Aquaporins - Expression, purification and characterization.

Aquaporin water channels facilitate the bi-directional flow of water and small, neutral solutes down an osmotic gradient in all kingdoms of life. Over the last two decades, the availability of high-quality protein has underpinned progress in the structural and functional characterization of these water channels. In particular, recombinant protein technology has guaranteed the supply of aquaporin samples that were of sufficient quality and quantity for further study. Here we review the features of successful expression, purification and characterization strategies that have underpinned these successes and that will drive further breakthroughs in the field. Overall, Escherichia coli is a suitable host for prokaryotic isoforms, while Pichia pastoris is the most commonly-used recombinant host for eukaryotic variants. Generally, a two-step purification procedure is suitable after solubilization in glucopyranosides and most structures are determined by X-ray following crystallization.

Affinity tag effect on the salt rejection potential of Halomonas elongata aquaporin incorporated in thin film nanocomposite membrane.

In this study, effect of affinity tags, Histidine (His) and Glutathione-S-Transferase (GST), on the activity of halophilic aquaporin was analyzed. The gene coding for H. elongata aquaporin was cloned into pET28a vector and expressed in E. coli BL21 successfully. Stopped flow light scattering measurements showed that His-tagged aquaporin is functional. The difference in the filtration parameters caused by affinity tags were determined by using thin film composite nano-filtration (NFC) membranes prepared with the aquaporins. At 100 mM salt concentration, water permeability (L/m2.h) and the % salt rejection of NFC membranes produced with the His-tagged aquaporin was found to be higher than that of the membrane with GST-tagged aquaporin. Salt rejection of His-tagged aquaporin-membrane was found to be 53% with a lower solute permeability value (B). Use of short affinity tag (His tag) for cloning resulted in higher solute rejection ability of TFC membranes prepared with H. elongata aquaporins.

Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System.

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

Protein-protein interactions in AQP regulation - biophysical characterization of AQP0-CaM and AQP2-LIP5 complex formation.

Protein-protein interactions play important roles in regulating human aquaporins (AQP) by gating as well as trafficking. While structural and functional studies have provided detailed knowledge of AQP transport mechanisms, selectivity as well as gating by conformational changes of loops or termini, the mechanism behind how protein-protein interactions control AQP-mediated water transport through cellular membranes remains poorly characterized. Here we explore the interaction between two human AQPs and regulatory proteins: the interaction between AQP0 and calmodulin, which mediates AQP0 gating, as well as the interaction between AQP2 and LIP5, which is involved in trafficking. Using microscale thermophoresis (MST) and fluorescence anisotropy, two methods that have the advantage of low sample consumption and detergent compatibility, we show that the interactions can be studied using both full-length AQPs and AQP peptides corresponding to the regulatory protein binding sites. However, full-length AQPs gave better reproducibility between methods and for the first time revealed that AQP0 binds CaM in a cooperative manner, which was not seen in experiments using peptides. Our study highlights that, while peptides are great tools for locating binding sites and pinpointing interacting residues, full-length proteins may give additional insights, such as binding mechanism, allostery and cooperativity, important parameters for understanding protein-protein mediated regulation in the cellular context. Our work provides a platform for further studies of AQP regulation that may be of interest for designing drugs that target AQP complexes as well as the development of artificial bio-mimetic water channels for water-purification purposes.

Applying bimolecular fluorescence complementation to screen and purify aquaporin protein:protein complexes.

Protein:protein interactions play key functional roles in the molecular machinery of the cell. A major challenge for structural biology is to gain high-resolution structural insight into how membrane protein function is regulated by protein:protein interactions. To this end we present a method to express, detect, and purify stable membrane protein complexes that are suitable for further structural characterization. Our approach utilizes bimolecular fluorescence complementation (BiFC), whereby each protein of an interaction pair is fused to nonfluorescent fragments of yellow fluorescent protein (YFP) that combine and mature as the complex is formed. YFP thus facilitates the visualization of protein:protein interactions in vivo, stabilizes the assembled complex, and provides a fluorescent marker during purification. This technique is validated by observing the formation of stable homotetramers of human aquaporin 0 (AQP0). The method's broader applicability is demonstrated by visualizing the interactions of AQP0 and human aquaporin 1 (AQP1) with the cytoplasmic regulatory protein calmodulin (CaM). The dependence of the AQP0-CaM complex on the AQP0 C-terminus is also demonstrated since the C-terminal truncated construct provides a negative control. This screening approach may therefore facilitate the production and purification of membrane protein:protein complexes for later structural studies by X-ray crystallography or single particle electron microscopy.

