Evolutionary conservation of a microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes.

Peroxisomes, glyoxysomes, glycosomes, and hydrogenosomes have each been classified as microbodies, i.e., subcellular organelles with an electron-dense matrix that is bound by a single membrane. We investigated whether these organelles might share a common evolutionary origin by asking if targeting signals used for translocation of proteins into these microbodies are related. A peroxisomal targeting signal (PTS) consisting of the COOH-terminal tripeptide serine-lysine-leucine-COOH has been identified in a number of peroxisomal proteins (Gould, S.J., G.-A. Keller, N. Hosken, J. Wilkinson, and S. Subramani. 1989. J. Cell Biol. 108:1657-1664). Antibodies raised to a peptide ending in this sequence (SKL-COOH) recognize a number of peroxisomal proteins. Immunocryoelectron microscopy experiments using this anti-SKL antibody revealed the presence of proteins containing the PTS within glyoxysomes of cells from Pichia pastoris, germinating castor bean seeds, and Neurospora crassa, as well as within the glycosomes of Trypanosoma brucei. Western blot analysis of purified organelle fractions revealed the presence of many proteins containing this PTS in both glyoxysomes and glycosomes. These results indicate that at least one of the signals, and therefore the mechanism, for protein translocation into peroxisomes, glyoxysomes, and glycosomes has been conserved, lending support to a common evolutionary origin for these microbodies. Hydrogenosomes, the fourth type of microbody, did not contain proteins that cross-reacted with the anti-PTS antibody, suggesting that this organelle is unrelated to microbodies.

Large microtubule-associated protein of T. brucei has tandemly repeated, near-identical sequences.

The parasitic protozoon Trypanosoma brucei contains a highly organized membrane skeleton, consisting of a dense array of parallel, singlet microtubules that are laterally interconnected and that are also in tight contact with the overlying cell membrane. A high molecular weight, heat-stable protein from this membrane skeleton was isolated that is localized along the microtubules. Protease digestion experiments and sequencing of a cloned gene segment showed that most of the protein is built up by more than 50 nearly identical tandem repeats with a periodicity of 38 amino acids.

The kinetoplast DNA of Trypanosoma equiperdum.

We have analyzed the kinetoplast DNA for Trypanosoma equiperdum (American Type Culture Collection 30019) and two dyskinetoplastic strains derived from it. The DNA networks from the kinetoplastic strain are made up of catenated mini-circles and maxi-circles, like the networks from the closely-related Trypanosoma brucei. The mini-circles of T. equiperdum lack the pronounced sequence heterogeneity of T. brucei mini-circles, as shown by the fragment distribution of restriction digests and by the predominance of well-matched duplexes in electron micrographs of renatured DNA. The electrophoretic analysis of kinetoplast DNA digested with various restriction endonucleases shows the maxi-circle of T. equiperdum to consist of circular DNA molecules of 8.4 x 10(6) daltons, without size or sequence heterogeneity or repetitious segments. A comparison of the sequence by restriction endonuclease fragmentation and hybridization shows extensive sequence homology. The size difference between both maxi-circles is due to the deletion of one continuous segment of 5.10(6) daltons. In the two dyskinetoplastic strains, we cannot detect DNA sequences that hybridize with kinetoplast DNA from T. brucei or from the kinetoplastic strain of T. equiperdum. In one of these strains, a 'low-density' DNA fraction contained a simple sequence DNA, cleaved by restriction endonuclease HindIII into fragments of 180 base-pairs and multimers of this. The relation of this DNA to kinetoplast DNA, if any, is unknown.

Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei.

A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, (Na+ +K+)-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in (3H-mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [3H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated Mr = 58000), there are several minor surface glycopeptides (Mr = 76000, 86000 and 92000-100000) which are apparent extrinsic membrane components, and two surface glycopeptides (Mr = 42000 and 130000) which are intrinsic membrane components.

African trypanosomes contain calmodulin which is distinct from host calmodulin.

