Impaired phosphate transport in SLC34A2 variants in patients with pulmonary alveolar microlithiasis

Variants and preparation of plasmid constructs

The variants investigated were two nonsense, one frameshift, and one in-frame deletion: c.910A > T (p.Lys304Ter) [24, 25, 29], c.1328delT (p.Leu443ArgfsTer6) [22, 30], c.1402_1404delACC (p.Thr468del) [26, 28], and c.1456C > T (p.Gln486Ter) [27]. These changes were introduced into human SLC34A2 cDNA and subcloned into a KSM vector. All constructs were fused N-terminally to c-Myc.

Mutagenic primers were designed using the Jellyfish Version 1.0 software programme. The variants were introduced into a commercially available c-Myc-DDK-tagged SLC34A2 vector, in which the c-Myc epitope was fused to the C-terminus of the cotransporter in a pCMV6 backbone (OriGene Technologies, Rockville, MD, USA) (NCBI GenBank accession no. NM_006424 and AF067196), using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The plasmids containing generated variants were isolated using QIAprep Spin Miniprep Kit (QIAGEN, 27106), and all variants were verified by sequencing (Eurofins Genomics, Ebersberg, Germany).

Because the C-terminus of other SLC34 transporters is required for proper sorting, plasmids containing the c-Myc epitope fused to the N-terminal tail of the transporter were then generated using the above constructs as templates. Thus, complementary oligonucleotides containing the Kozak consensus sequence followed by the sequence encoding the c-Myc (Microsynth AG, Balgach, Switzerland) were first annealed and purified with the QIAEX II Gel Extraction Kit (QIAGEN). The annealed fragment contained HindIII (upstream) and EcoRI (downstream) restriction sites. Upon annealing and digestion with HindIII and EcoRI (Thermo Scientific) the c-Myc sequence was ligated with T4 DNA Ligase (Thermo Scientific) into a KSM expression vector (previously digested with the same enzymes) that contains the 5′ and 3′ UTRs from Xenopus β-globin to optimise its expression in oocytes as described by Virkki et al. [38]. Hereafter, the ligation product was transfected into competent cells. Plasmid DNA was purified with QIAprep Spin Miniprep Kit (QIAGEN, 27106) and sequenced (Microsynth AG, Balgach, Switzerland) to confirm the presence of the c-Myc sequence.

The cDNAs encoding hNaPi-IIb (WT and mutants) were amplified from the original vector (pCMV6 backbone) by PCR. The sense primer contained an EcoRI site, whereas the antisense primer included a stop codon downstream of the last amino acid of the cotransporter followed by a BamHI site. After purification by use of the QIAquickPCR purification kit (QIAGEN), NaPi-IIb WT and mutants cDNAs were cut from the vector by EcoRI and BamHI digestion (Thermo Scientific). The isolated hNaPi-IIb inserts were then ligated into the c-Myc-tagged KSM vector (Additional file 1: Fig. S3) previously digested with the same enzymes and transfected into competent cells. After purification, KSM plasmids containing c-Myc-tagged WT and mutated hNaPi-IIb cDNA inserts were verified by DNA sequencing (Microsynth, AG, Balgach, Switzerland). For oocyte expression, plasmids were linearised with NotI (Thermo Scientific), and cRNA was synthesised in the presence of a cap analogue using the Megascript T3 kit (Ambion). The RNA was purified using the RNeasy Mini Kit (QIAGEN). After measuring the concentration by spectrophotometry, the samples were diluted to 0.2 µg/µl in nuclease-free water.

Expression of hNaPi-IIb wild type and mutants in Xenopus laevis oocytes

Oocytes were harvested from Xenopus laevis frogs anesthetised in MS222 (tricaine methansulphonate). Oocytes were treated with 1 mg/ml collagenase (Type I-A, Sigma-Aldrich, Buchs, Switzerland) in 100Na solution (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 10 mM HEPES, pH 7.4 adjusted with Tris) in the presence of 0.1 mg/ml trypsin inhibitor (Type III-O, Sigma-Aldrich, Buchs, Switzerland). The oocytes were stored in modified Barth’s solution (88 mM NaCl, 1 mM KCl, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.5 mM NaHCO3, 2 mM Ca(NO3)2, 7.5 mM HEPES, pH 7.5 adjusted with Tris) supplemented with antibiotics (doxycycline and gentamicin, 5 mg/l each). Healthy stage V–VI oocytes were selected and incubated in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, pH 7.4 adjusted with Tris) supplemented with antibiotics (doxycycline and gentamicin, 5 mg/l each). The oocytes were injected by use of the Nanoject II (Drummond Scientific Company, Broomall, USA), with 50 nl of in vitro synthesised cRNA (0.2 mg/ml) and maintained at 16 °C in ND96 solution supplemented with doxycycline and gentamicin (5 mg/l each). [32P] phosphate flux measurements were performed 3 days after injection. Non-injected and water-injected oocytes served as controls. All animal procedures were conducted following the Swiss Cantonal and Federal legislation for experiments involving animals. All standard reagents were obtained from Fluka (Buchs, Switzerland).

