title: Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites.
abstract: Menkes disease (MD) is an X-linked recessive disorder of copper metabolism. It is caused by mutations in the ATP7A gene encoding a copper-translocating P-type ATPase, which contains six N-terminal copper-binding sites (CBS1-CBS6). Most patients die in early childhood. We investigated the functional effect of a large frameshift deletion in ATP7A (including exons 3 and 4) identified in a patient with MD with unexpectedly mild symptoms and long survival. The mutated transcript, ATP7A(Delta ex3+ex4), contains a premature termination codon after 46 codons. Although such transcripts are generally degraded by nonsense-mediated mRNA decay (NMD), it was established by real-time PCR quantification that the ATP7A(Delta ex3+ex4) transcript was protected from degradation. A combination of in vitro translation, recombinant expression, and immunocytochemical analysis provided evidence that the ATP7A(Delta ex3+ex4) transcript was protected from degradation because of reinitiation of protein translation. Our findings suggest that reinitiation takes place at two downstream internal codons. The putative N-terminally truncated proteins contain only CBS5 and CBS6. Cellular localization and copper-dependent trafficking of the major part of endogenous and recombinant ATP7A(Delta ex3+ex4) proteins were similar to the wild-type ATP7A protein. Furthermore, the ATP7A(Delta ex3+ex4) cDNA was able to rescue a yeast strain lacking the homologous gene, CCC2. In summary, we propose that reinitiation of the NMD-resistant ATP7A(Delta ex3+ex4) transcript leads to the synthesis of N-terminally truncated and at-least-partially functional Menkes proteins missing CBS1-CBS4. This finding--that a mutation that would have been assumed to be null is not--highlights the need to examine the biochemical phenotype of patients to deduce the efficacy of copper therapy.
authors.affiliation: Kennedy Institute-National Eye Clinic, Glostrup, Denmark.
authors.forename: Marianne
authors.initials: M
authors.lastname: Paulsen
authors.forename: Connie
authors.initials: C
authors.lastname: Lund
authors.forename: Zarqa
authors.initials: Z
authors.lastname: Akram
authors.forename: Jakob R
authors.initials: JR
authors.lastname: Winther
authors.forename: Nina
authors.initials: N
authors.lastname: Horn
authors.forename: Lisbeth Birk
authors.initials: LB
authors.lastname: Møller
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000293:Adolescent; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism; D002474:Cell-Free System / Q000378:metabolism; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D006224:Cricetinae; D005347:Fibroblasts / Q000378:metabolism; D016368:Frameshift Mutation; D006801:Humans; D007223:Infant; D008297:Male; D007706:Menkes Kinky Hair Syndrome / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D017384:Sequence Deletion; D017741:Survivors
chemical_list: D027682:Cation Transport Proteins; D003300:Copper; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases