Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia

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

Functional characterisation of ANKH mutants in the pathology of CPPDD and CMD

Mutations at the terminals domains of ANKH are associated with calcium pyrophosphate deposition disease (CPPDD) and mutations within the central domains are associated with craniometaphyseal dysplasia (CMD). This study characterised the functional pathological effects of ANKH mutants in extracellular matrix biochemistry causing CPPDD and CMD. We first generated four ANKH mutant overexpression constructs containing either CPPDD linked P5L and E490del or CMD linked S375del and G389R mutations. Transfecting these constructs into CH-8 articular chondrocyte and HEK293 cells the dynamic differential localisation was monitored by confocal microscopy and the interactions with autophagy marker LC3 was investigated. Next we determined extracellular inorganic pyrophosphate (ePPi), mineralization, ENPP1 activity, expression of ENPP1, TNAP and PIT-1 to characterise mutant biochemical effects in the pathogenesis of CPPDD and CMD. Our data showed the P5L mutation delayed cell membrane localization but once the protein was recruited into the membrane it functioned to increase ePPi, mineralization and ENPP1 activity. The E490del mutated protein remained mostly cytoplasmic, forming punctate bodies that co-localized with LC3 and increased mineralization and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. The S375del mutation rendered ANKH completely cytoplasmic and trended to decrease ePPi and increase mineralization. The G389R mutation delayed cell membrane localisation of ANKH, trended to decrease ePPi and increased mineralization and also co-localized with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in severity of abnormal mineralization. Furthermore, mutant ANKH functions may be related to synthesis of defective proteins, PPi transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1