Aquaporin in Chondrosia reniformis Nardo, 1847 and Its Possible Role in the Interaction Between Cells and Engulfed Siliceous Particles.

The sponge Chondrosia reniformis selectively engulfs siliceous particles that, when in crystalline form, become quickly dissolved in its ectosome. The molecular mechanism, identity, and physiological significance of the cells involved in this process are not completely understood. In the present study, we applied light and electronic microscopic techniques to show how the quartz particles in C. reniformis are enveloped through collagen fibers and host cells near the surface of these organisms. As various aquaporins from bacteria, animals, and plants bidirectionally conduct metalloids-including silicon ions--through the cell membrane, the presence and potential involvement of aquaporins in quartz dissolution in C. reniformis have been investigated. An aquaporin-like transcript (CrAQP) was isolated according to the transcriptome sequencing results in C. reniformis The full-length CrAQP cDNA is 907 nucleotides long, with a 795-base pair (bp), open reading frame encoding a protein of 265 amino acids, a 29-bp, 5'-non-coding region, and a 83-bp, 3'-untranslated region. The Bayesian phylogenetic inference suggests that CrAqp is closely related to the Aqp8L grade, which is also implicated in H2O2 transport. Quantification of CrAQP mRNA through qPCR indicated that the transcript level was higher in the ectosome than in the choanosome. Immunofluorescence of a mammalian AQP8 in C. reniformis showed positivity in some cells near the quartz particles, a finding that may support the initial hypothesis of the potential involvement of CrAQP in quartz erosion. However, the features of the primary structure of this protein offer a new viewpoint about the functional role of these molecules in this process: that CrAQP may be involved in the permeation of H2O2 released during silica erosion.

The Hevea brasiliensis XIP aquaporin subfamily: genomic, structural and functional characterizations with relevance to intensive latex harvesting.

X-Intrinsic Proteins (XIP) were recently identified in a narrow range of plants as a full clade within the aquaporins. These channels reportedly facilitate the transport of a wide range of hydrophobic solutes. The functional roles of XIP in planta remain poorly identified. In this study, we found three XIP genes (HbXIP1;1, HbXIP2;1 and HbXIP3;1) in the Hevea brasiliensis genome. Comprehensive bioinformatics, biochemical and structural analyses were used to acquire a better understanding of this AQP subfamily. Phylogenetic analysis revealed that HbXIPs clustered into two major groups, each distributed in a specific lineage of the order Malpighiales. Tissue-specific expression profiles showed that only HbXIP2;1 was expressed in all the vegetative tissues tested (leaves, stem, bark, xylem and latex), suggesting that HbXIP2;1 could take part in a wide range of cellular processes. This is particularly relevant to the rubber-producing laticiferous system, where this isoform was found to be up-regulated during tapping and ethylene treatments. Furthermore, the XIP transcriptional pattern is significantly correlated to latex production level. Structural comparison with SoPIP2;1 from Spinacia oleracea species provides new insights into the possible role of structural checkpoints by which HbXIP2;1 ensures glycerol transfer across the membrane. From these results, we discuss the physiological involvement of glycerol and HbXIP2;1 in water homeostasis and carbon stream of challenged laticifers. The characterization of HbXIP2;1 during rubber tree tapping lends new insights into molecular and physiological response processes of laticifer metabolism in the context of latex exploitation.

Spreading of porous vesicles subjected to osmotic shocks: the role of aquaporins.

Aquaporin 0 (AQP0) is a transmembrane protein specific to the eye lens, involved as a water carrier across the lipid membranes. During eye lens maturation, AQP0s are truncated by proteolytic cleavage. We investigate in this work the capability of truncated AQP0 to conduct water across membranes. We developed a method to accurately determine water permeability across lipid membranes and across proteins from the deflation under osmotic pressure of giant unilamellar vesicles (GUVs) deposited on an adhesive substrate. Using reflection interference contrast microscopy (RICM), we measure the spreading area of GUVs during deswelling. We interpret these results using a model based on hydrodynamic, binder diffusion towards the contact zone, and Helfrich's law for the membrane tension, which allows us to relate the spread area to the vesicle internal volume. We first study the specific adhesion of vesicles coated with biotin spreading on a streptavidin substrate. We then determine the permeability of a single functional AQP0 and demonstrate that truncated AQP0 is no more a water channel.

One-step extraction of functional recombinant aquaporin Z from inclusion bodies with optimal detergent.

Aquaporins are integral membrane channel proteins found in all kingdoms of life. The Escherichia coli aquaporin Z (AqpZ) has been shown to solely conduct water at high permeability. Functional AqpZ is generally purified from the membrane fraction. However, the quantity of the purified protein is limited. In this study, a new method is developed to achieve high yield of bioactive AqpZ protein. A mild detergent n-dodecyl-ß-D-maltopyranoside (DDM) was used to solubilize the over-expressed insoluble AqpZ from inclusion bodies without a refolding process. The recovered AqpZ protein showed high water permeability comparable with AqpZ obtained from the membrane fraction. In this way, the total yield of bioactive AqpZ has been increased greatly, which will facilitate the structural and functional characterization and future applications of AqpZ.

Ectopic overexpression of a novel Glycine soja stress-induced plasma membrane intrinsic protein increases sensitivity to salt and dehydration in transgenic Arabidopsis thaliana plants.

Plasma membrane intrinsic proteins (PIPs) belong to the aquaporin family and facilitate water movement across plasma membranes. Existing data indicate that PIP genes are associated with the abilities of plants to tolerate certain stress conditions. A review of our Glycine soja expressed sequence tag (EST) dataset revealed that abiotic stress stimulated expression of a PIP, herein designated as GsPIP2;1 (GenBank_Accn: FJ825766). To understand the roles of this PIP in stress tolerance, we generated a coding sequence for GsPIP2;1 by in silico elongation and cloned the cDNA by 5'-RACE. Semiquantitative RT-PCR showed that GsPIP2;1 expression was stimulated in G. soja leaves by cold, salt, or dehydration stress, whereas the same stresses suppressed GsPIP2;1 expression in the roots. Transgenic Arabidopsis thaliana plants overexpressing GsPIP2;1 grew normally under unstressed and cold conditions, but exhibited depressed tolerance to salt and dehydration stresses. Moreover, greater changes in water potential were detected in the transgenic A. thaliana shoots, implying that GsPIP2;1 may negatively impact stress tolerance by regulating water potential. These results, deviating from those obtained in previous reports, provide new insights into the relationship between PIPs and abiotic stress tolerance in plants.

Immunological characterization of a chimeric form of Schistosoma mansoni aquaporin in the murine model.

Aquaporin (SmAQP) is the most abundant transmembrane protein in the tegument of Schistosoma mansoni. This protein is expressed in all developmental stages and seems to be essential in parasite survival since it plays a crucial role in osmoregulation, nutrient transport and drug uptake. In this study, we utilized the murine model to evaluate whether this protein was able to induce protection against challenge infection with S. mansoni cercariae. A chimeric (c) SmAQP was formulated with Freund's adjuvant for vaccination trial and evaluation of the host's immune response was performed. Our results demonstrated that immunization with cSmAQP induced the production of high levels of specific anti-cSmAQP IgG antibodies and a Th1/Th17 type of immune response characterized by IFN-γ, TNF-α and IL-17 cytokines. However, vaccination of mice with cSmAQP failed to reduce S. mansoni worm burden and liver pathology. Finally, we were unable to detect humoral immune response anti-cSmAQP in the sera of S. mansoni-infected human patients. Our results lead us to believe that SmAQP, as formulated in this study, may not be a good target in the search for an anti-schistosomiasis vaccine.

Enhanced functional expression of aquaporin Z via fusion of in situ cleavable leader peptides in Escherichia coli cell-free system.

Aquaporin Z (AqpZ) is a water channel protein from Escherichia coli and has attracted many attentions to develop the biomimetic water filtration technology. Cell-free protein synthesis (CFPS) system, one of the most complex multi-enzymatic systems, has the ability of producing the integral membrane protein in vitro. To enhance the synthesis of AqpZ in E. coli cell-free system, several natural leader peptides were respectively fused at the N-terminus and were verified to enhance the expression level significantly. Moreover, the supplementation of detergents or liposome could activate leader peptidase from the cell-free extract and provide hydrophobic environment for proper folding of AqpZ. Thus, the release of mature AqpZ via the in situ removal of leader peptide was achieved, with a specific water transport activity of (2.1 ± 0.1) × 10⁻¹4 cm³ s⁻¹ monomer⁻¹. Using this in situ removable leader peptide strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system, providing a convenient approach to enhance the expression of target proteins, especially those membrane proteins difficult to achieve.

