The ecto-nucleotide pyrophosphatase/ phosphodiesterase 1 (NPP1), encoded by the ENPP1 gene, is a plasma membrane protein that converts extracellular ATP to AMP and thereby generates PPi. PPi is a physiologic inhibitor of hydroxyapatite formation and is therefore important in the prevention of soft tissue calcification. In humans, mutations in ENPP1 are associated with general arterial calcification of infancy (GACI), a rare autosomal recessive condition causing a premature onset of severe calcification of the media of large and medium-sized arteries and intimal proliferation resulting in stenoses.
To determine the functional impact of missense mutations in ENPP1, we analyzed 12 putative pathogenic missense mutations and three known polymorphisms regarding their functional properties, i.e. expression, activity and localization and PPi generation. We transfected with cDNAs encoding wildtype (WT) or mutated ENPP1 into HEK293 cells and measured expression and NPP activity. Transfection of WT or mutated ENPP1 did not affect synthesis of NPP1 protein. Eight of the 12 variants tested completely lost NPP activity, whereas four mutants (Y471C, S504R, Y659C and H777R) showed residual but reduced NPP activity compared to WT NPP1. Interestingly, one putative pathologic variant showed normal NPP activity. HEK293 cells transfected with the known polymorphisms generated the same amount of PPi as cells transfected with WT NPP1. The five mutants with normal or residual NPP1 enzyme activity were still able to generate PPi. However, the PPi generation did not reach the PPi levels generated by WT NPP1. For cellular localization analyses, NPP1 mutants were expressed in COS7 cells. The WT NPP1 was present predominantly in the plasma membrane. Both the mutants and polymorphisms with residual enzyme activity were targeted to the plasma membrane. In this study, we showed that all ENPP1 polymorphisms tested have normal NPP1 function and we identified an additional functional polymorphism, which was expected to be pathogenic till now. We found, that missense mutations in strictly conserved amino acids lead to loss of function of NPP1. Loss of structure stability not necessarily leads to loss of function, but it could lead to reduced protein abundance due to misfolding during biosynthesis. Based on our results, we hypothesize that the four variants with residual NPP1 function (Y471C, S504R, Y659C and H777R) would be therapeutical targets for conformational stabilizing agents.