Quantification of the Intracellular Life Time of Water Molecules to Measure Transport Rates of Human Aquaglyceroporins
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Orthodox aquaporins are transmembrane channel proteins that facilitate rapid diffusion of water, while aquaglyceroporins facilitate the diffusion of small uncharged molecules such as glycerol and arsenic trioxide. Aquaglyceroporins play important roles in human physiology, in particular for glycerol metabolism and arsenic detoxification. We have developed a unique system applying the strain of the yeast Pichia pastoris, where the endogenous aquaporins/aquaglyceroporins have been removed and human aquaglyceroporins AQP3, AQP7, and AQP9 are recombinantly expressed enabling comparative permeability measurements between the expressed proteins. Using a newly established Nuclear Magnetic Resonance approach based on measurement of the intracellular life time of water, we propose that human aquaglyceroporins are poor facilitators of water and that the water transport efficiency is similar to that of passive diffusion across native cell membranes. This is distinctly different from glycerol and arsenic trioxide, where high glycerol transport efficiency was recorded.
KeywordsAquaporin Aquaglyceroporin Water transport NMR P. pastoris
Aquaporins (AQPs) are transmembrane proteins that are represented throughout all kingdoms of life, including animals and plants as well as in lower organisms such as yeast and bacteria. Their primary function is to facilitate water and glycerol transport across cell membranes (Verkman 2012). The aquaporin family is commonly divided into three sub-groups, the orthodox aquaporins (sole water facilitators), the aquaglyceroporins (that facilitate the transport of solutes such as glycerol, arsenic trioxide and urea), and the superaquaporins. There are 13 human aquaporins abbreviated AQP0-12, which are widely distributed in specific cell types in many organs and tissues (Day et al. 2014). AQP3, 7, 9, and 10 belong to the aquaglyceroporin sub-group, and in recent years, several reports suggest that the human aquaglyceroporins are essential players in human health and disease (Hara-Chikuma and Verkman 2008b; Maeda 2012). Commonly, the aquaglyceroporins are stated to have dual permeability, both for water and solutes such as glycerol (Laforenza et al. 2016). The transport specificity of the aquaglyceroporins is well-documented in the literature, frequently using either Xenopus laevis oocytes to measure the water transport rates, or artificial membranes creating liposomes with inserted proteins. In both these systems, the water transport is indirectly measured by detecting the swelling/shrinkage of the oocytes/liposomes upon an applied change in osmolality, and water transport is quantified in terms of the osmotic water permeability coefficient. Here, we apply a more direct method for studying water exchange over biological membrane by using nuclear magnetic resonance (NMR), which allows for the transport mechanisms to be studied in equilibrium conditions without applying an osmotic gradient (Eriksson et al. 2017).
We established a unique approach to measure transport of water, glycerol, and arsenic trioxide [As(III)] in a native cell membrane using the yeast Pichia pastoris. In this approach, human AQP3, AQP7, and AQP9 are highly expressed in an AQP deletion strain of P. pastoris, where both endogenous aquaporins (Aqy1 and Agp1) have been removed by standard cloning strategies. Spheroplasts of these strains have AQP-containing lipid bilayers without requiring elaborate protein purification schemes or need of exchanging detergent micelle-stabilized membrane proteins into the lipid bilayers, necessary in liposome-based approaches. We applied the diffusion NMR method that enables quantification of the rate of water exchange between intracellular and extracellular compartments on a millisecond time-scale (Eriksson et al. 2017). Interestingly, in our system, when human aquaglyceroporins are in their native environment (lipid bilayer) and the water transport rate is measured by the non-invasive NMR method, the water transport capability of the human aquaglyceroporins are not significantly different from the passive water diffusion across the plasma membrane of cells without aquaglyceroporins expressed. Still, all aquaglyceroporins investigated significantly facilitate the transport of glycerol and As(III) in the same system. Thus, we suggest that human aquaglyceroporins are poor water facilitators compared to orthodox aquaporins, and that their main function in the body is to facilitate transport of other solutes such as glycerol and arsenic trioxide.
