Abstract：
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.
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.
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.
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.
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.
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.
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.
摘要：
SLC34A2中编码钠依赖性磷酸盐转运蛋白2b（NaPi-IIb）的变体引起罕见的肺部疾病肺泡微石症（PAM）。PAM的特征在于肺泡中磷酸钙结石的沉积通常随时间进展。没有有效的治疗方法。到目前为止,已经报道了患者中的30个等位基因变体,但只有少数具有功能特征。本研究旨在确定细胞研究中选定的SLC34A2变体对转运蛋白表达和磷酸盐摄取的影响。
两个无义变体（c.910A>T和c.1456C>T），一个移码(c.1328delT)，选择先前在PAM患者中报告的一个帧内缺失（c.1402_1404delACC）进行研究。野生型和突变型c-Myc标记的人NaPi-IIb构建体在非洲爪狼卵母细胞中表达。用32Pi摄取测定研究转运功能。用免疫印迹法和免疫组织化学方法测定NaPi-IIb蛋白的表达和定位,分别。
与水注射的卵母细胞相比,用野生型人NaPi-IIb构建体注射的卵母细胞具有显著的32Pi转运。此外,蛋白质的分子量与预期的糖基化形式相同，它在卵母细胞膜中很容易检测到。尽管来自Thr468del构建体的蛋白质被合成并在卵母细胞膜中表达,磷酸盐转运与未注射对照卵母细胞相似.所有其他突变体都是无功能的，不在膜中表达，与过早终止密码子引起的截断的预期影响一致。
在四种分析的SLC34A2变体中，只有Thr468del在卵母细胞膜中显示出与野生型共转运蛋白相似的蛋白表达。所有突变转运蛋白都是无功能的，支持NaPi-IIb的功能障碍是PAM病理的基础。
两个无义变体（c.910A>T和c.1456C>T），一个移码(c.1328delT)，选择先前在PAM患者中报告的一个帧内缺失（c.1402_1404delACC）进行研究。野生型和突变型c-Myc标记的人NaPi-IIb构建体在非洲爪狼卵母细胞中表达。用32Pi摄取测定研究转运功能。用免疫印迹法和免疫组织化学方法测定NaPi-IIb蛋白的表达和定位,分别。
与水注射的卵母细胞相比,用野生型人NaPi-IIb构建体注射的卵母细胞具有显著的32Pi转运。此外,蛋白质的分子量与预期的糖基化形式相同，它在卵母细胞膜中很容易检测到。尽管来自Thr468del构建体的蛋白质被合成并在卵母细胞膜中表达,磷酸盐转运与未注射对照卵母细胞相似.所有其他突变体都是无功能的，不在膜中表达，与过早终止密码子引起的截断的预期影响一致。
在四种分析的SLC34A2变体中，只有Thr468del在卵母细胞膜中显示出与野生型共转运蛋白相似的蛋白表达。所有突变转运蛋白都是无功能的，支持NaPi-IIb的功能障碍是PAM病理的基础。