Studies were initiated to determine whether African trypanosomes utilize Ca2+ fluxes to coordinate complex morphological and biochemical life cycle changes. We have identified the ubiquitous intracellular Ca2+ receptor, calmodulin, in two developmental stages of Trypanosoma brucei rhodesiense. The transition from rapidly dividing, slender bloodstream trypomastigotes to slow growing procyclics in axenic culture was accompanied by changes in specific calmodulin content (3 micrograms/mg cell protein to 1 microgram/mg cell protein, respectively) and a shift in intracellular calmodulin distribution, Trypanosome calmodulin is physically and functionally distinct from that of host tissues, including bovine brain and rat erythrocytes. It is similar to but distinct from Tetrahymena calmodulin. Comparisons among these proteins isolated from the four sources were made using the following criteria: (1) mobility on sodium dodecyl sulfate discontinuous polyacrylamide gels; (2) Ca2+-induced conformational changes; (3) CNBr-cleavage fragments; (4) activation of bovine brain cyclic nucleotide phosphodiesterase in both a Ca2+-dependent and calmodulin-dependent manner; (5) activation of human erythrocyte (Ca2+ + Mg2+)-ATPase; and (6) inhibition of calmodulin activity by trifluoperazine and penfluridol. Trifluoperazine but not trifluoperazine sulfoxide was cytotoxic to trypanosomes in vitro. Half maximal effect occurred at 15 microM. We conclude that calmodulin is a functional component of Africal trypanosomes and suggest that it plays an important role in mediating the host-parasite relationship.

Characterization of satellite DNA in Trypanosoma brucei and Trypanosoma cruzi.

We have determined the properties of the simple-sequence satellite DNAs from two protozoa, Trypanosoma brucei and Trypanosoma cruzi. The T. brucei satellite DNA contains 29 mol% guanine plus cytosine and is made up of long tandem arrays of a 177 base-pair repeat. Sequence heterogeneity in these repeats is limited and restricted to certain positions as shown by sequence analysis, restriction enzyme digestion and two-dimensional analysis of nucleotides bordering the AluI and HhaI recognition sites in the repeat. The repeat contains two copies of a 19 base-pair sequence differing by a single base-pair substitution and several additional copies of part of this sequence. Sequence variants of the repeat are clustered in the DNA. Satellite DNA is not detectably linked to other DNA and no transcripts of this DNA are found in T. brucei. The T. cruzi satellite DNA repeat is 196 base-pairs long and contains 53 mol% guanine plus cytosine. Direct repetitions longer than eight base-pairs were not observed in the nucleotide sequence of this repeat. The nucleotide sequences of the satellites of T. brucei and T. cruzi are not related. In cell fractionation experiments, the T. brucei and T. cruzi satellite DNAs were recovered from the nuclear fraction. Micrococcal nuclease digestion of nuclear fractions yielded 193 and 197 base-pair nucleosomal oligomers in T. brucei and T. cruzi, respectively; these oligomers contained satellite DNA but not the extranuclear kinetoplast DNA. The 193 base-pair nucleosomal repeat of T. brucei is significantly different from the 177 base-pair satellite repeat. Satellite and nucleosomal repeats are, therefore, not in phase in T. brucei. These satellite DNAs are the first to be observed in protozoa, and we conclude that their properties are similar to those of satellites from animals or plants.

Crystallization and preliminary X-ray analysis of an intact soluble-form variant surface glycoprotein from the African trypanosome, Trypanosoma brucei.

The intact variant surface glycoprotein (VSG) ILTat 1.24 from Trypanosoma brucei has been crystallized. An amino-terminal domain of the protein comprising two thirds of the sequence had been crystallized previously after proteolytic digestion. Now intact VSG crystals have been grown from 50 mM-Mes (pH 6.5) containing 62% (w/v) saturated ammonium sulfate. The crystals are demonstrated to contain the intact VSG by h.p.l.c. gel filtration and reaction with an antibody to the inositol phosphate oligosaccharide on the VSG carboxy terminus. The space group of the crystals is P6(2)22 (or P6(4)22) with unit cell dimensions a = b = 184 A and c = 214 A. Preparative isoelectric focusing may have facilitated crystallization.

Secondary structure of the variant surface glycoproteins of trypanosomes.