32Pi uptake studies in Xenopus laevis oocytes

Non-injected control oocytes, oocytes expressing WT hNaPi-IIb, and mutants (7–10 oocytes per group) were first equilibrated in ice-cold ND96 without a tracer. After aspiration of the solution, the oocytes were incubated in ND96 containing 1 mM cold Pi and 32P (specific activity 10 mCi/mmol Pi) for 10 min (see below) at 25 °C. After incubation, the solution was removed, and oocytes were washed four times with ice-cold ND96. Each oocyte was transferred to a scintillation vial and lysed in 200 μl of 2% sodium dodecyl sulfate (SDS) for 45 min by shaking before the addition of 3 ml scintillation fluid (Emulsifier-Safe, PerkinElmer). The amount of radioactivity accumulated in each oocyte was measured by scintillation counting in a β-counter (Packard BioScience).

Na+-dependent Pi transport has been shown to be linear up to 60 min upon initiation of uptake, ensuring that uptake per unit time is a measure of transport velocity [39]. Preliminary experiments only with WT hNaPi-IIb and water-injected oocytes as negative controls were performed with an incubation time of 10 and 30 min (Additional file 1: Fig. S3). Based on these data, an incubation time of 10 min was chosen for subsequent experiments including mutants. Three independent experiments were performed for each mutant, always including oocytes expressing WT hNaPi-IIb and non-injected oocytes.

Western blot and immunocytochemistryOocyte preparation for western blot

Pools of three oocytes were lysed in 60 μl lysis buffer [100 mM NaCl, 20 mM Tris HCl pH 7.6, 1% Igepal CA 630 (Sigma-Aldrich, Buchs, CH)], by pipetting up and down. To pellet the yolk proteins, the lysates were centrifuged at 16,000g for 10 min at 4 °C. After the yolk-free supernatants were collected carefully, avoiding contamination with floating lipids, supernatants were mixed with Laemmli sample buffer (0.38 M Tris base, 8% SDS, 4 mM EDTA, 40% (v/v) glycerol, pH 6.8 with HCl, 4 mg/ml of Bromophenol Blue).

Immunoblotting of oocytes homogenates

A volume of the supernatant corresponding to one oocyte was loaded on a 10% acrylamide SDS gel for SDS-PAGE. Separated proteins were transferred from the gel to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore, Schaffhausen, Switzerland) in a standard tank system (Mini Trans-Blot, Bio-Rad). PVDF membranes were stained with 0.3% Ponceau in 3% Trichloroacetic acid (TCA) to confirm the transfer and subsequently blocked with Tris-buffered saline (TBS) containing 5% fat-free powder milk for 30 min at room temperature. Membranes were then incubated overnight at 4 °C with primary antibody against c-Myc (M4439, Sigma-Aldrich, Buchs, Switzerland) (1:4000) [40]. After washing with TBS three times followed by blocking, membranes were incubated with anti-mouse secondary antibody linked to horseradish peroxidase (HRP) (GE Healthcare, UK Limited) (1:10,000) for 3 h at room temperature. Membranes were then washed three times with TBS and subsequently exposed to HRP substrate (Western Chemiluminescence HRP Substrate, Millipore, Schaffhausen, Switzerland) for 5 min. Chemiluminescence was detected using a LAS-4000 camera system (Fujifilm).

Oocyte preparation for immunocytochemistry

Three days after injection, oocytes were washed with phosphate-buffered saline (PBS). For fixation, eggs were incubated in 3% paraformaldehyde (PFA) in PBS for 6 h at 4 °C. Oocytes were then washed with PBS and incubated in 30% sucrose in PBS overnight at 4 °C for cryoprotection. Hereafter, eggs were incubated in PBS for 15 min at room temperature and then transferred to cryomolds (Cryomold Biopsy, Sakura Finetek Germany GmbH, Staufen, Germany) filled with embedding medium (OCT embedding Matrix, Cell Path, Newtown, United Kingdom). The cryomolds with oocytes were immediately snap-frozen in liquid propane and stored at − 80 °C.

Immunostaining

Oocyte cryosections of 5 μm were mounted on slides (Superfrost Plus, Thermo Scientific) and incubated in PBS for 45 min. The slides were then blocked in 1% bovine serum albumin (BSA) in PBS for 15 min at room temperature. After blocking, the slides were incubated overnight at 4 °C with the primary antibody against c-Myc (M4439, Sigma-Aldrich, Buchs, Switzerland) (1:500, 1:1000) diluted in 0.02% sodium azide (NaN3) in PBS [41]. The slides were washed twice with hypertonic PBS (18 g NaCl in PBS) and once with PBS, followed by incubation with a donkey anti-mouse Alexa Fluor 594 secondary antibody (1:500, Invitrogen) for 1 h at room temperature. Then sections were washed twice with hypertonic PBS and once with PBS, and coverslips were then mounted with Glycergel (DakoCytomation, Baar, Switzerland). Fluorescence was detected using a Leica fluorescence microscope (Leica CTR600). The freeware programmes Leica AF lite and ImageJ version 1.52a were used to analyse the images.

Data analysis

The normality of the data was assessed by inspection of QQ-plots. Results are presented as the median and range. Differences between groups were tested with Mann–Whitney U-test or Kruskal–Wallis test by ranks. Post-hoc multiple comparisons were performed using Dunn's multiple comparison test. A p value < 0.05 was considered statistically significant. Data analysis was performed using Stata 11.2 (StataCorp 2009, College Station, TX, USA).

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