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.

Grantor dc:publisher

Oxford Brookes University

Year dc:date

2019

Author dc:creator

• Vijen, Sunny

Contributors dc:contributor

• Zhang, Yun
• Pink, Ryan

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/86wj-gw21

OAI identifier oai:identifier

tle:a3473120-ddec-4136-9dcc-407dd3a63a77:d6bd9758-527a-46cd-bfe2-c433766e8fca:1