Reducing isoform complexity of human tetraspanins by optimized expression in Dictyostelium discoideum enables high-throughput functional read-out.

The human tetraspanin family of scaffold proteins comprises 33 isoforms. Being integral membrane proteins, they organize a so-called tetraspanin web via homomeric and heteromeric protein-protein interactions with integrins, immunoglobulins, growth factors, receptor tyrosine kinases, proteases, signaling proteins, and viral capsid proteins. Tetraspanins promote cellular effects, such as adhesion, migration, invasion, signaling, membrane fusion, protein trafficking, cancer progression, and infections. The ubiquitous expression of multiple tetraspanin isoforms and partner proteins hampers specific interaction studies. Here, we evaluated Dictyostelium discoideum as a non-mammalian expression system for human tetraspanins. Using high-content imaging we quantified tetraspanins in D. discoideum via fusion with green fluorescent protein. Three human tetraspanins, CD9, CD81, and CD151, served as test cases for which optimizations were carried out. We swapped the GFP domain between the N- and C-termini, added a Kozak sequence, and partially or fully adapted of the codon usage. This way, CD81 and CD151 were successfully produced. A conformation specific antibody further confirmed correct folding of CD81 and flow cytometry indicated an intracellular localization. Based on these data, we envision a D. discoideum-based co-expression platform with human partner proteins for studying tetraspanin interactions and their selective druggability on a large scale without the interference of endogenous human proteins.

The C Isoform of Dictyostelium Tetraspanins Localizes to the Contractile Vacuole and Contributes to Resistance against Osmotic Stress.

Tetraspanins (Tsps) are membrane proteins that are widely expressed in eukaryotic organisms. Only recently, Tsps have started to acquire relevance as potential new drug targets as they contribute, via protein-protein interactions, to numerous pathophysiological processes including infectious diseases and cancer. However, due to a high number of isoforms and functional redundancy, knowledge on specific functions of most Tsps is still scarce. We set out to characterize five previously annotated Tsps, TspA-E, from Dictyostelium discoideum, a model for studying proteins that have human orthologues. Using reverse transcriptase PCRs, we found mRNAs for TspA-E in the multicellular slug stage, whereas vegetative cells expressed only TspA, TspC and, to a lesser extent, TspD. We raised antibodies against TspA, TspC and TspD and detected endogenous TspA, as well as heterologously expressed TspA and TspC by Western blot. N-deglycosylation assays and mutational analyses showed glycosylation of TspA and TspC in vivo. GFP-tagged Tsps co-localized with the proton pump on the contractile vacuole network. Deletion strains of TspC and TspD exibited unaltered growth, adhesion, random motility and development. Yet, tspC- cells showed a defect in coping with hypo-osmotic stress, due to accumulation of contractile vacuoles, but heterologous expression of TspC rescued their phenotype. In conclusion, our data fill a gap in Dictyostelium research and open up the possibility that Tsps in contractile vacuoles of e.g. Trypanosoma may one day constitute a valuable drug target for treating sleeping sickness, one of the most threatening tropical diseases.

Number and regulation of protozoan aquaporins reflect environmental complexity.

Protozoa are a diverse group of unicellular eukaryotes. Evidence has accumulated that protozoan aquaporin water and solute channels (AQP) contribute to adaptation in changing environments. Intracellular protozoan parasites live a well-sheltered life. Plasmodium spp. express a single AQP, Toxoplasma gondii two, while Trypanosoma cruzi and Leishamnia spp. encode up to five AQPs. Their AQPs are thought to import metabolic precursors and simultaneously to dispose of waste and to help parasites survive osmotic stress during transmission to and from the insect vector or during kidney passages. Trypanosoma brucei is a protozoan parasite that swims freely in the human blood. Expression and intracellular localization of the three T. brucei AQPs depend on the stage of differentiation during the life cycle, suggesting distinct roles in energy generation, metabolism, and cell motility. Free-living amoebae are in direct contact with the environment, encountering severe and sudden changes in the availability of nutrition, and in the osmotic conditions due to rainfall or drought. Amoeba proteus expresses a single AQP that is present in the contractile vacuole complex required for osmoregulation, whereas Dictyostelium discoideum expresses four AQPs, of which two are present in the single-celled amoeboidal stage and two more in the later multicellular stages preceding spore formation. The number and regulation of protozoan aquaporins may reflect environmental complexity. We highlight the gated AqpB from D. discoideum as an example of how life in the wild is challenged by a complex AQP structure-function relationship.

