Sequence alignment between mouse and human ATP7A proteins. Two murine isoforms (NP_001103227.1 (Mouse 1) and NP_033856.3 (Mouse 2) and two human isoforms (NP_000043.4 (Human 1) and NP_001269153.1 (Human 2) are shown. Functional domains: HMA—heavy-metal-associated domain, TM—transmembrane domain, A-domain—actuator domain, P-domain—phosphorylation domain and corresponding functional motifs: Cu-binding, phosphatase, phosphorylation, ATP-binding and di-leucine are marked. It should be noted, that the human isoform 2 is shorter than isoform 1 and lacks a part of TM2 in addition to TM3 and TM4. This product is a result of alternative splicing, leading to skipping of exon 10. Isoform 2 is expressed at low level, in normal healthy individuals, but has been observed as the major product in a patient with a IVS10 mutation. Because the patient had OHS, in contrast to classic Menkes Disease, it has been suggested that isoform 2 has partly copper transporting activity (Qi and Byers, 1998). The sequence alignment is performed using Clustal Omega software (http://www.ebi.ac.uk/Tools/msa/clustalo/). Characteristic protein domains are marked based on conserved domains database (Marchler-Bauer et al., 2015) and previously published ATP7A protein structures (Kaler, 2011; Tümer, 2013). C. cons. – Clustal Omega consensus. An “*” (asterisk) indicates positions with fully conserved residues, a “:” (colon) indicates conservation between groups of strongly similar properties and a (period) indicates conservation between groups of weakly similar properties.

Schematic presentation of the secondary structure of the ATP7A protein with location of the 15 mottled mutants indicated. ATP7A is a transmembrane protein anchored to the membrane of the Golgi apparatus with eight transmembrane domains. The CPC amino acid motif within the 6th transmembrane domain is assumed to play a direct role in copper ions translocation across the biological membrane. The N-terminal peptide contains six cytoplasmic copper binding domains. Cytoplasmic domains are involved in the catalytic cycle that mediates cupric ions active transport at the cost of ATP hydrolysis. In the catalytic cycle ATP was bind to the nucleotide binding domains (N) and after hydrolysis the γ-phosphate of ATP is transferred to the invariant aspartate residue in the in the phosphorylation domain (P). Energy released by ATP hydrolysis is utilized for ions transport across a membrane. The actuator domain (A) located between the 4th and 5th transmembrane domains plays a key role in the dephosphorylation of the phosphorylated protein. The amino terminal part of the protein contains a dileucine motif (LL) motif that is involved in retrograde transport to the trans Golgi network (TGN).

Interaction between copper (Cu) and iron (Fe) in young mosaic mice. Duodenal enterocytes can export copper across the basolateral membrane by ATP7A protein. Due to ATP7A gene mutation in mosaic mice, copper cannot be released to the serum and accumulates within the enterocytes in a complex with metallothionein (MT). Decreased serum Cu level entails Cu deficiency in red blood cells (RBC) and in consequence reduced activity/expression of Cu, Zn-superoxide dismutase (SOD1), which play a crucial role in RBC antioxidant defense. As the result, Cu-deficient RBC of mosaic mice display morphological abnormalities and undergo intravascular hemeolysis connected with hemoglobin (Hb) release to the serum and haptoglobin-dependent (Hp) elimination of free Hb from the circulation. When Hb is released from damaged RBC, it is instantly bound by haptoglobin (Hp) and forms a Hp–Hb high-affinity complex. This complex is then rapidly taken up from the circulation by the CD163 receptor present mainly on tissue macrophages (in the liver on Browicz-Kupffer cells). The CD163 receptor has no measurable affinity for free Hp. Thus, specific recognition of Hp–Hb by CD163 explains the decrease in Hp concentration in the serum during accelerated hemeolysis. The proteolytic Hb degradation in Browicz-Kupffer cells leads to the release of heme, which is then enzymatically decomposed by heme oxygenase 1 (HO-1) resulting in the formation of carbon monoxide (CO), biliverdin and Fe. Non-heme iron can be then stored as a complex with ferritin (Ft) or exported outside the cell by ferroportin (FPN), the sole cellular exporter of ionic iron known in mammalian cells. The content of hepatic non-heme Fe is elevated in mosaic mice, probably due to decreased expression of FPN. The concentration of cell surface Fpn largely depends on the post-translational regulation through internalization and degradation following hepcidin (Hepc) binding. Down-regulation of FPN expression in the liver of young mosaic mice is probably due to the concomitant up-regulation of hepatic hepcidin gene (Hamp), synthesized mainly in hepatocytes in response to systemic inflammation reported to occur in mosaic mice.