Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia
Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia
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DOI：
10.1038/s41598-020-63911-x
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发表时间：
2020-05-04
影响因子：
4.6
通讯作者：
Zhang, Yun
中科院分区：
文献类型：
--
作者：
Vijen, Sunny;Hawes, Chris;Zhang, Yun
ANKH突变与焦磷酸钙沉积病和颅骺发育不良有关。本研究调查了这些ANKH突变体对细胞定位和相关生物化学的影响。我们产生了四种ANKH过表达质粒，其含有焦磷酸钙沉积疾病或颅骺发育不良相关突变：P5 L，E490 del和S375 del，G389 R。转染CH-8关节软骨细胞和HEK 293细胞。ANKH突变体动态差异定位成像，我们研究了与自噬标记LC 3的相互作用。测定细胞外无机焦磷酸盐、矿化、ENPP 1活性、ENPP 1、TNAP和PIT-1的表达。P5 L延迟细胞膜定位，但一旦招募到膜中，它增加细胞外无机焦磷酸盐，矿化和ENPP 1活性。E490 del主要保持在细胞质中，与LC 3形成点状共定位，增加矿化，ENPP 1和ENPP 1活性，TNAP和PIT-1最初但不持续增加。S375 del有减少细胞外无机焦磷酸盐、增加矿化的趋势。G389 R延迟细胞膜定位，倾向于减少细胞外无机焦磷酸盐，增加矿化并与LC 3共定位。我们的研究结果表明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ANKH功能与缺陷蛋白质的合成、无机焦磷酸盐转运、ENPP 1活性以及ENPP 1、TNAP和PIT-1的表达有关。
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.