Diverse functional properties of Wilson disease ATP7B variants

Abstract

Background & aims: Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease.

Methods: We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper ((64)Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy.

Results: Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization.

Conclusions: Variants in ATP7B associated with Wilson disease disrupt the protein's transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype-phenotype correlation and mechanisms of disease pathogenesis.

Figure 1

(A) Catalytic cycle of ATP7B (the dotted frame highlights partial reactions, relevant for this study). (B) Transmembrane organization of ATP7B and representation of mutants and variants examined in this study. The 6 metal-binding sites in the N-terminal Cu-binding unit domain (NTD) of ATP7B are indicated by letters CxxC. ATP is bound to the nucleotide binding domain (N) and phosphorylated during ATP hydrolysis. The site of ATP-hydrolysis is the invariant residue D1027 (D1027A mutant = bold). The gene variants S406A, V456L, and K832R are presented in italic type. The CPC motif located in the sixth transmembrane domain is thought to form the intramembrane copper-binding site(s). A, actuator domain; P, phosphorylation domain.

Figure 2

(A) Copper transport by wt-ATP7B. Means of 10 independent experiments are shown. Copper uptake into vesicles was measured in nmol/mg protein during a time period of 240 minutes. Na₃VO₄ was added to the incubation solution to a final concentration of 200 µM. (B) Mutation of the phosphorylation site of ATP7B (D1027A) abolishes Cu transport. (C) Copper transport by ATP7B mutants or (D) variants; means of 3 independent experiments are shown (mutants were selected to represent different functional domains; data for more mutants are shown in Table 1 and Supplementary Figure 2).

Figure 3

Phosphorylation activity of ATP7B mutants (A–E) and variants (F): The protein gels show representative ³²P autoradiograms of phosphorylated wt-ATP7B, variants, and mutants. The phosphorylation activity (bar graphs) of variant and mutant ATP7B was calculated with respect to wt-ATP7B activity (100%). Averages of 4 to 6 independent experiments are shown.

Figure 4

ATP-dependent dephosphorylation of the A-domain mutants (A) and P-domain mutants (B) of ATP7B at a copper concentration of 1 µM. ATP was added after [γ-³²P] ATP phosphorylation to a final concentration of 1 mM and incubated for 10 minutes at room temperature before the reaction was stopped. The typical gels and the phosphorylation activities of mutant ATP7B calculated in respect to wt-ATP7B activity (100%) for 3 independent experiments are shown.

Figure 5

Expression and localization of ATP7B mutants in mammalian cells. Green fluorescent protein–tagged ATP7B variants were expressed in HEK 293 T REx cells. The expression pattern of wt- and mutant ATP7B is shown in the left column (green), organelle markers in the middle (red), and the superimposition of these images on the right (yellow). TGN46 is a TGN marker; Calnexin is marker for ER.

Figure 6

(A) Location of the characterized mutations in the functional domains of ATP7B. The structure of the P-domain was software-modeled using crystallographic structure of CopA (2B8E) as a template. For mutations within the N-domain, available nuclear magnetic resonance data (2ARF) were used. The nuclear magnetic resonance- based model for the A-domain was kindly provided by Dr Lucia Banci. (B) Summary of key properties of ATP7B (left panel) that are affected by various mutations examined in this study (right panel).