Identification of Novel Menkes Copper Atpase ( Atp7a ) Splice and Immunoreactive Protein Variants

The Menkes copper Atpase ( Atp7a ) protein is responsible for intestinal copper transport. We noted that the Atp7a gene is strongly induced in the intestine of iron deficient rats, suggesting an important physiological role during perturbations of iron homeostasis. Previous studies revealed novel 5′ splice variants of the rat Atp7a transcript leading us to hypothesize that different Atp7a protein variants existed. A series of Atp7a antibodies was thus developed, some specifically targeting the NH2 terminal region, which is predicted to be altered when translated from a particular splice variant. Further, RT‐PCR was use to amplify additional splice variants with combinations of 5′ end and 3′ end primers. Several novel splice variants were identified, with unusual combinations of upstream and downstream exons, some presumably producing novel Atp7a protein variants. Utilizing the array of Atp7a antibodies, which were extensively validated, different Atp7a proteins were detected from proteins isolated from membrane, cytosolic or nuclear cell fractions. The specificity of the immunoreactions was confirmed by shRNA knockdown of Atp7a transcripts, with a resulting attenuation of the bands detected in membrane preps. It is thus concluded that alternative splicing of the Atp7a transcript may leads to the production of novel protein variants in different subcellular locations. Grant Funding Source : 1R01 DK074867