Trypanosoma brucei: cis-aconitate and temperature reduction as triggers of synchronous transformation of bloodstream to procyclic trypomastigotes in vitro

Synchronous transformation of the monomorphic Trypanosoma brucei 427 variant clone MITat 1.4 (117) from bloodstream to procyclic trypomastigotes was studied in modified minimum essential medium plus 15% inactivated horse serum. Repression of variant surface glycoprotein synthesis, subsequent morphological transformation, and growth of procyclic cells was triggered by the simultaneous action of two signals: a reduction in temperature from 37 to 27 C and the addition of cis-aconitate. Repression of variant surface glycoprotein synthesis initiated by these two signals is reversible during the first hours, but becomes irreversible after about 1 day. Thereafter, cells are committed to differentiation at 27 C.     

Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-glycosylation and addition of a phosphatidylinositol membrane anchor

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are synthesized with a hydrophobic COOH-terminal peptide that is cleaved and replaced by a glycophospholipid, which anchors VSG to the surface membrane. The kinetics of VSG processing were studied by metabolic labeling with [35S]methionine and [3H]myristic acid. The COOH-terminal oligosaccharide-containing structure remaining after phospholipase removal of dimyristyl glycerol from membrane-form VSG could be detected serologically within 1 min of polypeptide synthesis in two T. brucei variants studied. Addition of the oligosaccharide-containing structure was resistant to tunicamycin. VSGs synthesized in the presence of tunicamycin displayed lower apparent molecular weights, consistent with the complete inhibition of N-glycosylation at one (variant 117), two (variant 221), or at least three (variant 118) internal asparagine sites. In most experiments, N-glycosylation appeared to occur during or immediately after polypeptide synthesis but in a few cases N-glycosylation was delayed or incomplete. In all cases, addition of the COOH-terminal oligosaccharide-containing structure occurred normally. In dual-labeling studies, cycloheximide caused rapid inhibition of both [35S]methionine and [3H]myristic acid incorporation, suggesting that myristic acid addition also occurs immediately after polypeptide synthesis. Our data suggest that the complex ethanolamine-glycosyl-dimyristylphosphatidylinositol structure of membrane-form VSG is added en bloc within 1 min of completion of the polypeptide.     

Glycosyl-sn-1,2-dimyristylphosphatidylinositol is covalently linked to Trypanosoma brucei variant surface glycoprotein

The COOH terminus of the externally disposed variant surface glycoprotein (VSG) of the eukaryotic pathogenic protozoan Trypanosoma brucei strain 427 variant MITat 1.4 (117) is covalently linked to a novel phosphatidylinositol-containing glycolipid. This conclusion is supported by analysis of the products of nitrous acid deamination or Staphylococcus aureus phosphatidylinositol-specific phospholipase C treatment of purified membrane-form VSG. Lysis of trypanosomes is accompanied by release of soluble VSG, catalyzed by activation of an endogenous phospholipase C. The only apparent difference between membrane-form VSG and soluble VSG is the removal of sn-1,2-dimyristylglycerol. The COOH-terminal glycopeptide derived by Pronase digestion of soluble VSG was characterized by chemical modification and digestion with alkaline phosphatase. The results are consistent with the single non-N-acetylated glucosamine residue being the reducing terminus of the oligosaccharide and in a glycosidic linkage to a myo-inositol monophosphate that is probably myo-inositol 1,2-cyclic monophosphate. A partial structure for the VSG COOH-terminal moiety is presented. This structure represents a new type of eukaryotic post-translational protein modification and membrane anchor. We discuss the relevance of this structure to observations that have been made with other eukaryotic membrane proteins.     

A role for the dynamic acylation of a cluster of cysteine residues in regulating the activity of the glycosylphosphatidylinositol-specific phospholipase C of Trypanosoma brucei

The glycosylphosphatidylinositol-specific phospholipase C or VSG lipase is the enzyme responsible for the cleavage of the glycosylphosphatidylinositol anchor of the variant surface glycoprotein (VSG) and concomitant release of the surface coat in Trypanosoma brucei during osmotic shock or extracellular acidic stress. In Xenopus laevis oocytes the VSG lipase was expressed as a nonacylated and a thioacylated form. This thioacylation occurred within a cluster of three cysteine residues but was not essential for catalytic activity per se. These two forms were also detected in trypanosomes and appeared to be present at roughly equivalent amounts. A reversible shift to the acylated form occurred when cells were triggered to release the VSG by either nonlytic acid stress or osmotic lysis. A wild type VSG lipase or a gene mutated in the three codons for the acylated cysteines were reinserted in the genome of a trypanosome null mutant for this gene. A comparative analysis of these revertant trypanosomes indicated that thioacylation might be involved in regulating enzyme access to the VSG substrate.     

