Intracellular localization of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei

1989 ◽  
Vol 93 (2) ◽  
pp. 233-240
Author(s):  
R. Bolow ◽  
G. Griffiths ◽  
P. Webster ◽  
Y.D. Stierhof ◽  
F.R. Opperdoes ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Cellular Localization ◽  
Intracellular Localization ◽  
Surface Glycoprotein ◽  
Glycosyl Phosphatidylinositol ◽  
Membrane Bound ◽  
Cyclic Phosphate ◽  
Inositol Ring ◽  
Cytoplasmic Side

Glycosyl-phosphatidylinositol-specific phospholipase C (GPI-PLC) is a membrane-bound enzyme of bloodstream forms of Trypanosoma brucei, which cleaves the GPI-membrane anchor of the variant surface glycoprotein forming diacylglycerol and 1,2-cyclic phosphate on the inositol ring. The cellular localization of the enzyme was studied by fractionation of sub-cellular organelles and immunofluorescence microscopy and was found to be primarily cytoplasmic. This was confirmed by immuno-electron microscopy using cryo-sections, which showed that the labelling was predominantly on the cytoplasmic side of intracellular membranes but was absent from the plasma membrane including the region lining the flagellar pocket. The significance of these results for the possible function of the phospholipase is discussed.

scholarly journals Site of palmitoylation of a phospholipase C-resistant glycosylphosphatidylinositol membrane anchor

1992 ◽  
Vol 284 (2) ◽  
pp. 297-300 ◽  
Author(s):  
M A J Ferguson
Keyword(s):  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Periodate Oxidation ◽  
Membrane Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Oxidation Analysis ◽  
Inositol Ring ◽  
Repetitive Protein

The site of palmitoylation of the phosphatidylinositol moiety of the glycosyl-phosphatidylinositol membrane anchor of Trypanosoma brucei procyclic acidic repetitive protein was studied by using periodate oxidation. Analysis of the products by g.c.-m.s. allowed the assignment of 40 and 60% of the palmitate to the 2-position and the 3-position respectively of the myo-inositol ring.

scholarly journals Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein.

1986 ◽  
Vol 103 (1) ◽  
pp. 255-263 ◽  
Author(s):  
J D Bangs ◽  
N W Andrews ◽  
G W Hart ◽  
P T Englund
Keyword(s):  
Cell Surface ◽  
Phospholipase C ◽  
Intracellular Transport ◽  
Signal Sequence ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Hydrophobic Peptide ◽  
Membrane Bound ◽  
A Site ◽  
Kinetics Of

After synthesis on membrane-bound ribosomes, the variant surface glycoprotein (VSG) of Trypanosoma brucei is modified by: (a) removal of an N-terminal signal sequence, (b) addition of N-linked oligosaccharides, and (c) replacement of a C-terminal hydrophobic peptide with a complex glycolipid that serves as a membrane anchor. Based on pulse-chase experiments with the variant ILTat-1.3, we now report the kinetics of three subsequent processing reactions. These are: (a) conversion of newly synthesized 56/58-kD polypeptides to mature 59-kD VSG, (b) transport to the cell surface, and (c) transport to a site where VSG is susceptible to endogenous membrane-bound phospholipase C. We found that the t 1/2 of all three of these processes is approximately 15 min. The comparable kinetics of these processes is compatible with the hypotheses that transport of VSG from the site of maturation to the cell surface is rapid and that VSG may not reach a phospholipase C-containing membrane until it arrives on the cell surface. Neither tunicamycin nor monensin blocks transport of VSG, but monensin completely inhibits conversion of 58-kD VSG to the mature 59-kD form. In the presence of tunicamycin, VSG is synthesized as a 54-kD polypeptide that is subsequently processed to a form with a slightly higher Mr. This tunicamycin-resistant processing suggests that modifications unrelated to N-linked oligosaccharides occur. Surprisingly, the rate of VSG transport is reduced, but not abolished, by dropping the chase temperature to as low as 10 degrees C.