The secondary structure of seven variant surface glycoproteins (VSGs) of trypanosomes has been determined by Raman spectroscopy. They are all predominantly alpha-helical, the alpha-helix content varying between 50 and 60%. The beta-strand content varies between 20 and 25%, and the content of beta-turn and nonregular structures is about 25%. For three VSGs the N-terminal domain obtained by proteolytic cleavage was found to have essentially the same secondary structure as the complete VSGs. For three VSGs a secondary structure prediction has been performed applying the rules of Chou and Fasman. In all cases, two long alpha-helices extending over about 50 residues or 80 A are predicted in agreement with the X-ray diffraction data of Freymann et al. [(1984) Nature 311, 167-169] and Metcalf et al. [(1987) Nature 325, 84-86]. The region between the two alpha-helical segments exhibits a high potential of beta-turns, suggesting that this segment may be exposed on the cell surface and carry major antigenic determinants.

A simple method for the production of specific antiserum to protein encoded in cloned genes. Immunization with precipitin lines.

A simple technique for raising specific antisera to protein encoded by cloned genes is described. The procedure involves preparation of an antiserum to Escherichia coli beta-galactosidase and the use of that serum to immunoprecipitate a fusion protein in a crossed immunoelectrophoresis gel followed by immunization with fusion protein precipitin arcs. An antiserum was prepared against protein encoded by an open reading frame in a dispersed repeated DNA sequence found in the protozoan Trypanosoma brucei. This serum recognized a polypeptide doublet of 33.5 and 32.5 kDa on immunoblots prepared from extracts of T. brucei. The method described should be applicable to other investigations where an immunochemical reagent against protein encoded by a cloned gene is desired.

6.5 S RNA; preliminary characterisation of unusual small RNAs in Trypanosoma brucei.

In this paper we report the preliminary characterisation of some unusual small rRNAs isolated from Trypanosoma brucei. These molecules correspond closely in size and properties to the 5 S and 5.8 S rRNAs of other eukaryotes and in addition there are two further small RNAs which we designate 6.5 S RNA. These have been characterised by sucrose gradient centrifugation and electrophoresis in denaturing gels. The two 6.5 S RNAs may be distinguished from each other on the basis of their size and the different conditions required for their dissociation from the ribosomes. They are present in the same abundance as 5.8 S RNA suggesting that they occur once per ribosome.

A Trypanosoma brucei small RNP particle containing the 5S rRNA.

In mammalian cells, approximately 50% of the 5S rRNA is found in ribosomes, and the remainder in a small particle, the 5S rRNA/ribosomal protein L5 complex, which is thought to be a precursor in ribosome assembly. Trypanosoma brucei, an African trypanosome, is one of the most primitive eukaryotic organisms which have been studied, and it likewise possesses a 5S rRNA species, a small proportion of which is found in an apparent ribonucleoprotein-(RNP) complex. Like the mammalian RNP particle, the T. brucei particle has a sedimentation coefficient of about 7S in sucrose gradients; unlike its mammalian counterpart, the complex is not disrupted by high salt and can be fractionated in cesium sulfate density gradients at a density characteristic of RNP complexes (1.45 g ml-1). Our studies demonstrate the the T. brucei 7S RNP contains 5S rRNA in association with a 36-kDa rRNA binding protein which not only shares molecular size, but also immunological determinants, with the yeast ribosomal protein YL3, and its mammalian homologue, L5. These results indicate that the RNP complex formed between the 5S rRNA and the 36-kDa ribosomal protein is conserved throughout great evolutionary distances between eukaryotic species.

Culture form and tsetse fly midgut form procyclic Trypanosoma brucei express common proteins.

Proteins expressed by culture form and tsetse fly midgut form procyclic trypanosomes were examined by polyacrylamide gel electrophoretic techniques. Analysis of the proteins of the two forms of procyclic organisms was performed by comparison of autoradiographs of high resolution two-dimensional polyacrylamide gels prepared using [35S]methionine-labelled parasites. Only eight spots were found to differ between autoradiographs of culture form and tsetse fly midgut form parasites. Seven of these differences were attributable to 35S-labelled non-trypanosomal proteins from the tsetse midgut. The other single spot difference was seen in one of two experiments and was present only in the autoradiograph of the material from trypanosome-infected tsetse fly midgut. Thus the cultivated procyclic organisms did not differ significantly from their tsetse-derived counterparts in protein composition and therefore their use as models for the natural stage is probably justified for most studies.