Challenges and achievements in the therapeutic modulation of aquaporin functionality.

Aquaporin (AQP) water and solute channels have basic physiological functions throughout the human body. AQP-facilitated water permeability across cell membranes is required for rapid reabsorption of water from pre-urine in the kidneys and for sustained near isosmolar water fluxes e.g. in the brain, eyes, inner ear, and lungs. Cellular water permeability is further connected to cell motility. AQPs of the aquaglyceroporin subfamily are necessary for lipid degradation in adipocytes and glycerol uptake into the liver, as well as for skin moistening. Modulation of AQP function is desirable in several pathophysiological situations, such as nephrogenic diabetes insipidus, Sjögren's syndrome, Menière's disease, heart failure, or tumors to name a few. Attempts to design or to find effective small molecule AQP inhibitors have yielded only a few hits. Challenges reside in the high copy number of AQP proteins in the cell membranes, and spatial restrictions in the protein structure. This review gives an overview on selected physiological and pathophysiological conditions in which modulation of AQP functions appears beneficial and discusses first achievements in the search of drug-like AQP inhibitors.

Bi-functionality of Opisthorchis viverrini aquaporins.

Aquaporins (AQP) are essential mediators of water regulation in all living organisms and members of the major intrinsic protein (MIP) superfamily of integral membrane proteins. They are potential vehicles or targets for chemotherapy, e.g. in Trypanosoma brucei melarsoprol and pentamidine uptake is facilitated by TbAQP-2. Transcriptome data suggests that there are at least three active aquaporins in the human liver fluke, Opisthorchis viverrini, OvAQP-1, 2 and 3, and crude RNA silencing of OvAQP-1 and 2 has recently been shown to affect parasite swelling in destilled water. In the present work we demonstrate that OvAQP-3 is a major water-conducting channel of the parasite, that it can be detected from the newly excysted juvenile to the adult stage and that it is present in major tissues of the parasite. Furthermore, a comparative functional characterization of the three parasite AQPs was performed by using Xenopus oocyte swelling and yeast phenotypic assays. OvAQP-1, OvAQP-2, and OvAQP-3 were found to conduct water and glycerol while only the latter two were also able to conduct urea. In addition, all OvAQPs were found to transport ammonia and methylamine. Our findings demonstrate that the sequence-based classification into orthodox aquaporins and glycerol-conducting aquaglyceroporins is not functionally conserved in the parasite and implicate a broder range of functions for these channels.

The amoeboidal Dictyostelium aquaporin AqpB is gated via Tyr216 and aqpB gene deletion affects random cell motility.

BACKGROUND INFORMATION: Regulation of aquaporin (AQPs) water channels mostly occurs on the transcriptional level or by intracellular trafficking. Direct AQP gating is comparatively rare and is described mainly for fungi and plants. Earlier, we identified a gated water-specific AQP in Dictyostelium discoideum, AqpB, which is opened by truncation of an extended intracellular loop D (AqpB Δ208-219). RESULTS: We show that Tyr216 of loop D is a key residue in the gating mechanism possibly involving phosphorylation. Mutation of Tyr216 to aspartate or glutamate initiated water permeability (Pf  = 69 µm/s) to the same extent as AqpB Δ208-219, whereas neither replacement of Tyr216 by a positive arginine (Y216R) nor introduction of a negative charge in a neighbouring position (T217D) opened the channel. We overexpressed AqpB wildtype and AqpB Δ208-219 as GFP fusion constructs in Dictyostelium amoebae and found that the truncated, permanently open AqpB yielded cells with reduced capability to cope with hypotonic stress. Genetic deletion of aqpB yielded a strain with significantly reduced speed of random motility (4.5 µm/min vs. 6.3 µm/min of wildtype cells). Yet, chemotaxis towards folate and cAMP was unaffected. In this context, we identified a second, so-far uncharacterised amoeboidal AQP, AqpD, whose role in directed cell locomotion needs to be established. CONCLUSION: Our data add to the completion of the Dictyostelium model for cell motility and show that knowledge on the expression, permeability properties and localisation of amoeboidal AQPs are crucial for further development of the system.

[Aquaporins--water channels in the cell membrane and therapeutic targets]. / Aquaporine. Wasserkanäle in der Zellmembran und therapeutische Zielstrukturen.

