Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites

Am J Hum Genet. 2006 Aug;79(2):214-29. doi: 10.1086/505407. Epub 2006 Jun 5.

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.

Publication types

  • Case Reports

MeSH terms

  • Adenosine Triphosphatases / genetics*
  • Adenosine Triphosphatases / metabolism
  • Adolescent
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • CHO Cells
  • Cation Transport Proteins / genetics*
  • Cation Transport Proteins / metabolism
  • Cell-Free System / metabolism
  • Cells, Cultured
  • Child
  • Child, Preschool
  • Copper / metabolism*
  • Copper-Transporting ATPases
  • Cricetinae
  • Fibroblasts / metabolism
  • Frameshift Mutation
  • Humans
  • Infant
  • Male
  • Menkes Kinky Hair Syndrome / genetics*
  • Menkes Kinky Hair Syndrome / metabolism
  • Molecular Sequence Data
  • Peptide Chain Initiation, Translational / genetics*
  • Sequence Deletion
  • Survivors

Substances

  • Cation Transport Proteins
  • Copper
  • Adenosine Triphosphatases
  • ATP7A protein, human
  • Copper-Transporting ATPases