Solution properties of the variant surface glycoprotein of Trypanosoma brucei.

The solution properties of the membrane form and soluble form of variant surface glycoproteins from Trypanosoma brucei have been compared. Solution cross-linking studies established that both forms are dimers, although dissociation of membrane-form variant surface glycoprotein can be promoted by certain ionic and zwitterionic detergents. Sedimentation coefficients were measured under a range of conditions, and the results were comparable with the results of solution cross-linking. Stokes radii were measured by gel filtration, allowing a value for the frictional coefficient to be calculated. The two forms show no differences other than those consistent with binding of detergent micelles to the hydrophobic moiety present on membrane form surface glycoprotein. This validates the use of soluble variant surface glycoprotein in X-ray crystallography experiments.

An improved method for the estimation of suramin in plasma and trypanosome samples.

An improved method for the chemical estimation of suramin is described in which the aromatic amines released from the drug by acid hydrolysis are diazotised and then coupled to N-(1-naphthyl)-ethylenediamine to form a pink coloured product (EmM545nm267 /+- 5) Provided certain precautions are followed, the assay method is highly reproducible (+/- 2%) and sufficiently sensitive to measure 2.5 nmol; suramin in mixtures with plasma (0.5 ml) or trypanosomes (200 mg wet wt.).

A glycolipid from Trypanosoma brucei related to the variant surface glycoprotein membrane anchor.

The variant surface glycoprotein (VSG) of Trypanosoma brucei is covalently linked to a phosphatidylinositol-containing glycolipid which serves as a membrane anchor. We previously identified a molecule, glycolipid A, which appears to be a biosynthetic precursor to the anchor [9]. In this paper we describe a related molecule, glycolipid C, which is similar to glycolipid A but which is more hydrophobic. Chromatographic analyses indicate that the polar head groups in glycolipids A and C are similar or identical. Both glycolipids contain phosphatidylinositol, but the inositol in glycolipid C is modified by a hydrophobic moiety. Since treatment of glycolipid C with mild alkali results in partial conversion to a molecule chromatographically identical to glycolipid A, it is likely that glycolipid C has an alkali-sensitive hydrophobic group, such as a fatty acid, linked to its inositol moiety.

A comparison of the glycosomes (microbodies) isolated from Trypanosoma brucei bloodstream form and cultured procyclic trypomastigotes.

Highly purified glycosomes were isolated from Trypanosoma brucei bloodstream forms and cultured procyclic trypomastigotes. A comparison of the specific activities of glycosomal enzymes revealed that glycosomes from insect stages had decreased levels of hexokinase, phosphoglucose isomerase, phospho-fructokinase, fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, but contained increased levels of adenylate kinase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. Glycosomes from bloodstream forms were almost totally devoid of the latter two activities. Comparison of the two types of glycosomes by sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed that bloodstream form glycosomes contained 3 prominent polypeptides (64, 46 and 40 kDa) which were hardly detectable in insect stage glycosomes, whereas the latter contained 3 insect stage specific bands with molecular weight of 34 000, 61 000 and 77 000 and 4 additional bands with molecular weights between 94 000 and 110 000. Both types of glycosome contained the phospholipids phosphatidylcholine and phosphatidylethanolamine. Insect stage glycosomes contained in addition also phosphatidylinositol and some phosphatidylserine.

Purification and cell-free translation of mRNA coding for a variant specific antigen from Trypanosoma brucei gambiense.

