Vijen Sunny, Hawes Chris, Runions John, Russell R Graham G, Wordsworth B Paul, Carr Andrew J, Pink Ryan C, Zhang Yun
Department of Biology and Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, UK.
Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford Institute of Musculoskeletal Sciences, Windmill Road, Oxford, OX3 7HE, UK.
Sci Rep. 2020 May 4;10(1):7408. doi: 10.1038/s41598-020-63911-x.
ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants dynamic differential localisations were imaged and we investigated the interactions with the autophagy marker LC3. Extracellular inorganic pyrophosphate, mineralization, ENPP1 activity expression of ENPP1, TNAP and PIT-1 were measured. P5L delayed cell membrane localisation but once recruited into the membrane it increased extracellular inorganic pyrophosphate, mineralization, and ENPP1 activity. E490del remained mostly cytoplasmic, forming punctate co-localisations with LC3, increased mineralization, ENPP1 and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. S375del trended to decrease extracellular inorganic pyrophosphate, increase mineralization. G389R delayed cell membrane localisation, trended to decrease extracellular inorganic pyrophosphate, increased mineralization and co-localised with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in mineralization. Furthermore, mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1.
ANKH 突变与焦磷酸钙沉积病和颅骨骨干发育异常有关。本研究调查了这些 ANKH 突变体对细胞定位及相关生物化学的影响。我们构建了四个含有与焦磷酸钙沉积病或颅骨骨干发育异常相关突变的 ANKH 过表达质粒：P5L、E490del 和 S375del、G389R。将它们转染到 CH-8 关节软骨细胞和 HEK293 细胞中。对 ANKH 突变体的动态差异定位进行成像，并研究其与自噬标记物 LC3 的相互作用。测量细胞外无机焦磷酸、矿化、ENPP1 活性以及 ENPP1、TNAP 和 PIT-1 的表达。P5L 延迟了细胞膜定位，但一旦被募集到细胞膜上，它会增加细胞外无机焦磷酸、矿化和 ENPP1 活性。E490del 大多保留在细胞质中，与 LC3 形成点状共定位，增加矿化、ENPP1 及 ENPP1 活性，同时 TNAP 和 PIT-1 最初有短暂增加但未持续。S375del 倾向于降低细胞外无机焦磷酸，增加矿化。G389R 延迟细胞膜定位，倾向于降低细胞外无机焦磷酸，增加矿化并与 LC3 共定位。我们的结果表明 ANKH 突变体的病理定位与不同程度的矿化之间存在联系。此外，突变型 ANKH 的功能与缺陷蛋白的合成、无机焦磷酸转运、ENPP1 活性以及 ENPP1、TNAP 和 PIT-1 的表达有关。