Expresión de aquaporinas en tejido pulmonar de pacientes con enfermedad pulmonar obstructiva crónica / Aquaporin expression in the lung tissue of copd patients

INTRODUCCIÓN: Estudio de la expresión de aquaporinas (AQP1 y AQP5) en el tejido bronquial y parénquima pulmo-nar de pacientes con enfermedad pulmonar obstructiva cróni-ca (EPOC) y fumadores sin la enfermedad. MÉTODO: Utilizando un diseño caso-control, se seleccionó un grupo de 15 pacientes con EPOC (93,3% varones, con una edad media de 68 años, una media de FEV1 del 72% y 26,7% con corticosteroides inhalados) y 15 fumadores sin la enfermedad, a los cuales se les sometió a cirugía de resección pulmonar por neoplasia pulmonar. Se estudió la expresión de AQP1 y AQP5 en el tejido bronquial y en parénquima pulmo-nar mediante reacción en cadena de la polimerasa en tiempo real.RESULTADOS: No encontramos diferencias en la expresión génica de estas AQPs en ambos territorios pulmonares entre los pacientes con EPOC y los fumadores sin la enfermedad. Sin embargo, en los pacientes EPOC, la expresión de AQP1 era 2,41 veces mayor en el parénquima comparado con los controles, mientras que la AQP5 mostraba un patrón inverso, con 7,75 veces mayor expresión en el tejido bronquial de los sujetos control.CONCLUSIÓN: Los resultados del presente trabajo proporcio-nan evidencia inicial respecto a la expresión de AQP1 y AQP5 en pacientes con EPOC

INTRODUCTION: Study of aquaporin expression (AQP1 and AQP5) in the bronchial tissue and lung parenchyma of pa-tients with chronic obstructive pulmonary disease (COPD) and smokers without the disease. METHOD: Using a case-control design, a group of 15 patients with COPD was selected (93.3% males, with an average age of 68 years, an average FEV1 of 72% and 26.7% with inha-led corticosteroids) and 15 smokers without the disease, who underwent lung resection surgery due to lung neoplasm. The expression of AQP1 and AQP5 in the bronchial tissue and in lung parenchyma was studied using real-time polymerase chain reaction (PCR). RESULTS: No differences were found in the gene expression of these AQPs in either lung territories between the patients with COPD and the smokers without the disease. Nevertheless, in the COPD patients, the expression of AQP1 was 2.41 times greater in the parenchyma compared with the controls, while the AQP5 showed an inverse pattern, with 7.75 times greater expression in the bronchial tissue of the control subject. CONCLUSION: The results of this study provide initial evidence regarding the expression of AQP1 and AQP5 in patient with COPD

In vivo studies of aquaporins 3 and 10 in human stratum corneum.

Aquaporins (AQPs) constitute one family of transmembrane proteins facilitating transport of water across cell membranes. Due to their specificity, AQPs have a broad spectrum of physiological functions, and for keratinocytes there are indications that these channel proteins are involved in cell migration and proliferation with consequences for the antimicrobial defense of the skin. AQP3 and AQP10 are aqua-glyceroporins, known to transport glycerol as well as water. AQP3 is the predominant AQP in human skin and has previously been demonstrated in the basal layer of epidermis in normal human skin, but not in stratum corneum (SC). AQP10 has not previously been identified in human skin. Previous studies have demonstrated the presence of AQP3 and AQP10 mRNA in keratinocytes. In this study, our aim was to investigate if these aquaporin proteins were actually present in human SC cells. This can be seen as a first step toward elucidating the possible functional role of AQP3 and AQP10 in SC hydration. Specifically we investigate the presence of AQP3 and AQP10 in vivo in human SC using "minimal-invasive" technique for obtaining SC samples. SC samples were obtained from six healthy volunteers. Western blotting and immunohistochemistry were used to demonstrate the presence of AQP3 as well as AQP10. The presence of AQP3 and AQP10 was verified by Western blotting, allowing for detection of proteins by specific antibodies. Applying immunohistochemistry, cell-like structures in the shape of corneocytes were identified in all samples by AQP3 and AQP10 antibodies. In conclusion, identification of AQP3 and AQP10 protein in SC in an in vivo model is new. Together with the new "minimal-invasive" method for SC collection presented, this opens for new possibilities to study the role of AQPs in relation to function of the skin barrier.

Expression analysis of aquaporins from desert truffle mycorrhizal symbiosis reveals a fine-tuned regulation under drought.

