Identification of Novel Menkes Copper Atpase (Atp7a) Protein Variants in the Cytoplasm and Nucleus of Intestinal Epithelial Cells

We previously noted alternative 5′ splice variants of the Atp7a transcript. To fully understand the splicing of this gene, we sought to identify additional “downstream” splice variants and to test the hypothesis that different protein variants exist. RT‐PCR was utilized with combinations of Atp7a primers spanning exons 1–23 and amplified products were subcloned and sequenced. Novel splice products which were missing various internal exons were identified. To identify Atp7a proteins potentially encoded by these splice variants, we utilized 3 polyclonal Atp7a antisera raised against different antigens: 1) against 2 C‐terminal peptides (called 54‐10), 2) against a fusion protein of the six, N‐terminal copper binding domains (called R17; a gift from Dr. Julian Mercer), and 3) against 2 peptides within the first 68 N‐terminal amino acids (called N‐term). The 54‐10 and R17 antisera, both proven to specifically react with Atp7a, detected a presumably full length Atp7a protein of ~180 kDa in rat intestine and IEC‐6 cells. The 54‐10 antiserum also detected a smaller protein band of ~160 kDa and a specific, ~97 kDa protein in the nuclear fraction of IEC‐6 cells. Moreover, a nuclear protein was detected by the N‐term antiserum, which was similar in size to the protein detected by the 54‐10 antiserum (~97 kDa). IP experiments and siRNA knockdowns provide additional evidence that these proteins may be novel Atp7a variants. Grant Funding Source : NIH