scholarly journals GPI-anchored Proteins and Free GPI Glycolipids of Procyclic Form Trypanosoma brucei Are Nonessential for Growth, Are Required for Colonization of the Tsetse Fly, and Are Not the Only Components of the Surface Coat

2006 ◽  
Vol 17 (12) ◽  
pp. 5265-5274 ◽  
Author(s):  
Maria Lucia Sampaio Güther ◽  
Sylvia Lee ◽  
Laurence Tetley ◽  
Alvaro Acosta-Serrano ◽  
Michael A.J. Ferguson
Keyword(s):  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Current Model ◽  
Apparent Molecular Weight ◽  
Tsetse Fly ◽  
Surface Coat ◽  
Procyclic Form ◽  
Cell Surface Biotinylation ◽  
A Cell

The procyclic form of Trypanosoma brucei exists in the midgut of the tsetse fly. The current model of its surface glycocalyx is an array of rod-like procyclin glycoproteins with glycosylphosphatidylinositol (GPI) anchors carrying sialylated poly-N-acetyllactosamine side chains interspersed with smaller sialylated poly-N-acetyllactosamine–containing free GPI glycolipids. Mutants for TbGPI12, deficient in the second step of GPI biosynthesis, were devoid of cell surface procyclins and poly-N-acetyllactosamine–containing free GPI glycolipids. This major disruption to their surface architecture severely impaired their ability to colonize tsetse fly midguts but, surprisingly, had no effect on their morphology and growth characteristics in vitro. Transmission electron microscopy showed that the mutants retained a cell surface glycocalyx. This structure, and the viability of the mutants in vitro, prompted us to look for non-GPI–anchored parasite molecules and/or the adsorption of serum components. Neither were apparent from cell surface biotinylation experiments but [3H]glucosamine biosynthetic labeling revealed a group of previously unidentified high apparent molecular weight glycoconjugates that might contribute to the surface coat. While characterizing GlcNAc-PI that accumulates in the TbGPI12 mutant, we observed inositolphosphoceramides for the first time in this organism.

scholarly journals Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei

2009 ◽  
Vol 8 (9) ◽  
pp. 1407-1417 ◽  
Author(s):  
Maria Lucia Sampaio Güther ◽  
Kenneth Beattie ◽  
Douglas J. Lamont ◽  
John James ◽  
Alan R. Prescott ◽  
...  
Keyword(s):  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Life Cycle Stage ◽  
Galactose Oxidase ◽  
Surface Coat ◽  
Wild Type ◽  
In The Wild ◽  
P Type

ABSTRACT A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB3H4 labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB3H4 labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.

scholarly journals Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei.

1989 ◽  
Vol 108 (2) ◽  
pp. 737-746 ◽  
Author(s):  
I Roditi ◽  
H Schwarz ◽  
T W Pearson ◽  
R P Beecroft ◽  
M K Liu ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Tsetse Fly ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Mammalian Host ◽  
Cylindrical Shape ◽  
Surface Coat ◽  
Insect Vector ◽  
Molecular Arrangement

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.

Phosphorylation of a major GPI-anchored surface protein of Trypanosoma brucei during transport to the plasma membrane

1999 ◽  
Vol 112 (11) ◽  
pp. 1785-1795 ◽  
Author(s):  
P. Butikofer ◽  
E. Vassella ◽  
S. Ruepp ◽  
M. Boschung ◽  
G. Civenni ◽  
...  
Keyword(s):  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Surface Protein ◽  
Surface Expression ◽  
Surface Coat ◽  
Placental Alkaline Phosphatase ◽  
Flagellar Pocket ◽  
Phosphorylated Form ◽  
Human Placental Alkaline Phosphatase

