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

Abstract

Background: Variants in SLC34A2 encoding the sodium-dependent phosphate transport protein 2b (NaPi-IIb) cause the rare lung disease pulmonary alveolar microlithiasis (PAM). PAM is characterised by the deposition of calcium-phosphate concretions in the alveoli usually progressing over time. No effective treatment is available. So far, 30 allelic variants in patients have been reported but only a few have been functionally characterised. This study aimed to determine the impact of selected SLC34A2 variants on transporter expression and phosphate uptake in cellular studies.

Methods: Two nonsense variants (c.910A > T and c.1456C > T), one frameshift (c.1328delT), and one in-frame deletion (c.1402_1404delACC) previously reported in patients with PAM were selected for investigation. Wild-type and mutant c-Myc-tagged human NaPi-IIb constructs were expressed in Xenopus laevis oocytes. The transport function was investigated with a ³²Pi uptake assay. NaPi-IIb protein expression and localisation were determined with immunoblotting and immunohistochemistry, respectively.

Results: Oocytes injected with the wild-type human NaPi-IIb construct had significant ³²Pi transport compared to water-injected oocytes. In addition, the protein had a molecular weight as expected for the glycosylated form, and it was readily detectable in the oocyte membrane. Although the protein from the Thr468del construct was synthesised and expressed in the oocyte membrane, phosphate transport was similar to non-injected control oocytes. All other mutants were non-functional and not expressed in the membrane, consistent with the expected impact of the truncations caused by premature stop codons.

Conclusions: Of four analysed SLC34A2 variants, only the Thr468del showed similar protein expression as the wild-type cotransporter in the oocyte membrane. All mutant transporters were non-functional, supporting that dysfunction of NaPi-IIb underlies the pathology of PAM.

Keywords: In-frame deletion, frameshift variant, nonsense variant; Pulmonary alveolar microlithiasis (PAM); SLC34A2; SLC34A2 mutations; SLC34A2 variants; Sodium-dependent phosphate transport protein 2B, NaPi-2b; Sodium-dependent phosphate transport protein 2B, NaPi-IIb; Xenopus laevis oocytes.

Fig. 1

Predicted model of the secondary topology of the human NaPi-IIb protein; the locations of the four investigated variants are indicated. The current model of SLC34 transporters based on homology modeling with the bacterial dicarboxylate cotransporter VcINDY as a template, was adapted from the model of the predicted structure of human NaPi-IIa by Fenollar-Ferrer et al. [12]. The Na1-binding site is presumed to be formed by the amino acids T197 (Thr), Q203 (Gln), D206 (Asp), N224 (Asn), and S461 (Ser), the Na2-binding site by S161 (Ser), T192 (Thr), S193 (Ser), and N196 (Asn), the Pi-binding site by S161 (Ser) and S433 (Ser), and the Na3-binding site by Q431 (Gln), S432 (Ser), S433 (Ser), T465 (Thr), and T468 (Thr) [13]. The protein sequences used for alignment in Clustal Omega version 1.2.4 [14]: Ensembl Transcript ID ENST00000382051.7 and Transcript ID ENST00000324417.5 release 92 (Human (GRCh38.p12) assembly), and Ensembl Transcript ID ENSRNOT00000033749.5 (Rat (Rnor_6.0) assembly). A (Ala), R (Arg), N (Asn), D (Asp), E (Glu), G (Gly), I (Ile), L (Leu), P (Pro), F (Phe), S (Ser), T (Thr), Y (Tyr)

Fig. 2

Evolutionary conservation of the K304 (Lys), L443 (Leu), T468 (Thr), and Q486 (Gln) residues. Partial amino acid sequences encoded by exon 8 (Panel A), exon 11 (Panel B), and exon 12 (Panel C and D) are shown. The residues mutated in the patients are boxed. A (Ala), R (Arg), N (Asn), D (Asp), C (Cys), Q (Gln), E (Glu), G (Gly), H (His), I (Ile), L (Leu), K (Lys), M (Met), F (Phe), P (Pro), S (Ser), T (Thr), W (Trp), Y (Tyr), V (Val)

Fig. 3

Phosphate transport activity in Xenopus laevis oocytes 3 days after injection of cRNA encoding wild-type (WT) or mutated c-Myc-tagged hNaPi-IIb constructs. NI is non-injected oocytes serving as additional controls. Data presented are values from 3 to 4 independent experiments with 7–10 oocytes per group incubated for 10 min in ND96 containing 1 mM cold Pi and ³²P. Vertical bars represent the median transport activity. There was a statistically significant between-group difference (p = 0.0001, Kruskal–Wallis test by ranks). Post-hoc analysis revealed a statistically significant difference between WT and both mutants and NI control oocytes (p < 0.05, Dunn's multiple comparison test). ³²Pi uptake levels in oocytes expressing mutants were similar to non-injected oocytes (p > 0.05, Dunn's multiple comparison test)

Fig. 4

Molecular identification of c-Myc hNaPi-IIb expression by Western Blot of lysates from Xenopus laevis oocytes injected with wild-type (WT) and mutated constructs. Each lane was loaded with a volume corresponding to one oocyte. Lanes correspond to (NI) non-injected control oocytes, (WT) wild type, Lys304Ter, Leu443fs, Thr468del, and Gln486Ter. Blots were incubated with a monoclonal c-Myc-specific antibody. The approximate protein molecular mass is indicated in kilodaltons (kDa). Specific immunoreactive protein bands are detected at ∼ 110–125 kDa for WT and Thr468del, at ∼ 35 kDa for Lys304Ter, and at ∼ 60 kDa for Leu443fs and Gln486Ter

Fig. 5

Immunofluorescence in Xenopus laevis oocytes expressing wild-type c-Myc-hNaPi-IIb (WT), mutants (Lys304Ter, Leu443fs, Thr468del, and Gln486Ter), non-injected (NI) control oocytes, and oocytes with no secondary antibody (NS). Immunostaining confirms cell membrane localisation of WT and Thr468del c-Myc-hNaPi-IIb constructs. Cryosections of oocytes were incubated with monoclonal c-Myc antibody. Scale bar: 30 µm. Magnification: × 63