Structural characterisation of two forms of procyclic acidic repetitive protein expressed by procyclic forms of Trypanosoma brucei

Treumann, Achim; Zitzmann, Nicole; Hülsmeier, Andreas J.; Prescott, Alan R.; Almond, Andrew; Sheehan, John K.; Ferguson, Michael A. J.

doi:10.1006/jmbi.1997.1066

Cited by 133 publications

(190 citation statements)

References 49 publications

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“…EP1-1 and EP1-2 are allelic genes; their products differ only in the number of EP repeats. The assignments suggest that each procyclin polypeptide carries a Hex 5 HexNAc 2 glycan, which corresponds to the previously characterized Man 5 GlcNAc 2 (9). No traces of the nonglycosylated EP2 and GPEET procyclins (m/z 8,344 and 6,142, respectively) were detected.…”

Section: Resultssupporting

confidence: 63%

“…Procyclins-For mass spectrometry, procyclins were purified from 2 ϫ 10 8 freeze-dried parasites by sequential extraction with organic solvents (7,9). To remove GPI anchors from the procyclins, the dry butanolic extracts were treated with 25 l of 48% aqueous hydrofluoric acid for 14 h at 0°C.…”

Section: Maldi-tof-ms Analysis Ofmentioning

confidence: 99%

“…In T. brucei Lister 427, the EP-procyclin isoforms are encoded by the EP1, EP2, and EP3 genes (4,8). Only the products of EP1 and EP3 contain a glycosylation site (Asn 29 ), which is modified exclusively by a Man 5 GlcNAc 2 glycan (7,9). Procyclins are important for survival in the tsetse (10 -12) but can be dispensed with in culture, after a period of adaptation that involves increased expression of free GPIs on the parasite surface (13).…”

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confidence: 99%

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Transposon Mutagenesis of Trypanosoma brucei Identifies Glycosylation Mutants Resistant to Concanavalin A

Leal

Acosta-Serrano

Morris

et al. 2004

Journal of Biological Chemistry

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We have engineered Trypanosoma brucei with a novel mariner transposition system that allows large populations of mutant cells to be generated and screened. As a proof of principle, we isolated and characterized two independent clones that were resistant to the cytotoxic action of concanavalin A. In both clones, the transposon had integrated into the locus encoding a homologue of human ALG12, which encodes a dolichyl-P-Man: Man 7 GlcNAc 2 -PP-dolichyl-␣6-mannosyltransferase. Conventional knock-out of ALG12 in a wild-type background gave an identical phenotype to the mariner mutants, and biochemical analysis confirmed that they have the same defect in the N-linked oligosaccharide synthesis pathway. To our surprise, both mariner mutants were homozygous; the second allele appeared to have undergone gene conversion by the marinertargeted allele. Subsequent experiments showed that the frequency of gene conversion at the ALG12 locus, in the absence of selection, was 0.25%. As we approach the completion of the trypanosome genome project, transposon mutagenesis provides an important addition to the repertoire of genetic tools for T. brucei.

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Section: Resultssupporting

confidence: 63%

Section: Maldi-tof-ms Analysis Ofmentioning

confidence: 99%

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confidence: 99%

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Transposon Mutagenesis of Trypanosoma brucei Identifies Glycosylation Mutants Resistant to Concanavalin A

Leal

Acosta-Serrano

Morris

et al. 2004

Journal of Biological Chemistry

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“…Like the coat of Leishmania, the membrane surface of T. brucei contains an abundance of GPI-linked proteins -procyclin in the insect form and variant surface glycoprotein (VSG) in the bloodstream form. However, neither procyclin nor VSG is associated with DRMs (Denny et al, 2001) (our unpublished work), probably owing to the unusual positions of the acyl chains on the inositol ring in the case of procyclin (Treumann et al, 1997) and the short acyl chain length of the anchor in VSG (Benting et al, 1999). Considering that the two related kinetoplastids both possess lipid rafts and synthesize ergosterol, but have GPI-anchored molecules with different biochemical properties, we wished to assess whether sphingolipid synthesis is similarly dispensable in T. brucei.…”