Release and purification of Trypanosoma brucei variant surface glycoprotein

Conditions affecting the solubilization of variant surface glycoprotein (VSG) from Trypanosoma brucei have been investigated. The results obtained form the basis for a convenient and efficient method for VSG purification. VSG release from the cell surface was temperature-dependent, following osmotic lysis at 0 degree C, and was inhibited by low concentrations of Zn2+ but not by tosyl-lysine chloromethyl-ketone (TLCK), phenylmethylsulfonylfluoride (PMSF), or iodoacetamide. These and other results eliminated the possibility that release was due to proteolytic cleavage of the C-terminal hydrophobic tail present on newly synthesized VSG. Bolton and Hunter reagent reacted with several components on living cells.     

Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein

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.     

Biosynthesis of rat liver pyruvate kinase. Measurement of enzyme lifetime and the rate of synthesis at weaning.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of immunoprecipitates of liver cytosol with anti-(L-type pyruvate kinase) serum revealed proteins of mol.wt. 56 000 and 42 000 in addition to the heavy and light chains. The ratio of the 56 000 mol.wt. to the 42 000 mol.wt. protein increased under dietary conditions that resulted in an increase in the apparent specific activity of hepatic pyruvate kinase. The 42 000 mol.wt. protein was removed from immunoprecipitates if the liver cytosol was partially purified by pH precipitation and (NH4)2SO4 fractionation before addition of the antiserum. This technique may be used to analyse the formation of pure L-type pyruvate kinase in liver. By using H14CO3-labelling, the t1/2 of L-type pyruvate kinase was estimated as 75 +/- 1.7 h in post-weaned high-carbohydrate-diet-fed rats. Before weaning there was little immunoreactive pyruvate kinase in rat liver cytosol. Induction began between 6 and 24 h after weaning and reached a maximum value 120 h after weaning. When clearly enhanced total pyruvate kinase activity was first observed at 24 h post-weaning, the apparent specific activity of hepatic pyruvate kinase was considerably lower than the specific activity of the pure isolated enzyme. When the induction of L-type pyruvate kinase was monitored by the incorporation of L-[4,5-3H]leucine, the maximum rate of synthesis occurred 24--48 h after weaning. After this period synthesis declined, indicating a relatively slow turnover of the enzyme once the enzyme concentration was established in the liver.     

Variant surface glycoprotein of Trypanosoma brucei brucei AnTat 1.1: influence of the isolation conditions upon the disulfide linked dimer/monomer ratio

1. Using the variant surface glycoprotein (VSG) isolation procedure described by Baltz et al. ([1976] Ann. Immunol. (Inst. Pasteur) 127 C, 761-774) which involves suspension of the trypanosomes in a pH 5.5 buffer, the Antwerpen trypanozoon antigenic type (AnTat) 1.1 VSG is mainly obtained as a disulfide linked dimeric form with a trace amount of a monomeric form. 2. The use of a parasite suspension buffer at pH 7.0 results in a slight decrease of the VSG dimer/monomer ratio. 3. pH 5.5 and 7.0 supernatants of centrifuged parasite suspensions were submitted to kinetic incubations at different temperatures and pH, and we found conditions involving transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer (shifting the pH 5.5 supernatant to pH 7.0 and incubation at room temperature). 4. This transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer is activated by the addition of 1 mM reduced glutathione, and is inhibited by the addition of 1 mM oxidized glutathione or 0.1 mM N-ethylmaleimide or cadmium acetate.     

A function for a specific zinc metalloprotease of African trypanosomes

The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains approximately 30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B (-/-) trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC (-/-) cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B (-/-) PLC (-/-) trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.     

Surface coat remodeling during differentiation of Trypanosoma brucei

African trypanosomes (Trypanosoma brucei) are digenetic parasites whose lifecycle alternates between the mammalian bloodstream and the midgut of the tsetse fly vector. In mammals, proliferating long slender parasites transform into non-diving short stumpy forms, which differentiate into procyclic forms when ingested by the tsetse fly. A hallmark of differentiation is the replacement of the bloodstream stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procylin. An undefined endoprotease and endogenous glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) have been implicated in releasing the old VSG coat. However, GPI hydrolysis has been considered unimportant because (i) GPI-PLC null mutants are fully viable and (ii) cytosolic GPI-PLC is localized away from cell surface VSG. Utilizing an in vitro differentiation assay with pleomorphic strains we have investigated these modes of VSG release. Shedding is initially by GPI hydrolysis, which ultimately accounts for a substantial portion of total release. Surface biotinylation assays indicate that GPI-PLC does gain access to extracellular VSG, suggesting that this mode is primed in the starting short stumpy population. Proteolytic release is up-regulated during differentiation and is stereoselectively inhibited by peptidomimetic collagenase inhibitors, implicating a zinc metalloprotease. This protease may be related to TbMSP-B, a trypanosomal homologue of Leishmania major surface protease (MSP) described in the accompanying paper (LaCount, D. J., Gruszynski, A. E., Grandgenett, P. M., Bangs, J. D., and Donelson, J. E. (2003) J. Biol. Chem. 278, 24658-24664). Overall, our results demonstrate that surface coat remodeling during differentiation has multiple mechanisms and that GPI-PLC plays a more significant role in VSG release than previously thought.     