Materials and Methods
Primers used for cloning. Nucleic acid sequences for primers used for creating the AGP1 deletion in P. pastoris GS115 aqy1Δ strain
Primer sequence (5′-3′ - direction)
Protein Expression and Western Blot Analysis
The human aquaporins AQP1, AQP3, AQP7, and AQP9 with a His6 purification tag were cloned into pPICZB vector using restriction enzyme EcoRI and XbaI. To identify clones that express the protein of interest, AQP transformants were spotted on YPD-agar plates supplemented with increasing zeocin concentrations (0, 0.5, 1.0, and 2.0 mg/ml) (Thermo Fisher, Life Technologies, USA). The five most zeocin-resistant clones of each AQP construct were further analyzed for expression levels. Cells were pre-grown in buffered glycerol complex medium (BMGY: 1% w/v yeast extract, 2% w/v peptone, 100 mM potassium phosphate pH 6.0, 1.34% w/v yeast nitrogen base, 0.4 mg/L biotin, 1% v/v glycerol) for 24 h, and protein production was induced with methanol to a final concentration of 0.25% v/v for 6 h (for AQP3, AQP7, AQP9) or 1 h (for AQP1). Cells were harvested at 2000×g for 5 min and washed in 20 mM HEPES pH 7.5. Plasma membranes were purified using the protocol of Panaretou and Piper (2006). Approximately, 300-μl cell pellet was harvested and re-suspended in an equal volume of breaking buffer (20 mM Tris–HCl pH 7.5, 0.4 M sucrose, 4 mM EDTA, 2 mM DTT) containing protease inhibitor (Roche Diagnostics) and glass beads. Cells were broken by Fast Prep (MP Biomedical, USA), cell debris was removed at 600×g for 10 min at 4 °C, and the total membrane was collected by centrifugation at 21,000×g for 30 min at 4 °C. Total membranes were re-suspended in membrane resuspension buffer (10 mM Tris–HCl pH 7.5, 2 mM EDTA). A sucrose gradient containing equal volumes of 2.25, 1.65, and 1.1 M sucrose in 10 mM Tris–HCl pH 7.5 and 2 mM EDTA was overlaid with the total membrane sample and spun overnight in a SW Ti60 centrifuge at 40,000 rpm at 4 °C. The plasma membrane fraction was removed from the 2.25/1.65 M interphase and diluted five times into membrane resuspension buffer and spun for 30 min at 20,000×g at 4 °C. Pellet was re-suspended in plasma membrane resuspension buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% v/v glycerol) with protease inhibitor. Purified plasma membrane samples were resolved on NuPAGE 4–12% Bis–Tris (Thermo Fisher, Life Technologies, USA) gels and blotted onto nitrocellulose membranes (Hoefer Inc, USA). Membranes were blocked with fish gelatin blocking buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 2% w/v Fish Gelatin, 1% w/v Ovalbumin). Antibodies were incubated with blocked membrane in 1:1 blocking buffer and 20 mM Tris–HCl pH 7.5, 100 mM NaCl with 0.1% v/v Tween-20 (TBS-T). The plasma membrane marker, Pma1, was detected using rabbit-anti-Pma1 antibody (Santa Cruz Biotechnology, USA; 1:1,000, #sc-33735) at 4 °C overnight and the His-tag was detected with the mouse-anti-His antibody (Sigma-Aldrich, USA; 1:5000) for 45 min at room temperature. Donkey-anti-mouse 680 nm (1:10,000) and donkey-anti-rabbit 800 nm (1:10,000) were used as secondary antibodies, and incubated for 45 min at room temperature (LI-COR, USA). Fluorescent signals were detected using Odyssey FC (LI-COR, USA). The endoplasmic reticulum (ER) marker, Sec61, antibody was used at 1:1000 and incubated over night at 4 °C. The signal was detected using an anti-rabbit horseradish peroxidase antibody and enhanced chemiluminescence (ECL). The signal was detected by the Gel DocTMsystem (BIO-RAD, USA).
Cells were grown and protein production induced as described above. Cells were harvested and washed with 20 mM HEPES pH 7.5, re-suspended in a 1:1 v/v solution with buffer, and transferred to 5-mm disposable NMR tubes and centrifuged at 1000×g for 10 min to achieve a pellet with high concentration of cells. The tubes were kept on ice until measurement. The NMR diffusion measurements were done at 0 °C for AQP1 and aqy1Δagp1Δ induced for 1 h and at 20 °C for strain AQP3, 7, and 9 and aqy1Δagp1Δ induced for 6 h. According to Eriksson et al., the temperatures and induction times were chosen so that the same set of mixing times (t m) allowed a precise quantification of exchange for all strains, aqy1Δagp1Δ, aqy1Δagp1Δ + hAQP1, and aqy1Δagp1Δ + AQP3,7,9 (Eriksson et al. 2017). Measurements were performed on a Bruker 200 MHz Avance-II spectrometer with a DIF-25 gradient probe capable of z-gradients up to 9.6 T/m. The NMR method used is described in Eriksson et al. (Eriksson et al. 2017) and identical experimental parameters were used here. The method combines a filter exchange spectroscopy (FEXSY) pulse sequence with a pulsed-gradient spin-echo (PGSE). The FEXSY experiment consists of two diffusion-encoding blocks separated by a mixing time, t m, and is specifically sensitive to exchange. The PGSE pulse sequence was applied with varying echo time, t E, to correlate the intra- and extracellular diffusion coefficients and T 2-relaxation times and was used to compensate for differences in T 2-relaxation between the intra- and extracellular water. The intra- to extracellular exchange rate was obtained by a constrained global fitting of both datasets.
Stopped Flow Measurements
Cells were isolated at 2000×g for 5 min and washed three times in 20 mM HEPES pH 7.5 and 1.2 M Sorbitol, and re-suspended to a final absorbance at A600 of 3. In the Stopped Flow Machine (SFM) procedure, cells were subjected to a hyperosmotic shock (20 mM HEPES pH 7.5 and 1.8 M Sorbitol) with a mixing rate of 7 ml/s in a ratio of 1:1 to a final volume of 148 µl. The cell response (shrinkage) was monitored as increased light-scattering intensity at 435 nm at an angle of 90° using a SFM-20 and MOS-450 spectrometer (BioLogic).