The surface coat of procyclic forms of Trypanosoma brucei consists of related, internally repetitive glycoproteins known as EP and GPEET procyclins. Previously we showed that the extracellular domain of GPEET is phosphorylated. We now show that phosphorylation of this glycosylphosphatidylinositol-anchored surface protein can be induced in vitro using a procyclic membrane extract. Using antibodies that recognize either the phosphorylated or unphosphorylated form of GPEET, we analyzed their expression during differentiation of bloodstream forms to procyclic forms. Unphosphorylated GPEET, together with EP, was detected in cell lysates 2–4 hours after initiating differentiation whereas phosphorylated GPEET only appeared after 24 hours. Surface expression of EP and both forms of GPEET occurred after 24–48 hours and correlated with the detection of phosphorylated GPEET on immuno-blots. Electron micrographs showed that unphosphorylated GPEET was predominantly in the flagellar pocket whereas the phosphorylated form was distributed over the cell surface. In contrast, expression of a membrane-bound human placental alkaline phosphatase in procyclic forms caused the accumulation of dephosphorylated GPEET on the cell surface, while the phosphorylated form was restricted to the flagellar pocket. A GPEET-Fc fusion protein, which was retained intracellularly, was not phosphorylated. We propose that unphosphorylated GPEET procyclin is transported to a location close to or at the cell surface, most probably the flagellar pocket, where it becomes phosphorylated. To the best of our knowledge, this study represents the first localization of phosphorylated and unphosphorylated forms of a GPI-anchored protein within a cell.

Capping of variable antigen on Trypanosoma brucei, and its immunological and biological significance

1979 ◽  
Vol 37 (1) ◽  
pp. 287-302
Author(s):  
J.D. Barry
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Surface Antigen ◽  
Mammalian Cells ◽  
Antigenic Variation ◽  
Surface Coat ◽  
Antibody Molecule ◽  
Variable Antigen

Pathogenic trypanosomes undergo antigenic variation, whereby the glycoprotein molecules constituting the cell coat are changed, the parasite thus evading the host's immune response. On application of homologous antiserum in indirect immunofluorescence to a given variable antigen type of Trypanosoma brucei, the surface variable antigen moves to the flagellar pocket region, which overlies the Golgi apparatus. This redistribution, or capping, is temperature-dependent, occurring at 37 degrees C but not at 0-4 degree C. Patching does not occur at either temperature. Immediately after capping no homologous or heterologous variable antigen, or host plasma or blood cell antigens, can be detected by immunofluorescence on the cell surface outside the cap; only trypanosome membrane common antigens can be found. It seems unlikely for two reasons that this antibody-induced redistribution is relevant to antigenic variation. Capping of the coat requires the indirect, rather than the direct, immunofluorescent method; a single layer of antibody, in nature, would appear to be ineffective. Also, capping of variable antigen of one type is followed within 3 h by appearance of antigen of the same, and not another, type. The necessity for 2 antibody layers is usually thought of as meaning that the individual molecules of the cell surface antigen are spaced further apart than the binding sites of an individual antibody molecule, so that the necessary cross-linked lattice cannot be formed, but on T. brucei the surface variable antigen molecules are very closely packed. It is proposed that one layer of antibody is ineffective for steric reasons; the dimensions of the exposed face of each variable antigen molecule may not permit the binding of more than one molecule of immunoglobulin, or perhaps the antigen molecules are so closely packed that most of the antigenic determinants are hidden from antibodies. To test this hypothesis, an attempt was made to cap variable antigen on trypanosomes transforming in vitro from the bloodstream to the procyclic (insect midgut) stage; such forms have a much less densely packed surface coat. Patching was observed, indicative of lattice formation, but these trypanosomes did not survive the in vitro manipulation long enough to permit any possible capping. T. brucei differs structurally from most other eukaryotic cells. It has no detectable microfilaments under the plasma membrane, except at the desmosomes in the region of flagellar binding, and it also has a pellicular cortex of microtubules. Capping of its surface antigen would appear then to differ from that on mammalian cells, either in the cellular components involved or in that specialized areas of the plasma membrane are involved.

Factors Controlling Electropermeabilisation of Cell Membranes

2002 ◽  
Vol 1 (5) ◽  
pp. 319-327 ◽  
Author(s):  
M. P. Rols ◽  
M. Golzio ◽  
B. Gabriel ◽  
J. Teissié
Keyword(s):  
Cell Surface ◽  
Electric Field ◽  
Pulse Duration ◽  
Cell Membranes ◽  
Physical Parameters ◽  
Local Exchange ◽  
Physical Mechanisms ◽  
A Cell

