scholarly journals Identification of glycosylphosphatidylinositol-specific phospholipases C in mouse brain membranes

1990 ◽  
Vol 269 (2) ◽  
pp. 321-327 ◽  
Author(s):  
F Fouchier ◽  
T Baltz ◽  
G Rougon
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Mouse Brain ◽  
The Other ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Acidic Ph ◽  
Lysosomal Compartment ◽  
Membrane Compartment ◽  
Trypanosoma Equiperdum

Using the membrane form of variant surface glycoprotein from Trypanosoma equiperdum labelled with [3H]myristate as a substrate, we identified two glycosylphosphatidylinositol phospholipase C enzymic activities in mouse brain. These activities were associated with particulate membrane fractions. They were characterized by their pH activity maxima and sensitivity to activators and ion chelators. One of the activities was maximal at acidic pH, stimulated by butanol, sensitive to cation chelator and insensitive to manganese. The activity of the other was maximal at neutral pH, stimulated by the detergent deoxycholate and independent of the presence of cation chelator or calcium. On membrane subfractionation, the acidic butanol-stimulated activity was found mainly associated with the lysosomal compartment, whereas the neutral deoxycholate-stimulated activity sediments with the myelin and plasma membrane compartment. These activities could be differentiated from particulate phosphatidylinositol phospholipases C, whose acidic lysosomal form is sensitive to manganese and insensitive to cation chelator or butanol, whereas the deoxycholate-activated enzymes are Ca2(+)-dependent.

DNA rearrangements and antigenic variation in Trypanosoma equiperdum: expression-independent DNA rearrangements in the basic copy of a variant surface glycoprotein gene

1983 ◽  
Vol 3 (3) ◽  
pp. 410-414
Author(s):  
S Longacre ◽  
A Raibaud ◽  
U Hibner ◽  
G Buck ◽  
H Eisen ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Dna Rearrangements ◽  
Present Evidence ◽  
Glycoprotein Gene ◽  
Copy Gene ◽  
Genomic Environment ◽  
Trypanosoma Equiperdum

Antigenic variation in Trypanosoma equiperdum is associated with the sequential expression of variant surface glycoprotein (VSG) genes in a process which involves gene duplication and transposition events. In this paper we present evidence that the genomic environment of the VSG-1 basic copy gene, the template for duplicated, expression-linked VSG-1 genes, differs in every trypanosome clone examined. This variation is thus independent of the expression of the VSG-1 gene, and it also appears to be restricted to the 3' genomic environment. It is also demonstrated that the DNA located 3' to the VSG-1 basic copy gene is moderately sensitive to digestion when the nuclei of either expressor or non-expressor trypanosomes are treated with DNase I.

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.

Identification of potential protein partners that bind to the variant surface glycoprotein in Trypanosoma equiperdum

Parasitology ◽  
2017 ◽  
Vol 144 (7) ◽  
pp. 923-936 ◽  
Author(s):  
LIOMARY M. CARRASQUEL ◽  
JOSÉ L. ESCALONA ◽  
ALVARO ACOSTA-SERRANO ◽  
YURONG GUO ◽  
JOSÉ BUBIS
Keyword(s):  
Antigenic Variation ◽  
Orthologous Gene ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Western Blots ◽  
Binding Partners ◽  
Protein Partners ◽  
Expression Site ◽  
Tryparedoxin Peroxidase ◽  
Trypanosoma Equiperdum

SUMMARYTrypanosoma equiperdum possesses a dense coat of a variant surface glycoprotein (VSG) that is used to evade the host immune response by a process known as antigenic variation. Soluble and membrane forms of the predominant VSG from the Venezuelan T. equiperdum TeAp-N/D1 strain (sVSG and mVSG, respectively) were purified to homogeneity; and antibodies against sVSG and mVSG were raised, isolated, and employed to produce anti-idiotypic antibodies that structurally mimic the VSG surface. Prospective VSG-binding partners were initially detected by far-Western blots, and then by immunoblots using the generated anti-idiotypic antibodies. Polypeptides of ~80 and 55 kDa were isolated when anti-idiotypic antibodies–Sepharose affinity matrixes were used as baits. Mass spectrometry sequencing yielded hits with various proteins from Trypanosoma brucei such as heat-shock protein 70, tryparedoxin peroxidase, VSG variants, expression site associated gene product 6, and two hypothetical proteins. In addition, a possible interaction with a protein homologous to the glutamic acid/alanine-rich protein from Trypanosoma congolense was also found. These results indicate that the corresponding orthologous gene products are candidates for VSG-interacting proteins in T. equiperdum.