Genomic organization, chromosomal localization, and developmentally regulated expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei

1990 ◽  
Vol 10 (2) ◽  
pp. 720-726
Author(s):  
K Mensa-Wilmot ◽  
D Hereld ◽  
P T Englund
Keyword(s):  
Phospholipase C ◽  
Blot Analysis ◽  
Developmental Regulation ◽  
Mrna Level ◽  
Immunoblot Analysis ◽  
Single Copy ◽  
Tsetse Fly ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Glycosyl Phosphatidylinositol

The surface of the bloodstream form of the African trypanosome, Trypansoma brucei, is covered with about 10(7) molecules of the variant surface glycoprotein (VSG), a protein tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) membrane anchor. This anchor is cleavable by an endogenous GPI-specific phospholipase C (GPI-PLC). GPI-PLC activity is down regulated when trypanosomes differentiate from the bloodstream form to the procyclic form found in the tsetse fly vector. We have mapped the GPI-PLC locus in the trypanosome genome and have examined the mechanism for this developmental regulation in T. brucei. Southern blot analysis indicates a single-copy gene for GPI-PLC, with two allelic variants distinguishable by two NcoI restriction fragment length polymorphisms. The gene was localized solely to a chromosome in the two-megabase compression region by contour-clamped homogeneous electric field gel electrophoresis. No rearrangement of the GPI-PLC gene occurs during differentiation to procyclic forms, which could potentially silence GPI-PLC gene expression. Enzymological studies give no indication of a diffusible inhibitor of GPI-PLC activity in procyclic forms, and Western immunoblot analysis reveals no detectable GPI-PLC polypeptide in these forms. Therefore, it is highly unlikely that the absence of GPI-PLC activity in procyclic forms is due to posttranslational control. Northern (RNA) blot analysis reveals barely detectable levels of GPI-PLC mRNA in procyclic forms; therefore, regulation of GPI-PLC activity in these forms correlates with the steady-state mRNA level.

scholarly journals Subcellular localization of a variable surface glycoprotein phosphatidylinositol-specific phospholipase-C in African trypanosomes.

1987 ◽  
Vol 105 (2) ◽  
pp. 737-746 ◽  
Author(s):  
D J Grab ◽  
P Webster ◽  
S Ito ◽  
W R Fish ◽  
Y Verjee ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Specific Activity ◽  
Surface Glycoprotein ◽  
Trypanosoma Vivax ◽  
Trypanosome Species ◽  
Phospholipase Activity ◽  
African Trypanosomes ◽  
Variable Surface Glycoprotein ◽  
Variable Surface

African trypanosomes contain a membrane-bound enzyme capable of removing dimyristylglycerol from the membrane-attached form of the variable surface glycoprotein (mfVSG; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968). Although mfVSG phospholipase-C has been implicated in the removal of the VSG from the trypanosome surface (Cardoso de Almeida, M. L., and M. J. Turner, 1983, Nature (Lond.)., 302:349-352; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968), its precise function and subcellular location have not been determined. We have developed a procedure for the separation of the cell fractions and organelles of Trypanosoma brucei brucei (and other trypanosome species) by differential sucrose and isopycnic PercollR centrifugation. These fractions were tested for mfVSG phospholipase activity using Trypanosoma brucei mfVSG labeled with 3H-myristic acid as substrate. The highest enzyme-specific activity was associated with the flagella and evidence is presented to suggest that it is localized in the flagellar pocket. Some activity was also associated with the Golgi complex. These results suggest that the mfVSG phospholipase is localized primarily in the membrane of the flagella pocket and possibly other membrane organelles derived from and associated with this structure, and may be part of the VSG-membrane recycling system in African trypanosomes. The activity of mfVSG phospholipase amongst various trypanosome species was determined. We show that, in contrast to the bloodstream forms of Trypanosoma brucei, cultured procyclic Trypanosoma brucei and bloodstream Trypanosoma vivax had little or no mfVSG phospholipase activity. The activity found in bloodstream forms of Trypanosoma congolense was intermediate between Trypanosoma vivax and Trypanosoma brucei.

scholarly journals Trypanosoma brucei Interaction with Host: Mechanism of VSG Release as Target for Drug Discovery for African Trypanosomiasis

2019 ◽  
Vol 20 (6) ◽  
pp. 1484 ◽  
Author(s):  
Cláudia Moreno ◽  
Adriana Temporão ◽  
Taffarel Torres ◽  
Marcelo Sousa Silva
Keyword(s):  
Drug Discovery ◽  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Mammalian Host ◽  
African Trypanosomiasis ◽  
Variable Surface Glycoprotein ◽  
Variable Surface ◽  
Major Surface