Glycerol Transport Measurement
Washed cells were re-suspended to an approximate density of 30 mg/ml, and subsequently the absorbance at A600 was measured and precisely adjusted for all strains. Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP) was added to a final concentration of 100 µM to the cell suspension to inhibit uptake of glycerol by active transporters, and equilibrated with the cells at 30 °C for 10 min prior to each measurement. Uptake studies were initiated by the addition of a glycerol mix (300 mM glycerol and 40 µM [14C] glycerol (142.7 mCi/mmol; Perkin Elmer)). The reaction was stopped by transferring the whole mixture to a pre-filled funnel with 5 ml pre-chilled 20 mM HEPES, pH 7.5, 500 mM glycerol. Cells were then washed and disintegration per minutes (dpm) was recorded as previously described (Tamas et al. 1999). The measured signal in dpm was converted to nmol glycerol per OD unit and plotted over time. For measurement carried out at pH 6.0, 20 mM MES pH 6.0 was used instead of 20 mM HEPES pH 7.5.
As(III) Transport and Phenotype Assay
For each individual measurement, 10 OD units of washed cells were re-suspended in 20 mM HEPES pH 7.5 and incubated at 30 °C for 5–10 min prior measurement. Uptake studies were initiated by the addition of NaAsO2 in 1:1 (v/v) ratio with a final concentration of 100 mM. The reaction was stopped by transferring the cell suspension to cold 20 mM HEPES buffer pH 7.5 and collected on a Whatman® glass microfiber filter Grade GF/C and washed twice in cold 20 mM HEPES buffer. Cells were re-suspended in water and disrupted by boiling. Cell debris was sedimented at 10,000×g for 10 min. Sodium arsenite-containing supernatants were sent to analysis by inductively coupled plasma mass spectrometry (ICP-MS). For the growth assay, cells were cultivated and protein expression was induced (as described above). Cells were diluted in 20 mM HEPES pH 7.5 to an absorbance at A600 of 0.2. Five microliters from 10 times dilution series were spotted on agar plates containing buffered methanol complex medium (BMMY: 1% w/v yeast extract, 2% w/v peptone, 100 mM potassium phosphate pH 6.0, 1.34% w/v yeast nitrogen base, 0.4 mg/l biotin, 0.5% v/v methanol, 2% w/v agar) with and without NaAsO2 (0 and 600 µM) and incubated at 30 °C for 3 days.
Comparisons between multiple groups were done in GraphPad Prism by one-way ANOVA followed by Tukey’s multiple comparisons test and the null hypothesis was rejected at the 0.05 confidence level.
Results and Discussion
The yeast P. Pastoris has been successfully used for heterologous protein production and, particularly, in recent years also for overexpressing transmembrane proteins. Thus, P. pastoris is a suitable system for measuring the activity of human transmembrane proteins. In this study, we aim at pinpointing the detailed specificity of the human aquaglyceroporins. The genome for P. pastoris GS115 has been public since 2009 (De Schutter et al. 2009) and contains two aquaporin genes encoding the previously characterized Aqy1 (Fischer et al. 2009) and a second putative aquaglyceroporin, Agp1 (AquaGlyceroPorin1).
A Novel System to Study Substrate Specificity of Human Aquaglyceroporins
The Human Aquaglyceroporins are Poor Water Facilitators
The Human Aquaglyceroporins Facilitate the Transport of Glycerol
AQP7 and AQP9 are Potent Arsenic Trioxide Facilitators
Human aquaglyceroporins are commonly referred to as channels facilitating the transport of glycerol in addition to water. Available methods for estimating the permeability of water through AQP channels are mainly restricted to non-polarizable cells (oocytes) or model membranes. This study uses a novel yeast-based system combined with a newly developed NMR method, and suggests that the main role of aquaglyceroporins is not related to water movement, but rather transport of glycerol and arsenic trioxide across the plasma membrane. Human aquaglyceroporins are likely not able to exclude water entirely, but the transport rates are not significantly different from passive diffusion of water across the plasma membrane, and hence likely of limited physiological relevance.
We would like to express our gratitude to Dr. Örjan Hansson at University of Gothenburg for his help with stopped flow analysis, Dr. Christoph Basse Karlsruhe at Institute of Technology for providing us with pSLNAT and Jennifer Carbrey, Duke University Medical Center for providing us with human AQP9 gene. Sec61 antibody was kindly provided by Prof. Thomas Sommer, Max-Delbrück-Center for Molecular Medicine/Humboldt-University of Berlin. This project was funded by the Swedish Research Council (2011-2891 and 2016-6214), the Cancer Foundation (2010/1171 and 2014/575), the Novo Nordisk Foundation (9807), AFA Försäkring, the Olle Engkvist Byggmästsre, VINNOVA and Crafoord Foundation (20160579), all received by Karin Lindkvist-Petersson.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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