGPI10 also rescued the inviability of GPI10-disrupted S. cerevisiae, indicating that TbGPI10 is the orthologue of PIG-B͞GPI10 that is involved in the transfer of the third mannose to GPI. The bloodstream form of T. brucei could not lose TbGPI10; therefore, GPI synthesis is essential for growth of mammalian stage parasites. Procyclic form cells (insect stage parasites) lacking the surface coat proteins because of disruption of TbGPI10 are viable and grow slower than normal, provided that they are cultured in nonadherent flasks. In regular flasks, they adhered to the plastic surface and died. Infectivity to tsetse flies is partially impaired, particularly in the early stage. Therefore, parasitespecific inhibition of GPI biosynthesis should be an effective chemotherapy target against African trypanosomiasis.T rypanosoma brucei is a protozoan parasite invading humans and other mammals by transmission via tsetse flies. It causes sleeping sickness in humans and nagana disease in domestic animals living in the ''tsetse belt'' in central Africa. These are serious medical and agricultural problems for which safe and effective therapeutic and protective measures are highly desirable (1, 2).T. brucei has two distinct proliferative stages, a bloodstream stage living free in mammalian blood and an insect stage (or procyclic form) living in the midguts of tsetse flies. The cell surface of both stages of this unicellular parasite is covered by a large amount of glycosylphosphatidylinositol (GPI)-anchored proteins (3, 4): 10 7 variant surface glycoproteins per cell for the bloodstream form of the parasite and 3 ϫ 10 6 to 6 ϫ 10 6 procyclins (or procyclic acidic repetitive proteins) per cell of the procyclic form of the parasite (4-6), corresponding to 10% and 1-3%, respectively, of total proteins in these parasite stages (7,8). T. brucei evades the host's immune response by expressing structurally different forms of variant surface glycoproteins (4). Procyclins are thought to protect procyclic cells from digestion by the digestive enzymes in the fly (4, 6). In addition, T. brucei expresses a number of other GPI-anchored proteins, such as transferrin receptors in the bloodstream form (3, 4). Thus, the importance of GPI anchors for the survival and infection of T. brucei has been suggested, leading to the notion that the GPI biosynthesis pathway may be a good target for chemo...

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Section: Tbgpi10 Is Not Essential But Important For Growth Of Procyclmentioning

confidence: 99%

mentioning

confidence: 99%

Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei

Nagamune

Nozaki

Maeda

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

167

162

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“…Following ingestion in a blood meal, the stumpy trypomastigote form differentiates into the dividing procyclic form that colonizes the tsetse midgut. The procyclic trypanosomes express a radically different cell surface coat made up of about 3 ϫ 10 6 procyclin glycoproteins (3)(4)(5)(6) and a smaller number of free glycoinositol phospholipids (GIPLs) 1 (7)(8)(9). The procyclins are polyanionic, rod-like (6, 10) proteins encoded by four procyclin genes: GPEET, which encodes a protein with five or six Gly-Pro-Glu-Glu-Thr repeats, and EP1, EP2, and EP3, which encode proteins with 18 -30 Glu-Pro repeats (11).…”

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confidence: 99%

The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number

Roper¹,

Güther

MacRae³

et al. 2005

Journal of Biological Chemistry

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Galactose metabolism is essential in bloodstream form Trypanosoma brucei and is initiated by the enzyme UDP-Glc 4-epimerase. Here, we show that the parasite epimerase is a homodimer that can interconvert UDPGlc and UDP-Gal but not UDP-GlcNAc and UDP-GalNAc. The epimerase was localized to the glycosomes by immunofluorescence microscopy and subcellular fractionation, suggesting a novel compartmentalization of galactose metabolism in this organism. The epimerase is encoded by the TbGALE gene and procyclic form T. brucei single-allele knockouts, and conditional (tetracycline-inducible) null mutants were constructed. Under non-permissive conditions, conditional null mutant cultures ceased growth after 8 days and resumed growth after 15 days. The resumption of growth coincided with constitutive re-expression epimerase mRNA. These data show that galactose metabolism is essential for cell growth in procyclic form T. brucei. The epimerase is required for glycoprotein galactosylation. The major procyclic form glycoproteins, the procyclins, were analyzed in TbGALE single-allele knockouts and in the conditional null mutant after removal of tetracycline. The procyclins contain glycosylphosphatidylinositol membrane anchors with large poly-N-acetyl-lactosamine side chains. The single allele knockouts exhibited 30% reduction in procyclin galactose content. This example of haploid insufficiency suggests that epimerase levels are close to limiting in this life cycle stage. Similar analyses of the conditional null mutant 9 days after the removal of tetracycline showed that the procyclins were virtually galactose-free and greatly reduced in size. The parasites compensated, ultimately unsuccessfully, by expressing 10-fold more procyclin. The implications of these data with respect to the relative roles of procyclin polypeptide and carbohydrate are discussed.

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“…The bloodstream form is sheathed by a dense variant cell surface glycoprotein (VSG) coat which is lost upon differentiation into the procyclic form. The procyclic trypanosome is characterized by an abundant cell surface protein, the procyclic acidic repetitive protein (PARP) or procyclin (7,(27)(28)(29)(33)(34)(35). The Transcription of many protein-coding genes in trypanosomes and related species is believed to be polycistronic, generating large precursor RNAs (pre-mRNAs) containing multiple protein-coding genes.…”

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confidence: 99%