The protozoan Trypanosoma brucei, responsible for animal and human trypanosomiasis, has a family of major surface proteases (MSPs) and phospholipase-C (PLC), both involved in some mechanisms of virulence during mammalian infections. During parasitism in the mammalian host, this protozoan is exclusively extracellular and presents a robust mechanism of antigenic variation that allows the persistence of infection. There has been incredible progress in our understanding of how variable surface glycoproteins (VSGs) are organised and expressed, and how expression is switched, particularly through recombination. The objective of this manuscript is to create a reflection about the mechanisms of antigenic variation in T. brucei, more specifically, in the process of variable surface glycoprotein (VSG) release. We firstly explore the mechanism of VSG release as a potential pathway and target for the development of anti-T. brucei drugs.

scholarly journals A new method for the rapid purification of both the membrane-bound and released forms of the variant surface glycoprotein from Trypanosoma brucei

1985 ◽  
Vol 230 (1) ◽  
pp. 195-202 ◽  
Author(s):  
D G Jackson ◽  
M J Owen ◽  
H P Voorheis
Keyword(s):  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Aqueous Salt Solution ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
High Yield ◽  
Dependent Manner ◽  
Wide Range ◽  
Membrane Bound ◽  
Rapid Purification

A simple new technique was developed for the rapid purification of either the membrane-bound or the released forms of the variant surface glycoprotein of Trypanosoma brucei in high yield. Whole cells were used as the source of the membrane-bound form, and the supernatant of benzyl alcohol-treated cells was used as the source of the released form. The technique was based on extraction of the acid-treated protein into chloroform/methanol, followed by selective re-partition into aqueous salt solution. The yield of purified protein was found to be dependent critically on a low pH during the extraction/re-partition stages. This finding and the ability to cycle the protein repeatedly through organic and aqueous phases in a strictly pH-dependent manner suggested that the protein could undergo fully reversible denaturation/renaturation only while in an extensively protonated form. The yield was independent of the polarity of the organic phase and the protein concentration over a wide range. After purification, both forms retain their ability to react with specific antibody raised against the authentic native protein purified by conventional means. The amino acid composition and the identity of the N-terminal amino acid was the same for both forms of the protein. In addition, both forms had blocked C-terminal residues. There were determined to be 1.13 × 10(7) copies of the variant surface glycoprotein per cell.

scholarly journals Expression of a variant surface glycoprotein of Trypanosoma gambiense in procyclic forms of Trypanosoma brucei shows that the cell type dictates the nature of the glycosylphosphatidylinositol membrane anchor attached to the glycoprotein

1997 ◽  
Vol 324 (3) ◽  
pp. 885-895 ◽  
Author(s):  
Françoise PATURIAUX-HANOCQ ◽  
Nicole ZITZMANN ◽  
Jacqueline HANOCQ-QUERTIER ◽  
Luc VANHAMME ◽  
Sylvie ROLIN ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Degradation Products ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Mrna Levels ◽  
Membrane Anchor ◽  
Gpi Anchor ◽  
Intact Membrane ◽  
Membrane Bound ◽  
Trypanosoma Gambiense

Procyclic forms of Trypanosoma brucei have been genetically modified to express the major metacyclic variant surface glycoprotein (VSG variant AnTat 11.17) of Trypanosoma gambiense. The VSG is expressed in an intact membrane-bound form that can be detected over the entire plasma membrane, together with procyclin, and as a series of lower-molecular-mass fragments that are mostly soluble degradation products. The presence of degraded VSG in the cells and the culture medium suggests that VSG is not efficiently processed and/or efficiently folded when expressed in procyclic cells. The level of procyclin expressed on the surface of these cells is slightly reduced, although there is no difference in procyclin mRNA levels. The intact membrane-bound form of the VSG is N-glycosylated with oligomannose structures and contains a glycosylphosphatidylinositol (GPI) membrane anchor that can be biosynthetically labelled with [3H]ethanolamine. The anchor is sensitive to mammalian GPI-specific phospholipase D but, like the anchor of procyclin, it is resistant to the action of bacterial phosphatidylinositol-specific phospholipase C. This pattern of phospholipase sensitivity suggests that the GPI anchor acquired by VSG when expressed in procyclics is acylated on the inositol ring and therefore resembles a procyclic procyclin-type anchor rather than a trypomastigote VSG-type anchor with respect to the lipid structure. The VSG expressed in procyclics was sensitive to the action of a mixture of sialidase, β-galactosidase and β-hexosaminidase, suggesting that the VSG GPI anchor also contains a sialylated polylactosamine side-chain modification similar to that described for procyclin. These results indicate that the nature of the protein expressed has little influence on the post-translational modifications performed in the secretory pathway of procyclic trypanosomes.

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

1988 ◽  
Vol 167 (2) ◽  
pp. 300-314 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document