Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

Tina Skjørringe,Lisbeth Birk Møller,Poul Nissen,Pontus Gourdon,Sidsel Salling Thorborg,Per Amstrup Pedersen

doi:10.1038/s41598-017-00618-6

Abstract

Menkes disease (MD) is caused by mutations in ATP7A, encoding a copper-transporting P-type ATPase which exhibits copper-dependent trafficking. ATP7A is found in the Trans-Golgi Network (TGN) at low copper concentrations, and in the post-Golgi compartments and the plasma membrane at higher concentrations. Here we have analyzed the effect of 36 ATP7A missense mutations identified in phenotypically different MD patients. Nine mutations identified in patients with severe MD, virtually eliminated ATP7A synthesis, in most cases due to aberrant RNA splicing. A group of 21 predominantly severe mutations led to trapping of the protein in TGN and displayed essentially no activity in a yeast-based functional assay. These were predicted to inhibit the catalytic phosphorylation of the protein. Four mutants showed diffuse post-TGN localization, while two displayed copper dependent trafficking. These six variants were identified in patients with mild MD and typically displayed activity in the yeast assay. The four post-TGN located mutants were presumably affected in the catalytic dephosphorylation of the protein. Together these results indicate that the severity of MD correlate with cellular localization of ATP7A and support previous studies indicating that phosphorylation is crucial for the exit of ATP7A from TGN, while dephosphorylation is crucial for recycling back to TGN.

Highlights

Menkes disease (MD; OMIM: 309400), including the milder form Occipital Horn Syndrome (OHS; OMIM: 304150), is a rare (1:300.000), X-linked, multisystemic lethal disorder of copper metabolism linked to mutations in the ATP7A gene (OMIM: 300011)
We analyzed the effect of 36 ATP7A missense mutations on mRNA splicing, mRNA accumulation, protein accumulation, Cu-ATPase activity, cellular localization and copper-dependent trafficking
For eight of the 36 predicted amino acid substitutions - E628VC, K633RC, K802NC, R844HC, G876RC, K1037NC, G1369RC and A1373PC - no ATP7A protein was detected, and RT-PCR analyses revealed that all eight variants, except R844HC, affected splicing of the ATP7A transcript

Summary

Introduction

Menkes disease (MD; OMIM: 309400), including the milder form Occipital Horn Syndrome (OHS; OMIM: 304150), is a rare (1:300.000), X-linked, multisystemic lethal disorder of copper metabolism linked to mutations in the ATP7A gene (OMIM: 300011). The symptoms of MD derive from a lack of dietary copper absorption and copper transfer across the basolateral membrane of intestinal enterocytes into the portal circulation, and from impaired reabsorption of copper in the kidney This leads to accumulation of copper in intestinal cells and kidney, and lack of copper in vital organs such as heart, liver and brain, and reduced activity of essential cuproenzymes[1,2,3]. At low cellular copper concentrations wild-type ATP7A is located in the Trans-Golgi Network (TGN), whereas at higher intracellular copper levels, the steady state distribution of ATP7A shifts to cytosolic vesicles and to the plasma membrane (Fig. 1c)[18, 19]. The copper-mediated trafficking response is reversible; if the intracellular copper level is reduced ATP7A returns to TGN19

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