Mervyn J. Turner

Complete nucleotide sequence of an unusual mobile element from trypanosoma brucei

The complete nucleotide sequence of a mobile element from Trypanosoma brucei is presented along with the sequence of its target site, which shows that the insertion has generated a 7 base pair direct repeat. The cloned copy of the element is a dimeric structure, one end of each monomer consisting of a stretch of 14 A residues preceded by a putative trypanosome polyadenylation signal. Six base pairs of DNA of unknown origin are found in the dimer between the two copies of the element. Evidence suggests that the element is present in the genome mainly as a monomer whose sequence is conserved across several species of trypanosome. The element contains an open reading frame encoding the same 160 amino acid protein in both sequenced copies and is extensively transcribed from both strands.

Biosynthesis, Attachment and Release of Variant Surface Glycoproteins of the African Trypanosome

Antigenic variation in the African trypanosome is a consequence of changes in the composition of the surface coat which covers the entire surface of the trypanosome, when it is in the blood stream of its mammalian host (reviewed by Vickerman and Barry 1982). The surface coat is made up of a matrix of about 107 identical glycoprotein molecules which are known as the variant surface glycoproteins (VSGs), because each antigenically distinct variant can be characterised by the immunochemical profile of the VSG which is expressed at its surface. Much is known about the biochemistry of the VSGs, which are notable for the remarkable diversity within their amino acid sequences (reviewed in Turner 1982 a; Cross 1984). However, it is clear that all VSGs must have certain features in common. Thus, they must all be capable of forming a compact surface coat, about 12- to 15-mm thick (Vickerman 1969), be impermeable to soluble mediators of the immune system, such as antibody and complement, and be anchored firmly to the plasma membrane. Furthermore, all the VSGs are presumably produced through a common intracellular pathway, utilising the same biosynthetic machinery. Thus it is to be expected that this combination of structural demands and synthetic restraints will be reflected in conserved structural elements, and interference with the synthesis of such elements should therefore be a potent form of chemotherapy. It is the purpose of this review to discuss what is known about the biosynthesis of VSGs, and also to review our knowledge of one of the most intriguing features of VSG biochemistry, namely the unusual mode of attachment to, and ready release from, the plasma membrane.

The purification of sporocysts and sporozoites from Eimeria tenella oocysts using Percoll density gradients.

A protocol is presented for the purification of sporozoites from sporulated oocysts of Eimeria tenella. Two Percoll density gradients are the basis of the purification. The first gradient is used after glass-bead grinding to purify undamaged sporocysts; 87% of the sporocysts loaded onto the gradient were recovered in the pellet. The second gradient is used after excystation to purify sporozoites; 98% of the sporozoites loaded onto the gradient were recovered in the pellet. The sporozoites are 99% pure with a final recovery of about three sporozoites per oocyst.

Identification of an acid-lipase in human serum which is capable of solubilizing glycophosphatidylinositol-anchored proteins.

A lipase has been identified in human serum which can convert the membrane form of the variant surface glycoprotein of Trypanosoma brucei to a water soluble form. The conversion can be monitored by loss of [3H] myristic acid incorporated into the diacylglycerol of the glycophosphatidylinositol membrane anchor of the protein, but does not lead to the exposure of the antigenic determinant in the polar head group of the glycolipid. The serum lipase is a glycoprotein, and is optimally active at pH 5.4. Treatment at 62 degrees for one hour does not inactivate the enzyme, which is inhibited by chelating agents.

Identification and characterization of cDNA clones encoding antigens of Eimeria tenella

An Eimeria tenella cDNA library was constructed in the expression vector lambda gt11 from poly (A+) RNA extracted from sporulating oocysts. The library was screened with rabbit antiserum raised against antigens extracted from fully sporulated oocysts. All of the antigen-expressing plaque-purified clones were initially characterized by cross screening with antisera raised against different stages of the E. tenella life cycle, as well as with antiserum raised against sporozoites of a related species, namely E. acervulina. A selected number of clones were further characterized by antibody selection coupled with immunoblotting and DNA cross hybridization. Three different E. tenella antigens were identified. All three appear to be constitutively expressed at the protein level during sporogony.

Biosynthesis of Trypanosoma brucei variant surface glycoprotein. I. Synthesis, size, and processing of an N-terminal signal peptide

An N-terminal signal peptide has been identified on a precursor of the variant surface glycoprotein (VSG) of Trypanosoma brucei, synthesised both in vitro and in vivo. An ordered cyanogen bromide (CNBr) cleavage map was constructed for both mature VSG and for precursor VSG, and this allowed a size estimate of the signal peptide to be made by comparison of the N-terminal CNBr peptides produced from each. The signal peptide contains 30-40 amino acids. Analysis of the precursor VSGs produced when nascent polypeptide chains on T. brucei polysomes were completed in the absence of reinitiation showed that the signal peptide is removed before translation is complete and that the same translational start signal is utilised in the trypanosome and in the rabbit reticulocyte protein synthesis system.

Biosynthesis of Trypanosoma brucei variant surface glycoproteins — Analysis of carbohydrate heterogeneity and timing of post-translational modifications

The variant surface glycoproteins from two cloned populations of Trypanosoma brucei brucei which were known to migrate as multiple bands on SDS gels have been studied. The heterogeneity present was located in those oligosaccharide side chains the addition of which is tunicamycin-sensitive. The time required for the trypanosome to synthesize and express a variant surface glycoprotein molecule in vitro was found, from pulse-chase and limited trypsinisation experiments, to be approximately 40 min. In the light of these data, pulse-chase experiments on the two antigens known to have heterogeneity in their oligosaccharide side chains demonstrated that the heterogeneity probably arose by two different mechanisms. Pulse-chase experiments on three different clones of trypanosomes have also been used to investigate the timing of cleavage of the carboxyl-terminal extension, known to be encoded on variant surface glycoprotein mRNA. Similar pulse-chase experiments followed by immunoprecipitation using affinity purified antiserum have been used to investigate the addition of the cross-reacting determinant. The timing of both these events has been discussed in relation to the time necessary for the synthesis and expression of the variant surface glycoprotein on the surface of the trypanosome.

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.

An assay of membrane-bound Trypanosoma brucei phospholipase using an integral membrane protein substrate and detergent phase separation.

The technique of phase separation in a solution of the non-ionic detergent Triton X-114 was used to measure the enzymatic conversion of a membrane protein to a soluble product via removal of a hydrophobic moiety. The substrate was the major surface protein (p63), of Leishmania promastigotes and the enzyme was a phospholipase C purified from Trypanosoma brucei. This membrane-bound enzyme is responsible for the cleavage of the hydrophobic lipid membrane anchor of the variant surface glycoprotein (VSG), of T. brucei. The assay is fast, simple and uses small amounts of reagents. It has been used to determine the pH optimum, thermal resistance, and the sensitivity to inhibitors of the trypanosomal phospholipase.

The biosynthesis of Trypanosoma brucei variant surface glycoproteins — in vitro processing of signal peptide and glycosylation using heterologous rough endoplasmic reticulum vesicles

The ability of stripped microsomal dog pancreas vesicles to process precursors of the variant glycoprotein of Trypanosoma brucei has been investigated. The vesicles were shown to be capable of cleaving a peptide precursor variant surface glycoprotein, identified as the N-terminal signal peptide in the case of one variant. The vesicles were also shown to add carbohydrate to precursor variant surface glycoprotein. This carbohydrate, which was sensitive to the action of endo-H was located within a C-terminal CNBr fragment in one variant. However, the vesicles were not capable of adding the oligosaccharide carrying the variant surface glycoprotein cross-reacting determinant.