Patterns of proteins synthesized by non-proliferating murine L cells

When L cells were plated at high density (2 × 10⁵/cm²), proliferation ceased within 3 days, but the cells remained viable and capable of reinitiating DNA synthesis for at least 16 days. SDS-polyacrylamide gel electrophoresis of [³⁵S]methionine labeled polypeptides at various intervals after plating revealed distinct changes in the relative abundance of major cellular proteins beginning 9 days after DNA synthesis ceased. An 84 000 mol. wt polypeptide increased markedly in amount while a polypeptide of 94 000 mol. wt disappeared. Autoradiograms following pulse-labeling showed that these changes were due to increased synthesis of the 84 000 mol. wt polypeptide and decreased synthesis of the 94 000 mol. wt polypeptide. Increased synthesis of a 109 000 mol. wt polypeptide occurred without a concomitant change in its relative abundance. These alterations in the pattern of proteins synthesized revert to normal after feeding with serum-free medium even though DNA synthesis does not resume. Therefore, it appears that even though no abrupt changes in the synthesis of major cellular proteins occurred upon cessation of proliferation, eventually a distinct adaptive pattern of protein synthesis develops in response to the changing culture conditions.     

Degradation, recycling, and shedding of Trypanosoma brucei variant surface glycoprotein

Trypanosoma brucei bloodstream forms express a densely packed surface coat consisting of identical variant surface glycoprotein (VSG) molecules. This surface coat is subject to antigenic variation by sequential expression of different VSG genes and thus enables the cells to escape the mammalian host's specific immune response. VSG turnover was investigated and compared with the antigen switching rate. Living cells were radiochemically labeled with either 125I-Bolton-Hunter reagent or 35S-methionine, and immunogold-surface labeled for electron microscopy studies. The fate of labeled VSG was studied during subsequent incubation or cultivation of labeled trypanosomes. Our data show that living cells slowly released VSG into the medium with a shedding rate of 2.2 +/- 0.6% h-1 (t1/2 = 33 +/- 9 h). In contrast, VSG degradation accounted for only 0.3 +/- 0.06% h-1 (t1/2 = 237 +/- 45 h) and followed the classical lysosomal pathway as judged by electron microscopy. Since VSG uptake by endocytosis was rather high, our data suggest that most of the endocytosed VSG was recycled to the surface membrane. These results indicate that shedding of VSG at a regular turnover rate is sufficient to remove the old VSG coat within one week, and no increase of the VSG turnover rate seems to be necessary during antigenic variation.     

A cell-free assay for glycosylphosphatidylinositol anchoring in African trypanosomes. Demonstration of a transamidation reaction mechanism.

We established an in vitro assay for the addition of glycosyl-phosphatidylinositol (GPI) anchors to proteins using procyclic trypanosomes engineered to express GPI-anchored variant surface glycoprotein (VSG). The assay is based on the premise that small nucleophiles, such as hydrazine, can substitute for the GPI moiety and effect displacement of the membrane anchor of a GPI-anchored protein or pro-protein causing release of the protein into the aqueous medium. Cell membranes containing pulse-radiolabeled VSG were incubated with hydrazine, and the VSG released from the membranes was measured by carbonate extraction, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis/fluorography. Release of VSG was time- and temperature-dependent, was stimulated by hydrazine, and occurred only for VSG molecules situated in early compartments of the secretory pathway. No nucleophile-induced VSG release was seen in membranes prepared from cells expressing a VSG variant with a conventional transmembrane anchor (i.e. a nonfunctional GPI signal sequence). Pro-VSG was shown to be a substrate in the reaction by assaying membranes prepared from cells treated with mannosamine, a GPI biosynthesis inhibitor. When a biotinylated derivative of hydrazine was used instead of hydrazine, the released VSG could be precipitated with streptavidin-agarose, indicating that the biotin moiety was covalently incorporated into the protein. Hydrazine was shown to block the C terminus of the released VSG hydrazide because the released material, unlike a truncated form of VSG lacking a GPI signal sequence, was not susceptible to proteolysis by carboxypeptidases. These results firmly establish that the released material in our assay is VSG hydrazide and strengthen the proof that GPI anchoring proceeds via a transamidation reaction mechanism. The reaction could be inhibited with sulfhydryl alkylating reagents, suggesting that the transamidase enzyme contains a functionally important sulfhydryl residue.     

