Background/Purpose: Biallelic mutations in ACP5, encodng tartrate-resistant acid phosphatase (TRAP), result in the immuno-osseous disorder spondyloenchondrodysplasia (SPENCD), characterized by a variety of autoimmune phenotypes – most particularly systemic lupus erythematosus (SLE). Importantly, patients with SPENCD demonstrate an upregulation of type 1 interferon (IFN) stimulated genes (ISGs – an “interferon signatures”) similar to that observed in SLE. Since very little is known about the function of TRAP in immune cells, the objectives of our study were: a) to determine the consequences of TRAP deficiency in human immune cells, B) to identify substrates of TRAP, and c) to determine whether ACP5 mutations occur in ‘idiopathic’ SLE.

Methods: Unbiased substrates of TRAP were queried using a yeast 2 hybrid (Y2H) screen in a human macrophage cDNA library, and interaction of a candidate substrate, osteopontin (OPN) with TRAP was determined by confocal microscopy and immunoprecipitation-western blot analysis (IP-western). TRAP overexpression / knockdown was performed by transfection with cDNA / lentivirus shRNA, respectively. Expression of ISGs was determined by quantitative PCR (qPCR). Phosphatase activity was quantified by colorimetry, and dephosphorylation of substrates by Liquid Chromatogram-tandem Mass Spectrometer (LC-MS/MS). Sequencing of ACP5 coding exons was undertaken in patients with SLE.

Results: OPN is a substrate for TRAP in Macrophase and pDC. TRAP interacted and co-localized with OPN as determined by Y2H, confocal microscopy and by IP-western. Consistent with these data, recombinant human TRAP (rhTRAP) dephosphorylated recombinant human OPN (rhOPN) by the release of free phosphate in an in vitro assay. LC-MS/MS demonstrated rhTRAP dephosphorylated rhOPN at two serine residues in vitro. To relate the functional significance of TRAP deficiency to IFN-α production, we knocked down TRAP expression in pDC and observed that TRAP specific shRNA, but not scrambled shRNA, increased the expression of ISGs as well as IL-6 following TLR9 stimulation. This was associated with increased nuclear translocation of IRF7 and P50 in TRAP KD pDC cells compared to control cells. Sequencing of ACP5 in 865 SLE patients and 511 controls revealed an excess of heterozygous ACP5 possibly pathogenic missense variants in SLE patients (11 adults in the lupus cohort compared to 2 in controls). These variants were predicted as pathogenetic since they were: coding, non-synonymous, occurred at a frequency of less than 1/300 in controls, were in residues conserved in mammalian species and were predicted to destabilize protein on in silico testing. Transient transfection of several mutants and patient serum assays revealed a significant reduction in TRAP enzyme activity.

Conclusion: Our findings indicate that TRAP and OPN co-localize, and that OPN is a substrate for TRAP in immune cells. Significantly, TRAP deficiency in pDCs leads to increased IFN-α production, providing at least a partial explanation for why ACP5 mutations cause lupus in the context of SPENCD. Detection of ACP5 missense variants in lupus patients suggests that impaired function of TRAP may play a role increasing susceptibility to adult-onset idiopathic lupus.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/molecular-pathogenesis-and-genetics-of-tartrate-resistant-acid-phosphatase-deficiency-in-systemic-lupus-erythematosus/