We isolated cDNA fragments from four human cell lines that had sequences for the Menkes Cu-transporting ATPase (ATP7A). Primers designed to generate a 4.8 kb cDNA with the complete open reading frame generated a 1.9 kb cDNA in addition to the expected 4.8 kb product. Sequence analysis revealed that the 1.9 kb cDNA encoded one of the six Cu-binding sites and two of the eight transmembrane domains of ATP7A. Stop and start codons were also present. More striking, however, was an unusual union between exons 2 and 16 that retained an in-frame reference to exon 23. The 1.9 kb cDNA thus appeared to be a truncated Menkes mRNA that coded for an ATP7A variant that lacked exons 3–15. A 530 bp probe specific for exon 23 that avoided sequences in the exon 3–15 region hybridized to a 5.5 kb band on Northern blot analysis. Western blotting provided immunochemical evidence for the presence of both a 170 kDa and a 57 kDa protein with ATP7A sequences in detergent extracts of Caco-2 and induced BeWo cells. Extracts from non-induced BeWo cells, which lack the capacity to express the Menkes gene (MNK), showed neither protein. In a cell-free reticulocyte lysate, a plasmid containing the 1.9 kb cDNA insert directed the synthesis of a 59 kDa protein with antigenic properties of ATP7A. These studies provide evidence that non-Menkes cells have the capacity to synthesize more than one MNK mRNA. The one characterized in this report codes for a 57–59 kDa protein that lacks the core structure of the ATP7A protein. The smaller variant could be an alternative spliced form of MNK mRNA.
- The Biochemical Society, London © 1998