Section: Introductionmentioning

confidence: 87%

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Fridberg

Olson

Nakayasu

et al. 2008

Journal of Cell Science

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Sphingolipids and their metabolites have been thought crucial for cell growth and cell cycle progression, membrane and protein trafficking, signal transduction, and formation of lipid rafts; however, recent studies in trypanosomes point to the dispensability of sphingolipids in some of these processes. In this study, we explore the requirements for de novo sphingolipid biosynthesis in the insect life cycle stage of the African trypanosome Trypanosoma brucei by inhibiting the enzyme serine palmitoyltransferase (SPT2) by using RNA interference or treatment with a potent SPT2 inhibitor myriocin. Mass spectrometry revealed that upon SPT2 inhibition, the parasites contained substantially reduced levels of inositolphosphorylceramide. Although phosphatidylcholine and cholesterol levels were increased to compensate for this loss, the cells were ultimately not viable. The most striking result of sphingolipid reduction in procyclic T. brucei was aberrant cytokinesis, characterized by incomplete cleavage-furrow formation, delayed kinetoplast segregation and emergence of cells with abnormal DNA content. Organelle replication continued despite sphingolipid depletion, indicating that sphingolipids act as second messengers regulating cellular proliferation and completion of cytokinesis. Distention of the mitochondrial membrane, formation of multilamellar structures within the mitochondrion and near the nucleus, accumulation of lipid bodies and, less commonly, disruption of the Golgi complex were observed after prolonged sphingolipid depletion. These findings suggest that some aspects of vesicular trafficking may be compromised. However, flagellar membrane targeting and the association of the flagellar membrane protein calflagin with detergent-resistant membranes were not affected, indicating that the vesicular trafficking defects were mild. Our studies indicate that sphingolipid biosynthesis is vital for cell cycle progression and cell survival, but not essential for the normal trafficking of flagellar membrane-associated proteins or lipid raft formation in procyclic T. brucei.

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“…Thus the H5 band ( Figure 5B Figure 5B, lanes 8 and 9), the H5 band was sensitive to human Figure 5B, lane 4). This suggests that there may be some heterogeneity in the acyl chain attached to the inositol ring, as has been observed in human erythrocyte acetylcholinesterase [24], T. brucei glycolipid C [25] and T. brucei procyclic acid repetitive protein [26]. This conclusion would be broadly consistent with the ability of the mammalian inositolacyltransferase to use different acyl-CoA donors [27] but it is at variance with the study of Sevlever et al [28] on the GlcN-(acyl)PI that accumulates in HeLa S3 cells, which suggests that, in that case, the acyl chain is exclusively palmitate.…”

Section: Comparative Studies On the Substrate Specificity Of The Helamentioning

confidence: 93%

Substrate specificity of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol membrane anchor biosynthesis in African trypanosomes and human cells

Sharma

Smith

Crossman

et al. 1997

Biochemical Journal

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De-N-acetylation of N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) is the second step of glycosylphosphatidylinositol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent mannosylation of glucosaminyl-phosphatidylinositol (GlcN-PI) which leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. The substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells were studied with respect to the N-acyl groups (R) that could be removed from a series of GlcNR-PI substrates, where R = acetyl (Ac), propionyl (Pr), butyryl (Bu), isobutyryl (iBu), pentanoyl (Pen) or hexanoyl (Hex). The data show that the trypanosomal and HeLa enzymes had similar specificities and that the turnover of GlcNR-PIs by the trypanosomal enzyme was in the order GlcNAc-PI > GlcNPr-PI GlcNBu - PI ≈ GlcNiBu - PI ≈ GlcNPen - PI GlcNHex - PI. The trypanosome and HeLa de-N-acetylases were unable to de-N-acetylate mannosylated GlcNAc-PI intermediates, which explains why de-N-acetylation must precede mannosylation in the GPI biosynthetic pathway.

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Structural characterisation of two forms of procyclic acidic repetitive protein expressed by procyclic forms of Trypanosoma brucei

Cited by 133 publications

References 49 publications

Transposon Mutagenesis of Trypanosoma brucei Identifies Glycosylation Mutants Resistant to Concanavalin A

Transposon Mutagenesis of Trypanosoma brucei Identifies Glycosylation Mutants Resistant to Concanavalin A

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Substrate specificity of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol membrane anchor biosynthesis in African trypanosomes and human cells

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