We have performed the isolation, functional characterization, and expression analysis of aquaporins in roots and leaves of Helianthemum almeriense, in order to evaluate their roles in tolerance to water deficit. Five cDNAs, named HaPIP1;1, HaPIP1;2, HaPIP2;1, HaPIP2;2, and HaTIP1;1, were isolated from H. almeriense. A phylogenetic analysis of deduced proteins confirmed that they belong to the water channel proteins family. The HaPIP1;1, HaPIP2;1, and HaTIP1;1 genes encode functional water channel proteins, as indicated by expression assays in Saccharomyces cerevisiae, showing divergent roles in the transport of water, CO2, and NH3. The expression patterns of the genes isolated from H. almeriense and of a previously described gene from Terfezia claveryi (TcAQP1) were analyzed in mycorrhizal and nonmycorrhizal plants cultivated under well-watered or drought-stress conditions. Some of the studied aquaporins were subjected to fine-tuned expression only under drought-stress conditions. A beneficial effect on plant physiological parameters was observed in mycorrhizal plants with respect to nonmycorrhizal ones. Moreover, stress induced a change in the mycorrhizal type formed, which was more intracellular under drought stress. The combination of a high intracellular colonization, together with the fine-tuned expression of aquaporins could result in a morphophysiological adaptation of this symbiosis to drought conditions.

Immunolocalization of water channel aquaporins in human knee articular cartilage with intact and early degenerative regions.

Aquaporins (AQPs), a family of water channel proteins expressed in various cells and tissues, serve as physiological pathways of water and small solute transport. Articular cartilage is avascular tissue with unique biomechanical structure, a major component of which is "water". Our objective is to investigate the immunolocalization and expression pattern changes of AQPs in articular cartilage with normal and early degenerative regions in the human knee joint, which is the joint most commonly involved in osteoarthritis (OA). Two isoforms (AQPs 1 and 3) of AQPs were examined by immunohistochemical analyses using isoform-specific antibodies with cartilage samples from OA patients undergoing total knee arthroplasty. AQP 1 and AQP 3 were expressed in human knee articular cartilage and were localized in chondrocytes, both in the intact and early degenerative cartilage regions. Compared to the intact cartilage, both AQP 1 and AQP 3 immunopositive cells were observed at the damaged surface area in the degenerative region. These findings suggest that these AQPs play roles in metabolic water regulation in articular cartilage of load bearing joints and that they are responsible for OA onset.

Isolation and characterization of three maize aquaporin genes, ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4 involved in urea transport.

Urea-based nitrogen fertilizer was widely utilized in maize production, but transporters involved in urea uptake, translocation and cellular homeostasis have not been identified. Here, we isolated three maize aquapoin genes, ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4, from a cDNA library by heterogeneous complementation of a urea uptake-defective yeast. ZmNIP2;1 and ZmNIP2;4 belonged to the nodulin 26-like intrinsic proteins (NIPs) localized at plasma membrane, and ZmTIP4;4 belonged to the tonoplast intrinsic protein (TIPs) at vacuolar membrane. Quantitative RT-PCR revealed that ZmNIP2;1 was expressed constitutively in various organs while ZmNIP2;4 and ZmTIP4;4 transcripts were abundant in reproductive organs and roots. Expression of ZmTIP4;4 was significantly increased in roots and expanded leaves under nitrogen starvation, while those of ZmNIP2;1 and ZmNIP2;4 remained unaffected. Functions of maize aquapoin genes in urea transport together with their distinct expression manners suggested that they might play diverse roles on urea uptake and translocation, or equilibrating urea concentration across tonoplast.

Reconstitution of water channel function and 2D-crystallization of human aquaporin 8.

Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of three human aquaporins (HsAQP1, HsAQP4, and HsAQP5) have been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP1, HsAQP4 and HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3Å.

Novel fatty acid acylation of lens integral membrane protein aquaporin-0.

Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0. LC/MS/MS proteomic analysis of hydrophobic tryptic peptides from lens membrane proteins revealed both N-terminal and C-terminal peptides modified by 238 and 264 Da which were subsequently assigned by accurate mass measurement as palmitoylation and oleoylation, respectively. Specific sites of modification were the N-terminal methionine residue and lysine 238 revealing, for the first time, an oleic acid modification via an amide linkage to a lysine residue. The specific fatty acids involved reflect their abundance in the lens fiber cell plasma membrane. Imaging mass spectrometry indicated abundant acylated AQP0 in the inner cortical region of both bovine and human lenses and acylated truncation products in the lens nucleus. Additional analyses revealed that the lipid-modified forms partitioned exclusively to a detergent-resistant membrane fraction, suggesting a role in membrane domain targeting.