Arno Tiedtke

Secretion of functional human enzymes by Tetrahymena thermophila

BackgroundThe non-pathogenic ciliate Tetrahymena thermophila is one of the best-characterized unicellular eucaryotes used in various research fields. Previous work has shown that this unicellular organism provides many biological features to become a high-quality expression system, like multiplying to high cell densities with short generation times in bioreactors. In addition, the expression of surface antigens from the malaria parasite Plasmodium falciparum and the ciliate Ichthyophthirius multifiliis suggests that T. thermophila might play an important role in vaccine development. However, the expression of functional mammalian or human enzymes remains so far to be seen.ResultsWe have been able to express a human enzyme in T. thermophila using expression modules that encode a fusion protein consisting of the endogenous phospholipase A1 precursor and mature human DNaseI. The recombinant human enzyme is active, indicating that also disulfide bridges are correctly formed. Furthermore, a detailed N-glycan structure of the recombinant enzyme is presented, illustrating a very consistent glycosylation pattern.ConclusionThe ciliate expression system has the potential to become an excellent expression system. However, additional optimisation steps including host strain improvement as wells as measures to increase the yield of expression are necessary to be able to provide an alternative to the common E. coli and yeast-based systems as well as to transformed mammalian cell lines.

Magnetic Separation of Phagosomes of Defined Age from Tetrahymena thermophila

ABSTRACT. We describe a new mass isolation procedure for both pure and stage‐specific phagosomes from Tetrahymena thermophila. We prepared magnetic iron dextran particles about 1 μm in diameter to label the phagosomes. The oral apparatus of the cells concentrated these particles so readily that after 1 min the majority of the cells had formed a single phagosome. A short wash removed non‐ingested particles, enabling us to follow the age‐dependent changes of a single labeled phagosome through the cell. Phagosomes of different ages, including very young and nascent phagosomes, were removed easily from the non‐magnetic cell debris of mechanically homogenized cells by means of a permanent magnet. The isolated phagosomes are pure as tested by enzymatic assays and light and electron microscopy. Since the yield of pure phagosomes of all ages is high (∼ 90%), this method could be generally applied for phagosome isolation from ciliates.

The Golgi apparatus of Tetrahymena thermophila.

ABSTRACT. Electron microsocpic investigations reveal that the Golgi apparatus of Tetrahymena thermophila consists of numerous tiny dictyosomes, each consisting of one or two cisternae. the dictyosomes are localized predominantly in the cell cortex closely associated with the mitochondria, arranged in meridians alternating with the ciliary meridians. We estimated about 300‐400 of these dictyosomes in the periphery of a cell, a value corresponding to the number of somatic cilia per cell. Cytochemical assays of thiamine pyrophosphatase and acid phosphatase, both marker enzymes of trans Golgi cisternae, resulted in deposits of lead or cerium phosphate in the outermost cisternae of the dictyosomes. In addition, cisternae located at the bases of the basal body/parasomal sac arrangements are stained. This indicates that these cisternae may belong to the Golgi apparatus of the cell.

Biochemical and molecular characterisation of Tetrahymena thermophila extracellular cysteine proteases

BackgroundOver the last decades molecular biologic techniques have been developed to alter the genome and proteome of Tetrahymena thermophila thereby providing the basis for recombinant protein expression including functional human enzymes. The biotechnological potential of Tetrahymena has been proved in numerous publications, demonstrating fast growth, high biomass, fermentation in ordinary bacterial/yeast equipment, up-scalability, existence of cheap and chemical defined media. For these reasons Tetrahymena offers promising opportunities for the development of a high expression system. Yet optimised high yield strains with protease deficiency such as commonly used in yeast and bacterial systems are not available.ResultsThis work presents the molecular identification of predominant proteases secreted into the medium by Tetrahymena thermophila. A one-step purification of the proteolytic enzymes is described.ConclusionThe information provided will allow silencing of protease activity by either knock out methods or by Tetrahymena specific antisense-ribosome-techniques. This will facilitate the next step in the advancement of this exciting organism for recombinant protein production.

Growth requirements of a new food-vacuole-less mutant of Tetrahymena

The growth requirements of the ciliate Tetrahymena thermophila have been known for a long time. In this study we have investigated the additional requirements induced in a cell line defective in phagocytosis. The mutant cell line of T. thermophila, II 8 G, derived from the wildtype, T. thermophila, CU 399, has apparently no oral structures and forms no food vacuoles at 37°C, but has normal oral structures and the ability to form food vacuoles at normal rates at 28°C. Taking advantage of a synthetic nutrient medium, we observed that the mutant cell line shows increased requirement for Fe(III), Cu(++) and folic acid under restrictive conditions. Phagocytosis obviously plays a role in utilization of low concentrations of the three substances, whereas all other required substances can enter fast enough to support short generation times.

Biochemical analysis of membrane proteins from an early maturation stage of phagosomes

We used an improved technique for pulse‐chase labeling of phagosomes using custom‐made magnetic microparticles. With the help of a permanent magnet we purified both newly formed, nascent and early matured (i.e., 5‐min‐old) condensed phagosomes in high amounts. The protein patterns of membrane proteins of newly formed phagosomes and 5‐min‐old condensed ones were compared by two‐dimensional (2‐D) electrophoresis. The protein patterns allowed the detection of protein spots that changed in abundance between these two stages. Three protein spots abundant in condensed phagosomes only and one spot well‐stained in both stages were collected from ten preparative Coomassie brilliant blue‐stained 2‐D gels. Following microdigestion, selected purified oligopeptides were sequenced by Edman degradation. While the oligopeptide sequences of proteins from two spots showed high homology to an already sequenced 25 kDa calcium binding protein, the other two showed no significant homology to protein sequences available in sequence databases. Presently polymerase chain reaction (PCR) and cloning experiments are set up to reveal the cDNAs of these proteins in order to study their function by knock‐out and gene replacement experiments.

