Menkes Protein Contributes to the Function of Peptidylglycine α-Amidating Monooxygenase

Abstract

Menkes protein (ATP7A) is a P-type ATPase involved in copper uptake and homeostasis. Disturbed copper homeostasis occurs in patients with Menkes disease, an X-linked disorder characterized by mental retardation, neurodegeneration, connective tissue disorders, and early childhood death. Mutations in ATP7A result in malfunction of copper-requiring enzymes, such as tyrosinase and copper/zinc superoxide dismutase. The first step of the two-step amidation reaction carried out by peptidylglycine alpha-amidating monooxygenase (PAM) also requires copper. We used tissue from wild-type rats and mice and an ATP7A-specific antibody to determine that ATP7A is expressed at high levels in tissues expressing high levels of PAM. ATP7A is largely localized to the trans Golgi network in pituitary endocrine cells. The Atp7a mouse, bearing a mutation in the Atp7a gene, is an excellent model system for examining the consequences of ATP7A malfunction. Despite normal levels of PAM protein, levels of several amidated peptides were reduced in pituitary and brain extracts of Atp7a mice, demonstrating that PAM function is compromised when ATP7A is inactive. Based on these results, we conclude that a reduction in the ability of PAM to produce bioactive end-products involved in neuronal growth and development could contribute to many of the biological effects associated with Menkes disease.

Supplying Copper to the Cuproenzyme Peptidylglycine α-Amidating Monooxygenase

“…Although normal levels of PAM protein were found in Atp7a mice, levels of amidated Joining Peptide and ␣-melanocyte stimulating hormone were diminished in the pituitary, and levels of amidated cholecystokinin were diminished in the cerebral cortex (67). Peptide amidation was reduced, but not eliminated, in the Atp7a mouse.…”

Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme

“…The generation of the following antibodies was previously described: Atp7a (C terminus) (17); C-STOP (18); exon 16 (19); PHM (20); POMC (21); ACTH (22); JP-NH 2 (23); and TGN38 (24). The specificity of the Atp7a antibody for Western blotting and for immunostaining was established previously (25).…”

“…The addition of a Gly residue to the C terminus of this peptide (JP(12-19)) reduced its cross-reactivity with this antiserum by 10,000-fold (23). For Western blot analysis, a fraction enriched in immunoglobulin by precipitation with ammonium sulfate (45% saturation) was dialyzed into 100 mM sodium phosphate, pH 7.4, and incubated with JP (12)(13)(14)(15)(16)(17)(18)(19) linked to Affi-Gel15 beads (4 mg of peptide/2 ml of resin) for 1 h at 4°C to remove any antibodies capable of recognizing this peptide. The unbound fraction was applied to a column of JP(12-18)NH 2 linked to Affi-Gel10 beads (5 mg of peptide/2 ml of resin); bound antibodies were eluted with 0.2 M glycine HCl, 0.1 M NaCl, 0.1% Triton X-100, pH 2.3; the eluate was rapidly neutralized with 3 M Tris-HCl, pH 8.0, and BSA (final concentration in eluate ϭ 0.2 mg/ml) was added before dialysis against 100 mM sodium phosphate, pH 7.4.…”

“…1) (16,17). Following cleavage at the C terminus of ACTH, cleavage at its N terminus reveals a potential amidation site (-Glu-Gly-Lys-Arg) at the C terminus of 18-kDa fragment; PAM cannot act until carboxypeptidase E or D has removed the C-terminal basic amino acids, revealing a C-terminal glycine, but these cleavages are followed quickly by amidation (21).…”

“…Although ATP7A trafficking in neuroendocrine cells has not been extensively explored, studies in other cell types predict an essential role for AP-1 (6,14,16,17). AtT-20 cells produce proopiomelanocortin (POMC), a prohormone that is cleaved sequentially into a well characterized set of biosynthetic intermediates and product peptides that are either released constitutively or stored in secretory granules for release in response to secretagogue (Fig.…”