Messenger RNA (mRNA) coding for a variant specific surface antigen (VSA) from Trypanosoma brucei gambiense was isolated from total trypanosomal polyribosomes by indirect immunoprecipitation. An IgG fraction of antisera to purified VSA was obtained by ion exchange chromatography and Protein A-agarose affinity chromatography. These antibodies were then subjected to affinity chromatography on a VSA-agarose column to remove non-specific IgG. Polyribosomes from the same antigenic variant of T. b. gambiense were isolated from a cell lysate and those polysomes bearing nascent VSA were bound to the IgG by gentle mixing and the complexes formed were retrieved by precipitation with fixed Staphylococcus aureus cells. The VSA-specific mRNA was separated from these complexes by dissociation of the polysomes, deproteinization, and affinity chromatography on oligo(dT)-cellulose. The mRNA isolated in this way was shown to be undegraded, active in protein synthesis and homogeneous electrophoretically. The products of the cell-free translation of this mRNA were precipitable by specific IgG but not by antiserum to a heterologous VSA. The translation product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography. The molecular weight of the mRNA was measured by electron microscopy, agarose and polyacrylamide gel electrophoresis. Enough highly purified mRNA can be isolated in this manner to be used for hybridization analysis of the VSA gene(s).

Kinetoplast DNA and RNA of Trypanosoma brucei.

Kinetoplast DNA (kDNA) and kinetoplast RNA (kRNA) were isolated from bloodstream and procyclic culture forms of two clonal strains of Trypanosoma brucei. No differences were observed in kDNA (maxicircle) restriction profiles between bloodstream or procyclic culture forms of the same strain. Some differences were observed in kDNA maxicircle restriction sites between the two strains. Buoyant density analysis of Pst I digested kDNA showed the release of a minor low density band representing unit length linearized maxicircle DNA. Pst I or Bam H1-linearized maxicircle DNA was isolated by the Hoechst 33258 dye--CsCl method and a restriction enzyme map of the maxicircle was constructed. Closed monomeric minicircles released from kDNA networks by sonication sedimented with a molecular size of around 1100 base pairs. A substantial minor length heterogeneity was evident in acrylamide gel electrophoresis of once cut minicircles. Several minicircle sequence classes and two Hind III maxicircle fragments representing approx. 50% of the maxicircle were cloned in the bacterial plasmid, pBR322, in Escherichia coli. A purified kinetoplast-mitochondrion fraction was isolated from procyclic culture forms by the Renografin flotation method. The major kRNA components were two small RNAs which comigrated with Leishmania tarentolae 9 and 12 S kRNAs in denaturing gels. These RNAs hybridized to the maxicircle component of the kDNA, specifically to the smaller cloned Hind III maxicircle fragment. This cloned fragment had substantial sequence homology with the cloned maxicircle fragment from L. tarentolae which contains the 9 and 12 S RNA genes, implying an evolutionary conservation of the 9 and 12 S gene sequences. Identical kRnAs were observed in cultured bloodstream forms of T. brucei.

Identification of two integral glycosomal membrane proteins in Trypanosoma brucei.

Glycosomal membranes of bloodstream form Trypanosoma brucei were purified to apparent homogeneity by sodium carbonate treatment and found to contain two major integral membrane proteins of 26 kDa (band 10) and 24 kDa (band 11). The procyclic-form glycosomal membranes isolated by the same procedure also resulted in a homogeneous preparation, but each piece of membrane thus purified was associated with an electron-dense granule. Procyclic membranes also contained primarily bands 10 and 11. These two proteins were selectively reduced by protease treatment of intact glycosomes, suggesting surface exposed domain(s) of the two proteins accessible to proteolytic digestion. They and band 8 protein also vanished in glycosomal lysates by apparent proteolysis, implying the presence of a specific protease which degrades the integral membrane proteins upon the loss of membrane integrity. The two proteins, hydrophobic in nature and apparently unglycosylated, have no known biological functions, but their possible involvement in translocating precursor proteins from the cytoplasm into the glycosome of T. brucei is postulated.

Purification and characterisation of membrane-form variant surface glycoproteins of Trypanosoma brucei.

Membrane-form variant surface glycoprotein of Trypanosoma brucei can be prepared in the presence of para-chloromercuriphenylsulphonic acid. The membrane-bound enzyme that usually cleaves a lipid from this glycoprotein, thus producing the soluble variant surface glycoprotein, is inhibited by a range of sulphydryl reagents. The effect of such inhibitors, both on cell lysates and on semi-purified enzyme, reveals that the enzyme may have a sulphydryl at or near its active site. Fatty acid analysis and isoelectric point measurements of membrane form and soluble form are presented.