ABSTRACT
The ancestral gamete fusion protein, HAP2/GCS1, plays an essential role in fertilization in a broad range of taxa. To identify factors that may regulate HAP2/GCS1 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2/gcs1 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2, whose products are necessary for membrane pore formation following mating type recognition and adherence. GFU2 is predicted to be a single-pass transmembrane protein, while GFU1, though lacking obvious transmembrane domains, has the potential to interact directly with membrane phospholipids in the cytoplasm. Like Tetrahymena HAP2/GCS1, expression of GFU1 is required in both cells of a mating pair for efficient fusion to occur. To explain these bilateral requirements, we propose a model that invokes cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in Tetrahymena's multiple mating type system.
ABSTRACT
A transmission-blocking vaccine targeting the sexual stages of Plasmodium species could play a key role in eradicating malaria. Multiple studies have identified the P. falciparum proteins Pfs25 and Pfs48/45 as prime targets for transmission-blocking vaccines. Although significant advances have been made in recombinant expression of these antigens, they remain difficult to produce at large scale and lack strong immunogenicity as subunit antigens. We linked a self-assembling protein, granule lattice protein 1 (Grl1p), from the ciliated protozoan, Tetrahymena thermophila, to regions of the ectodomains of either Pfs25 or Pfs48/45. We found that resulting protein chimera could be produced in E. coli as nanoparticles that could be readily purified in soluble form. When produced in the E. coli SHuffle strain, fusion to Grl1p dramatically increased solubility of target antigens while at the same time directing the formation of particles with diameters centering on 38 and 25â¯nm depending on the antigen. In a number of instances, co-expression with chaperone proteins and induction at a lower temperature further increased expression and solubility. Based on Western blotting and ELISA analysis, Pfs25 and Pfs48/45 retained their transmission-blocking epitopes within E. coli-derived particles, and the particles themselves elicited strong antibody responses in rabbits when given with an aluminum-based adjuvant. Antibodies against Pfs25-containing nanoparticles blocked parasite transmission in standard membrane-feeding assays. In conclusion, fusion to Grl1p can act as a solubility enhancer for proteins with limited solubility while retaining correct folding, which may be useful for applications such as the production of vaccines and other biologics.
Subject(s)
Antibodies, Protozoan/biosynthesis , Calcium-Binding Proteins/genetics , Malaria Vaccines/genetics , Malaria, Falciparum/prevention & control , Membrane Glycoproteins/genetics , Plasmodium falciparum/chemistry , Protozoan Proteins/genetics , Tetrahymena thermophila/chemistry , Animals , Antigens, Protozoan/administration & dosage , Antigens, Protozoan/chemistry , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Biological Assay , Calcium-Binding Proteins/administration & dosage , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/immunology , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Immunogenicity, Vaccine , Malaria Vaccines/administration & dosage , Malaria, Falciparum/immunology , Malaria, Falciparum/parasitology , Membrane Glycoproteins/administration & dosage , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/immunology , Mosquito Vectors/parasitology , Nanoparticles , Plasmodium falciparum/immunology , Protein Folding , Protozoan Proteins/administration & dosage , Protozoan Proteins/chemistry , Protozoan Proteins/immunology , Rabbits , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Proteins/administration & dosage , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Solubility , Tetrahymena thermophila/immunologyABSTRACT
Identifying monoclonal antibodies that block human voltage-gated ion channels (VGICs) is a challenging endeavor exacerbated by difficulties in producing recombinant ion channel proteins in amounts that support drug discovery programs. We have developed a general strategy to address this challenge by combining high-level expression of recombinant VGICs in Tetrahymena thermophila with immunization of phylogenetically diverse species and unique screening tools that allow deep-mining for antibodies that could potentially bind functionally important regions of the protein. Using this approach, we targeted human Kv1.3, a voltage-gated potassium channel widely recognized as a therapeutic target for the treatment of a variety of T-cell mediated autoimmune diseases. Recombinant Kv1.3 was used to generate and recover 69 full-length anti-Kv1.3 mAbs from immunized chickens and llamas, of which 10 were able to inhibit Kv1.3 current. Select antibodies were shown to be potent (IC50<10 nM) and specific for Kv1.3 over related Kv1 family members, hERG and hNav1.5.
