Abstract
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.
Highlights
ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia
At least four proteins influence PPi homeostasis in the extracellular matrix (ECM): these include the transmembrane pyrophosphate transporter ANKH, ENPP1, TNAP and PIT-1; ENPP1 readily hydrolyses extracellular adenosine triphosphate into extracellular PPi and extracellular AMP, whilst ANKH exports intracellular PPi into the ECM, TNAP hydrolyses ePPi into extracellular Pi(ePi), and PIT-1 imports ePi into the cell via a sodium exchange channel to be used in metabolic pathways[1,2]
We investigated the involvement of autophagy for potential mutated ANKH protein recycling in the pathogenesis of calcium pyrophosphate dihydrate deposition disease (CPPDD) and CMD
Summary
ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. 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. 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. Mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1. G389R (p.Gly389Arg, NM_054027.4:c.1165 G > A), reported in several cases of CMD and recently in CPPDD, where it was predicted to be a loss-of-function variant[4,12]
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