Aquaporins are membrane proteins that facilitate the rapid movement of water and other small solutes across biological membranes. They have been found in all kingdoms of life. In humans thirteen aquaporin isoforms exist with specific tissue and cell distribution. Aquaporins are linked to physiological functions like maintaining water homeostasis in the whole body as well as to patho-physiological conditions like cancer formation and spreading. Increasing knowledge about the structure and function of aquaporins led to new approaches using aquaporins as diagnostics and drug targets.

Functional characterization of a novel aquaporin from Dictyostelium discoideum amoebae implies a unique gating mechanism.

The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA, and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wild-type AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB, hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride, confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot analysis in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.

The aquaporin gene family of the ectomycorrhizal fungus Laccaria bicolor: lessons for symbiotic functions.

Soil humidity and bulk water transport are essential for nutrient mobilization. Ectomycorrhizal fungi, bridging soil and fine roots of woody plants, are capable of modulating both by being integrated into water movement driven by plant transpiration and the nocturnal hydraulic lift. Aquaporins are integral membrane proteins that function as gradient-driven water and/or solute channels. Seven aquaporins were identified in the genome of the ectomycorrhizal basidiomycete Laccaria bicolor and their role in fungal transfer processes was analyzed. Heterologous expression in Xenopus laevis oocytes revealed relevant water permeabilities for three aquaporins. In fungal mycelia, expression of the corresponding genes was high compared with other members of the gene family, indicating the significance of the respective proteins for plasma membrane water permeability. As growth temperature and ectomycorrhiza formation modified gene expression profiles of these water-conducting aquaporins, specific roles in those aspects of fungal physiology are suggested. Two aquaporins, which were highly expressed in ectomycorrhizas, conferred plasma membrane ammonia permeability in yeast. This indicates that these proteins are an integral part of ectomycorrhizal fungus-based plant nitrogen nutrition in symbiosis.

Functional analysis of novel aquaporins from Fasciola gigantica.

Fascioliasis, caused by liver flukes of the genus Fasciola, is an important disease of ruminants. In order to identify a potential new drug target we have studied aquaporin (AQP) in Fasciola gigantica. AQPs facilitate the transport of water, glycerol and other small solutes across biological membranes. The structure, function, and pathology of AQPs have been extensively studied in mammals but data for AQPs from trematodes is still limited. In the present study, we have functionally characterized two closely related AQP isoforms, FgAQP-1 and FgAQP-2, from the trematode F. gigantica. Immunohistochemical analysis located the FgAQPs in the tegumental cells, their processes and the tegument itself. In addition, they were present in the epithelial linings of testes and ovary. Expression in Xenopus oocytes of these FgAQPs increased osmotic water permeability 3-4-fold but failed to increase glycerol and urea permeability. AQPs have two highly conserved NPA motifs that are important for the function of the channel pore. In FgAQP-1 and FgAQP-2 the first NPA motif is changed to TAA. Substitution of Thr with Asn in the TAA motif of FgAQP-1 increased its water permeability twofold but did not affect urea and glycerol impermeability while the substitution at the pore mouth of Cys204 by Tyr caused loss of water permeability. In addition, the FgAQPs did not increase methylamine and ammonia permeability after expression in yeast. In comparison to rat AQP-1 the described FgAQPs showed low water permeability and further in vivo analyses are necessary to determine their contribution to osmoregulation in Fasciola.

In vitro analysis and modification of aquaporin pore selectivity.

Aquaporins enable the passage of a diverse set of solutes besides water. Many novel aquaporin permeants, such as antimonite and arsenite, silicon, ammonia, and hydrogen peroxide, have been described very recently. By the same token, the number of available aquaporin sequences has rapidly increased. Yet, sequence analyses and structure models cannot reliably predict permeability properties. Even the contribution to pore selectivity of individual residues in the channel layout is not fully understood. Here, we describe and discuss established in vitro assays for water and solute permeability. Measurements of volume change due to flux along osmotic or chemical gradients yield quantitative biophysical data, whereas phenotypic growth assays can hint at the relevance of aquaporins in the physiological setting of a certain cell. We also summarize data on the modification of pore selectivity of the prototypical water-specific mammalian aquaporin-1. We show that replacing residues in the pore constriction region allows ammonia, urea, glycerol, and even protons to pass the aquaporin pore.