scholarly journals Formation and Remodeling of Inositolphosphoceramide during Differentiation of Trypanosoma cruzi from Trypomastigote to Amastigote

2003 ◽  
Vol 2 (4) ◽  
pp. 756-768 ◽  
Author(s):  
Maria Laura Salto ◽  
Laura E. Bertello ◽  
Mauricio Vieira ◽  
Roberto Docampo ◽  
Silvia N. J. Moreno ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Trypanosoma Cruzi ◽  
Palmitic Acid ◽  
Phospholipase C ◽  
Phospholipase A ◽  
Acidic Ph ◽  
Acid Hydrolase ◽  
Aureobasidin A

ABSTRACT Differentiation of Trypanosoma cruzi trypomastigotes to amastigotes inside myoblasts or in vitro, at low extracellular pH, in the presence of [3H]palmitic acid or [3H]inositol revealed differential labeling of inositolphosphoceramide and phosphatidylinositol, suggesting that a remodeling process takes place in both lipids. Using 3H-labeled inositolphosphoceramide and phosphatidylinositol as substrates, we demonstrated the association of at least five enzymatic activities with the membranes of amastigotes and trypomastigotes. These included phospholipase A1, phospholipase A2, inositolphosphoceramide-fatty acid hydrolase, acyltransferase, and a phospholipase C releasing either ceramide or a glycerolipid from the inositolphospholipids. These enzymes may be acting in remodeling reactions leading to the anchor of mature glycoproteins or glycoinositolphospholipids and helping in the transformation of the plasma membrane, a necessary step in the differentiation of slender trypomastigotes to round amastigotes. Synthesis of inositolphosphoceramide and particularly of glycoinositolphospholipids was inhibited by aureobasidin A, a known inhibitor of fungal inositolphosphoceramide synthases. The antibiotic impaired the differentiation of trypomastigotes at acidic pH, as indicated by an increased appearance of intermediate forms and a decreased expression of the Ssp4 glycoprotein, a characteristic marker of amastigote forms. Aureobasidin A was also toxic to differentiating trypomastigotes at acidic pH but not to trypomastigotes maintained at neutral pH. Our data suggest that inositolphosphoceramide is implicated in T. cruzi differentiation and that its metabolism could provide important targets for the development of antiparasitic therapies.

scholarly journals Cloning and characterization of a variant surface glycoprotein expression site from Trypanosoma equiperdum.

1986 ◽  
Vol 6 (8) ◽  
pp. 2950-2956 ◽  
Author(s):  
A Raibaud ◽  
G Buck ◽  
T Baltz ◽  
H Eisen
Keyword(s):  
Trypanosoma Brucei ◽  
Southern Blot Analysis ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
African Trypanosomes ◽  
Active Expression ◽  
Expression Site ◽  
Trypanosoma Equiperdum ◽  
Dna Fragment

Variant surface glycoprotein (VSG) genes of African trypanosomes are expressed when they are inserted into one of several telomere-linked expression sites. We cloned and characterized an 11-kilobase (kb) DNA fragment located upstream of an expressed VSG gene. A DNA sequence of 1.8 kb that is located immediately upstream of the inserted VSG gene contains sequences homologous to the 76-base-pair repeats described as being upstream of VSG genes in Trypanosoma brucei (D. A. Campbell, M. P. Van Bree, and J. C. Boothroyd, Nucleic Acids Res. 12:2759-2774). There are no such sequences elsewhere in the 11-kb cloned region. Southern blot analysis using probes from the cloned region revealed multiple unlinked copies of the same or very similar regions. At least three of these are located near telomeres, and two have been shown to be used for the expression of known Trypanosoma equiperdum VSG genes. Like VSG genes, the upstream sequences themselves can be duplicated and deleted. The choice of expression site to be used by a duplicated VSG gene is nonrandom; the site used for expression of the parental VSG gene is strongly favored for use in the daughter variant. Furthermore, even when the parental expression site is not used, the VSG gene occupying it is replaced. Thus, an active expression site is a preferential target for gene conversion in the next variation event.