Purification and properties of secreted N-acetyl-β--hexosaminidase of Tetrahymena thermophila

1. 1. N-acetyl-μ-d-hexosaminidase secreted by the ciliate Tetrahymena thermophila was purified 260-fold with 41% yield by chromatography on Sephacryl S-300 and DEAE-cellulose. 2. 2. On a polyacrylamide gel, the enzyme appeared as a single peak of protein and activity. The molecular weight of the denatured and reduced enzyme was 55,000 as estimated on SDS-PAGE. 3. 3. The pH-optima were at pH 3.6 and 4.7. 4. 4. The enzyme hydrolysed p-nitrophenyl N-acetyl-β-d-glucosaminide with Km 0.49 mM and Vmax 0.45 μmol/min/mg protein. It was active on N,N′-diacetylchitobiose. 5. 5. Thermal stability and the effect of various metal ions on the enzyme activity were investigated.

Processing and Secretion of Lysosomal Acid α-Glucosidase In Tetrahymena Wild Type and Secretion-Deficient Mutant Cells

ABSTRACT. The proteolytic processing and secretion of a lysosomal enzyme, acid α‐glucosidase, was studied by pulse‐chase labeling with [35S]methionine in Tetrahymena thermophila CU‐399 cells treated with ammonium chloride. This cell secreted a large amount of acid α‐glucosidase into the cultured medium during starvation. the secretion was found to be repressed by addition of ammonium chloride (NH4Cl). Acid α‐glucosidase was produced as a precursor form (108 kDa) and then processed to a mature polypeptide (105 kDa) within 60 min. This mature enzyme was secreted into the media within 2‐3 h after chase, whereas the precursor form was not secreted by either control cells or NH4Cl‐treated cells. NH4Cl did not affect the processing of the precursor acid α‐glucosidase. Processing profile of this enzyme was apparently indistinguishable from that of the mutant MS‐1 defective in lysosomal enzyme secretion. Furthermore, the purified extracellular (CU‐399) and intracellular (MS‐1) acid a‐glucosidases were the same in molecular mass (105 kDa) and enzymatic properties. They contained no mannose 6‐phosphate residues in N‐linked oligosaccharides. These results suggested that unlike mammalian cells, Tetrahymena acid α‐glucosidase may be transferred to lysosomes by a mannose 6‐phosphate receptor‐independent mechanism, and also that low pH was not essential for the proteolytic processing of precursor polypeptide.

Deficiency in lysosomal enzyme secretion is associated with upregulation of phosphatidylinositol 4-phosphate in tetrahymena.

ABSTRACT. A variety of lower eukaryotes and certain mammalian cells are known to constitutively secrete lysosomal hydrolases. Recent studies in Tetrahymena have shown that phosphatidylinositol 3‐phosphate regulates the proper secretion of lysosomal enzymes at the level of phagolysosome formation. We extend these findings by studying the secretion‐deficient strain MS‐1 of Tetrahymena thermophila, which possess phosphatidylinositol levels similar to wild type. However, steady‐state levels of phosphatidylinositol 4‐phosphate (PtdIns4P) were found to be doubled in this strain compared with wild type as shown by in vivo [3H]inositol labeling and high‐performance liquid chromatography analysis. The increased PtdIns4P levels in MS‐1 cells were unrelated to the upregulation of total phosphatidylinositol phosphate induced by hyperosmotic stress because this treatment resulted in a similar increase of PtdIns4P in MS‐1 and wild‐type cells. Hyperosmotic stress did not affect secretion in either of the two types of cells. On the other hand, under conditions of wortmannin‐induced hypersecretion in wild‐type cells, MS‐1 cells developed a highly vacuolated phenotype while secretion was not induced. Importantly, comparative analysis of wild‐type and MS‐1 cells under wortmannin treatment showed that PtdIns4P levels were differentially regulated in the two strains. These results collectively suggest that PtdIns4P turnover in Tetrahymena is linked to lysosome secretion.

The bifunctional dihydrofolate reductase thymidylate synthase of Tetrahymena thermophila provides a tool for molecular and biotechnology applications

BackgroundDihydrofolate reductase (DHFR) and thymidylate synthase (TS) are crucial enzymes in DNA synthesis. In alveolata both enzymes are expressed as one bifunctional enzyme.ResultsLoss of this essential enzyme activities after successful allelic assortment of knock out alleles yields an auxotrophic marker in ciliates. Here the cloning, characterisation and functional analysis of Tetrahymena thermophilas DHFR-TS is presented. A first aspect of the presented work relates to destruction of DHFR-TS enzyme function in an alveolate thereby causing an auxotrophy for thymidine. A second aspect is to knock in an expression cassette encoding for a foreign gene with subsequent expression of the target protein.ConclusionThis system avoids the use of antibiotics or other drugs and therefore is of high interest for biotechnological applications.