Electric field pulses are a new approach for drug and gene delivery for cancer therapy. They induce a localized structural alteration of cell membranes. The associated physical mechanisms are well explained and can be safely controlled. A position dependent modulation of the membrane potential difference is induced when an electric field is applied to a cell. Electric field pulses with an overcritical intensity evoke a local membrane alteration. A free exchange of hydrophilic low molecular weight molecules takes place across the membrane. A leakage of cytosolic metabolites and a loading of polar drugs into the cytoplasm are obtained. The fraction of the cell surface which is competent for exchange is a function of the field intensity. The level of local exchange is strongly controlled by the pulse duration and the number of successive pulses. The permeabilised state is long lived. Its lifetime is under the control of the cumulated pulse duration. Cell viability can be preserved. Gene transfer is obtained but its mechanism is not a free diffusion. Plasmids are electrophoretically accumulated against the permeabilised cell surface and form aggregates due to the field effect. After the pulses, several steps follow: translocation to the cytoplasm, traffic to the nucleus and expression. Molecular structural and metabolic changes in cells remain mostly poorly understood. Nevertheless, while most studies were established on cells in culture ( in vitro), recent experiments show that similar effects are obtained on tissue ( in vivo). Transfer remains controlled by the physical parameters of the electrical treatment.

scholarly journals A Cell Surface Biotinylation Assay to Reveal Membrane-associated Neuronal Cues: Negr1 Regulates Dendritic Arborization

2013 ◽  
Vol 13 (3) ◽  
pp. 733-748 ◽  
Author(s):  
Francesca Pischedda ◽  
Joanna Szczurkowska ◽  
Maria Daniela Cirnaru ◽  
Florian Giesert ◽  
Elena Vezzoli ◽  
...  
Keyword(s):  
Cell Surface ◽  
Dendritic Arborization ◽  
Cell Surface Biotinylation ◽  
A Cell

scholarly journals Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N -Acetyltransferase from Trypanosoma brucei

2011 ◽  
Vol 10 (7) ◽  
pp. 985-997 ◽  
Author(s):  
Karina Mariño ◽  
M. Lucia Sampaio Güther ◽  
Amy K. Wernimont ◽  
Wei Qiu ◽  
Raymond Hui ◽  
...  
Keyword(s):  
Crystal Structure ◽  
High Resolution ◽  
Trypanosoma Brucei ◽  
Protein Glycosylation ◽  
Surface Glycoprotein ◽  
Surface Coat ◽  
Content Type ◽  
Phosphate Analysis ◽  
Novel Drug

ABSTRACT A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N -acetyltransferase ( TbGNA1 ; EC 2.3.1.4) was cloned and expressed in Escherichia coli . The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly- N -acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed.

Trypanosome sociology and antigenic variation

Parasitology ◽  
1989 ◽  
Vol 99 (S1) ◽  
pp. S37-S47 ◽  
Author(s):  
K. Vickerman
Keyword(s):  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Growth Characteristics ◽  
Tsetse Fly ◽  
Antigenic Specificity ◽  
Surface Coat ◽  
Humoral Immune ◽  
Variable Antigen ◽  
Minority Variants ◽  
Stimulation Of

SUMMARYSurvival of the trypanosome (Trypanosoma brucei) population in the mammalian body depends upon paced stimulation of the host's humoral immune response by different antigenic variants and serial sacrifice of the dominant variant (homotype) so that minority variants (heterotypes) can continue the infection and each become a homotype in its turn. New variants are generated by a spontaneous switch in gene expression so that the trypanosome puts on a surface coat of a glycoprotein differing in antigenic specificity from its predecessor. Homotypes appear in a characteristic order for a given trypanosome clone but what determines this order and the pacing of homotype generation so that the trypanosome does not quickly exhaust its repertoire of variable antigens, is not clear. The tendency of some genes to be expressed more frequently than others may reflect the location within the genome and mode of expression of the genes concerned and may influence homotype succession. Differences in the doubling time of different variants or in the rate at which trypanosomes belonging to a particular variant differentiate into non-dividing (vector infective) stumpy forms have also been invoked to explain how a heterotype's growth characteristics may determine when it becomes a homotype. Recent estimations of the frequency of variable antigen switching in trypanosome populations after transmission through the tsetse fly vector, however, suggest a much higher figure (0·97–2·2 × 10−3switches per cell per generation) than that obtained for syringe-passed infections (10−5–10−7switches per cell per generation) and it seems probable that most of the variable antigen genes are expressed as minority variable antigen types very early in the infection. Instability of expression is a feature of trypanosome clones derived from infective tsetse salivary gland (metacyclic) trypanosomes and it is suggested that high switching rates in tsetse-transmitted infections may delay the growth of certain variants to homotype status until later in the infection.