Subject(s)
Antibodies, Monoclonal , Drug Discovery/methods , Kv1.3 Potassium Channel/antagonists & inhibitors , Animals , Camelids, New World , Chickens , Humans , Recombinant Proteins , Tetrahymena thermophilaABSTRACT
Here we demonstrate that ciliated protozoa can jettison mitochondria as intact organelles, releasing their contents to the extracellular space either in a soluble form, or in association with membrane vesicles at the cell periphery. The response is triggered by lateral clustering of GPI-anchored surface antigens, or by heat shock. In the first instance, extrusion is accompanied by elevated levels of intracellular calcium and is inhibited by Verapamil and BAPTA-AM arguing strongly for the involvement of calcium in triggering the response. Cells survive mitochondrial discharge raising the interesting possibility that extrusion is an early evolutionary adaptation to cell stress.
Subject(s)
Calcium/metabolism , Ciliophora/radiation effects , Mitochondria/radiation effects , Hot TemperatureABSTRACT
Metallothioneins (MTs) are ubiquitous, cysteine-rich, metal-binding proteins whose transcriptional activation is induced by a variety of stimuli, in particular heavy metals such as cadmium, copper and zinc. Here we describe the sequence and organization of a novel copper-inducible metallothionein gene (MTT2) from Tetrahymena thermophila. Based on its deduced sequence, the gene encodes a protein 108 amino acids, containing 29 cysteine residues (30%) arranged in motifs characteristic of vertebrate and invertebrate MTs. We demonstrate that the 5'-region of the MTT2 gene can act as an efficient promoter to drive the expression of heterologous genes in the Tetrahymena system. In the latter case, a gene for a candidate vaccine antigen against Ichthyophthirius multifiliis, a ubiquitous parasite of freshwater fish, was expressed at high levels in transformed T. thermophila cell lines. Moreover, the protein was properly folded and targeted to the plasma membrane in its correct three-dimensional conformation. This new copper-inducible MT promoter may be an attractive alternative to the cadmium-inducible MTT1 promoter for driving ectopic gene expression in Tetrahymena and could have a great impact on biotechnological perspectives.
Subject(s)
Copper Sulfate/pharmacology , Gene Expression Regulation , Metallothionein/genetics , Tetrahymena thermophila/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Cadmium Chloride/metabolism , Cadmium Chloride/pharmacology , Cloning, Molecular , Copper Sulfate/metabolism , Metallothionein/immunology , Metallothionein/metabolism , Molecular Sequence Data , Organisms, Genetically Modified , RNA, Messenger/metabolism , Recombinant Proteins , Sequence Analysis, Protein , Tetrahymena thermophila/immunology , Tetrahymena thermophila/metabolismABSTRACT
The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific, directed by a conserved chromosome breakage sequence (Cbs element). An accompanying article in this issue reports the development of a successful scheme for the genome-wide cloning and identification of functional chromosome breakage sites. This article reports the physical and genetic characterization of 30 functional chromosome breakage junctions. Unique sequence tags and physical sizes were obtained for the pair of macronuclear chromosomes generated by fragmentation at each Cbs. Cbs-associated polymorphisms were used to genetically map 11 junctions to micronuclear linkage groups and macronuclear coassortment groups. Two pairs of junctions showed statistically significant similarity of the sequences flanking the Cbs, suggestive of relatively recent duplications of entire Cbs junctions during Tetrahymena genome evolution. Two macronuclear chromosomes that lose at least one end in an age-related manner were also identified. The whole-genome shotgun sequencing of the Tetrahymena macronucleus has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from natural ends of macronuclear chromosomes, Cbs junctions will provide useful sequence tags for relating macro- and micronuclear genetic, physical, and whole-genome sequence maps.