The variable surface glycoproteins of Trypanosoma equiperdum are phosphorylated.

The phosphoproteins from three Trypanosoma equiperdum variants were studied by labelling the parasites in vivo with 32P. Phosphoprotein analysis reveals the presence of a 58 000 mol. wt. phosphoprotein ( pp58 ) which is absent when live trypanosomes are pre-treated with proteinase K under conditions where only the surface coat containing the variable surface glycoprotein (VSG) is removed. Immunological and fingerprint analysis on labelled pp58 , purified from these variants by affinity chromatography on Concanavalin A-Sepharose, clearly identify this component as the VSG. Furthermore, the VSGs seem to be phosphorylated to the extent of 1 mol phosphate per mol glycoprotein. The phosphorylated region is located in the extreme C-terminal region representing approximately 10% of the total molecule. The phosphorylated residue is not an aliphatic or aromatic ester of serine, threonine, or tyrosine, nor an acyl phosphate involving an aspartyl or glutamyl residue, nor phosphohistidine. The evidence that VSGs are phosphorylated could have considerable implications for the transfer and function of these structures.     

Biosynthesis of the epidermal growth factor receptor in A431 cells.

A monoclonal antibody R1 against the human epidermal growth factor receptor has been used to study biosynthesis in the carcinoma cell line A431. Two glycoproteins of apparent mol. wts. 95 000 and 160 000 were immunoprecipitated from cells labelled for short times with [35S]methionine or [3H]mannose. Pulse-chase studies show the 160 000 mol. wt. glycoprotein to be a precursor of the 175 000 mol. wt. receptor, but do not establish a precursor role for the 95 000 mol. wt. glycoprotein. Limited proteolysis, peptide mapping, endoglycosidase digestion and the use of monensin and tunicamycin show that the 95 000 mol. wt. glycoprotein is structurally related to the 160 000 mol. wt. glycoprotein and that both glycoproteins have approximately 22 000 - 28 000 mol. wt. of oligosaccharide side chains. Monensin blocks conversion of the 160 000 to the 175 000 mol. wt. mature receptor, a process which involves complexing several of its N-linked oligosaccharide chains. Pulse-chase studies showed that an immunoprecipitable polypeptide of 115 000 mol. wt., or 95 000 mol. wt., in the presence of monensin, was secreted into the medium at late chase times. The possible mechanisms for the origins of all the receptor-related polypeptides are discussed.     

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37 degrees C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.     

Early and late molecular and morphologic changes that occur during the in vitro transformation of Trypanosoma cruzi metacyclic trypomastigotes to amastigotes

The amastigogenesis primary of T. cruzi occurs naturally when metacyclic trypomastigotes transform into amastigotes within the cells of the mammalian host. The in vitro study of the macromolecular changes that occur over several days during the transformation process should provide significant indications of how the parasite adapts to the mammalian host environment. We show here that metacyclic trypomastigotes pre-incubated at 37 degrees C in a protein-rich medium reach a high degree of transformation to amastigotes when re-incubated in the fresh medium. Giemsa-stained smears show that during the pre-incubation phase, the metacyclic trypomastigotes undergo lengthening at the posterior end and a thinning out of the entire body. SDS-PAGE analysis of polypeptides and glycopeptides or Western blot with stage-specific antisera analyses indicate that the in vitro primary amastigogenesis is associated with abrupt changes in protein, glycoprotein, and stage-specific antigens that occur simultaneously during the first 24 hours of pre-incubation. Since the differentiating system consists of a rich media at 37 degrees C, temperature and medium constitution must trigger a macromolecular differentiation to amastigotes that precedes the morphological transformation by several days. This transformation is associated with the rearrangement of stage-specific antigens and takes place when the culture medium is changed.     

Purification and some properties of nitrate reductase (EC 1.7.99.4) from Escherichia coli K12.

1. Nitrate reductase was purified 134-fold from Escherichia coli K12. The purification procedure involves the release by Triton X-100 of the enzyme from the cell envelope. i. The purified enzyme exists in aqueous solution either as a monomer (mol. wt. about 220 000) or as an associated form (probably a tetramer; mol.wt. about 880 000). 3. The purified enzyme has three subunits with apparent mol.wts. of 150 000, 67000 and 65000. An additional subunit of apparent mol.wt. 20000 is present in a haem-containing fraction that is also produced by the preparative procedure described. 4. None of the enzyme subunits is present in the cell envelope of cells grown in the absence of nitrate. 5. Reversible changes in the activity of nitrate reductase in vitro with FMNH2 as reductant can be induced under circumstances which are without effect on the reduced Benzyl Viologen-NO3-activity.