scholarly journals An endogenous glycosylphosphatidylinositol-specific phospholipase D releases basic fibroblast growth factor-heparan sulfate proteoglycan complexes from human bone marrow cultures

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2115-2125 ◽  
Author(s):  
G Brunner ◽  
CN Metz ◽  
H Nguyen ◽  
J Gabrilove ◽  
SR Patel ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Heparan Sulfate Proteoglycan ◽  
Biologically Active ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Sulfate Proteoglycan ◽  
Fibroblast Growth ◽  
Gpi Anchor

Basic fibroblast growth factor (bFGF) is a hematopoietic cytokine that stimulates stromal and stem cell growth. It binds to a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan on human bone marrow (BM) stromal cells. The bFGF- proteoglycan complex is biologically active and is released by addition of exogenous phosphatidylinositol-specific phospholipase C. In this study, we show the presence of an endogenous GPI-specific phospholipase D (GPI-PLD) that releases the bFGF-binding heparan sulfate proteoglycan and the variant surface glycoprotein (a model GPI-anchored protein) from BM cultures. An involvement of proteases in this process is unlikely, because released proteoglycan contained the GPI anchor component, ethanol-amine, and protease inhibitors did not diminish the release. The mechanism of release is likely to involve a GPI-PLD and not a GPI-specific phospholipase C, because the release of variant surface glycoprotein did not reveal an epitope called the cross- reacting determinant that is exposed by phospholipase C-catalyzed GPI anchor cleavage. In addition, phosphatidic acid (which is specifically a product of GPI-PLD-catalyzed anchor cleavage) was generated during the spontaneous release of the GPI-anchored variant surface glycoprotein. We also detected GPI-PLD-specific enzyme activity and mRNA in BM cells. Therefore, we conclude that an endogenous GPI-PLD releases bFGF-heparan sulfate proteoglycan complexes from human BM cultures. This mechanism of GPI anchor cleavage could be relevant for mobilizing biologically active bFGF in BM. An endogenous GPI-PLD could also release other GPI-anchored proteins important for hematopoiesis and other physiologic processes.

scholarly journals Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes

2015 ◽  
Vol 112 (21) ◽  
pp. E2803-E2812 ◽  
Author(s):  
Igor Cestari ◽  
Ken Stuart
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Inositol Phosphate ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Activator Protein 1 ◽  
African Trypanosomes ◽  
Polymerase I

African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing.

scholarly journals ATPases, ion exchangers and human sperm motility

Reproduction ◽  
2015 ◽  
Vol 149 (5) ◽  
pp. 475-484 ◽  
Author(s):  
Rubén D Peralta-Arias ◽  
Carmen Y Vívenes ◽  
María I Camejo ◽  
Sandy Piñero ◽  
Teresa Proverbio ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Sperm Motility ◽  
Plasma Membranes ◽  
Semen Analysis ◽  
Human Sperm ◽  
The Other ◽  
Computer Assisted ◽  
Acidic Ph ◽  
Α Subunit

Human sperm has several mechanisms to control its ionic milieu, such as the Na,K-ATPase (NKA), the Ca-ATPase of the plasma membrane (PMCA), the Na+/Ca2+-exchanger (NCX) and the Na+/H+-exchanger (NHE). On the other hand, the dynein-ATPase is the intracellular motor for sperm motility. In this work, we evaluated NKA, PMCA, NHE, NCX and dynein-ATPase activities in human sperm and investigated their correlation with sperm motility. Sperm motility was measured by Computer Assisted Semen Analysis. It was found that the NKA activity is inhibited by ouabain with twoKi(7.9×10−9and 9.8×10−5 M), which is consistent with the presence of two isoforms of α subunit of the NKA in the sperm plasma membranes (α1 and α4), being α4 more sensitive to ouabain. The decrease in NKA activity is associated with a reduction in sperm motility. In addition, sperm motility was evaluated in the presence of known inhibitors of NHE, PMCA and NCX, such as amiloride, eosin, and KB-R7943, respectively, as well as in the presence of nigericin after incubation with ouabain. Amiloride, eosin and KB-R7943 significantly reduced sperm motility. Nigericin reversed the effect of ouabain and amiloride on sperm motility. Dynein-ATPase activity was inhibited by acidic pH and micromolar concentrations of Ca2+. We explain our results in terms of inhibition of the dynein-ATPase in the presence of higher cytosolic H+and Ca2+, and therefore inhibition of sperm motility.

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