scholarly journals PTP1B Modulates the Association of β-Catenin with N-cadherin through Binding to an Adjacent and Partially Overlapping Target Site

2002 ◽  
Vol 277 (51) ◽  
pp. 49989-49997 ◽  
Author(s):  
Gang Xu ◽  
Carlos Arregui ◽  
Jack Lilien ◽  
Janne Balsamo
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Tyrosine Phosphatase ◽  
Target Site ◽  
Target Domain ◽  
Chick Retina ◽  
Cell Permeable Peptide ◽  
A Cell ◽  
Relationship Of

The nonreceptor tyrosine phosphatase PTP1B associates with the cytoplasmic domain of N-cadherin and may regulate cadherin function through dephosphorylation of β-catenin. We have now identified the domain on N-cadherin to which PTP1B binds and characterized the effect of perturbing this domain on cadherin function. Deletion constructs lacking amino acids 872–891 fail to bind PTP1B. This domain partially overlaps with the β-catenin binding domain. To further define the relationship of these two sites, we used peptides to competein vitrobinding. A peptide representing the most NH2-terminal 8 amino acids of the PTP1B binding site, the region of overlap with the β-catenin target, effectively competes for binding of β-catenin but is much less effective in competing PTP1B, whereas two peptides representing the remaining 12 amino acids have no effect on β-catenin binding but effectively compete for PTP1B binding. Introduction into embryonic chick retina cells of a cell-permeable peptide mimicking the 8 most COOH-terminal amino acids in the PTP1B target domain, the region most distant from the β-catenin target site, prevents binding of PTP1B, increases the pool of free, tyrosine-phosphorylated β-catenin, and results in loss of N-cadherin function. N-cadherin lacking this same region of the PTP1B target site does not associate with PTP1B or β-catenin and is not efficiently expressed at the cell surface of transfected L cells. Thus, interaction of PTP1B with N-cadherin is essential for its association with β-catenin, stable expression at the cell surface, and consequently, cadherin function.

Trypanosoma brucei vacuolar protein sorting 41 (VPS41) is required for intracellular iron utilization and maintenance of normal cellular morphology

Parasitology ◽  
2007 ◽  
Vol 134 (11) ◽  
pp. 1639-1647 ◽  
Author(s):  
S. LU ◽  
T. SUZUKI ◽  
N. IIZUKA ◽  
S. OHSHIMA ◽  
Y. YABU ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Iron Metabolism ◽  
Protein Sorting ◽  
Tsetse Fly ◽  
Cellular Morphology ◽  
Yeast Cells ◽  
Intracellular Iron ◽  
Procyclic Form ◽  
Vacuolar Protein ◽  
Iron Utilization

SUMMARYProcyclic forms of Trypanosoma brucei brucei remain and propagate in the midgut of tsetse fly where iron is rich. Additional iron is also required for their growth in in vitro culture. However, little is known about the genes involved in iron metabolism and the mechanism of iron utilization in procyclic-form cells. Therefore, we surveyed the genes involved in iron metabolism in the T. b. brucei genome sequence database. We found a potential homologue of vacuole protein sorting 41 (VPS41), a gene that is required for high-affinity iron transport in Saccharomyces cerevisiae and cloned the full-length gene (TbVPS41). Complementation analysis of TbVPS41 in ΔScvps41 yeast cells showed that TbVPS41 could partially suppress the inability of ΔScvps41 yeast cells to grow on low-iron medium, but it could not suppress the fragmented vacuole phenotype. Further RNA interference (RNAi)-mediated gene knock-down in procyclic-form cells resulted in a significant reduction of growth in low-iron medium; however, no change in growth was observed in normal culture medium. Transmission electron microscopy showed that RNAi caused T. b. brucei cells to have larger numbers of small intracellular vesicles, similar to the fragmented vacuoles observed in ΔScvps41 yeast cells. The present study demonstrates that TbVPS41 plays an important role in the intracellular iron utilization system as well as in the maintenance of normal cellular morphology.

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