The CPPDD-associated ANKH M48T mutation interrupts the interaction of ANKH with the sodium/phosphate cotransporter PiT-1.
OBJECTIVE
Numerous dominant human homolog of progressive ankylosis (ANKH) mutations have been identified in familial calcium pyrophosphate dihydrate crystal deposition disease (CPPDD). Due to the dominant nature of these mutations, we investigated whether ANKH interacts with other proteins; and if so, whether any CPPDD-associated ANKH mutation might disrupt such protein interactions.


METHODS
Stable ATDC5 ANKH wt- and ANKH M48T-transfectants were generated. Lysates from these transfectants were used to identify candidate protein interaction with ANKH by coimmunoprecipitation followed by Western blot analysis. The effect of high phosphate on the expression of genes involved in modulating Pi (inorganic phosphate)/PPi (inorganic pyrophosphate) homeostasis in these transfectants was assessed.


RESULTS
We showed that ANKH protein associates with the sodium/phosphate cotransporter PiT-1, and that ANKH M48T mutant protein failed to interact with PiT-1. We also showed that upon high phosphate treatment, the normally coordinated upregulation of endogenous Ank and PiT1 transcript expression was disrupted in ANKH M48T transfectants.


CONCLUSIONS
Our results suggested that there is a coordinated interrelationship between 2 key participants of Pi and PPi metabolism, ANKH and PiT-1.
Toronto Western Research Institute, University Health Network, University of Toronto, and Toronto Western Hospital, Mc14-419, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada.
John
J
Wang
Hing Wo
HW
Tsui
Frank
F
Beier
Florence W L
FW
Tsui
D000818:Animals; D002131:Calcium Pyrophosphate / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D002805:Chondrocalcinosis / Q000235:genetics* / Q000378:metabolism; D019902:Chondrocytes / Q000166:cytology / Q000502:physiology; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D009154:Mutation; D028061:Phosphate Transport Proteins / Q000235:genetics* / Q000378:metabolism; D050817:Transcription Factor Pit-1 / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D002131:Calcium Pyrophosphate; D008565:Membrane Proteins; D028061:Phosphate Transport Proteins; D050817:Transcription Factor Pit-1; C498770:ANKH protein, human; C494187:POU1F1 protein, human; C494185:Pou1f1 protein, mouse; C410982:ank protein, mouse