Deletion of peptide amidation enzymatic activity leads to edema and embryonic lethality in the mouse

Abstract

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of peptide hormones. We previously had found high expression of PAM in several regions of the developing rodent. To determine the function of PAM during mouse embryogenesis, we produced a null mutant of the PAM gene. Homozygous mutants die in utero between e14.5 and e15.5 with severe edema that is likely due to cardiovascular deficits. These defects include thinning of the aorta and carotid arteries and are very similar to those of the recently characterized adrenomedullin (AM) gene KO despite the presence of elevated immunoreactive AM in PAM KO embryos. No peptide amidation activity was detected in PAM mutant embryos, and there was no moderation of the AM-like phenotype that could be expected if any alternative peptide amidation mechanism exists in the mouse. Despite the proposed contribution of amidated peptides to neuronal cell proliferation, no alteration in neuroblast proliferation was observed in homozygous mutant embryos prior to lethality. Mice heterozygous for the mutant PAM allele develop normally and express wildtype levels of several amidated peptides despite having one half the wildtype levels of PAM activity and PAM protein. Nonetheless, both an increase in adiposity and a mild glucose intolerance developed in aged (>10 months) heterozygous mice compared to littermate controls. Ablation of PAM thus demonstrates an essential function for this gene during mouse development, while alterations in PAM activity in the adult may underlie more subtle physiologic effects.

Early eukaryotic origins for cilia-associated bioactive peptide-amidating activity

“…Amidation, often essential for bioactivity, can reduce peptide turnover and increase receptor affinity. PAM is essential in metazoans; mice and flies lacking PAM die at mid-gestation or mid-larval stages, respectively (Kolhekar et al, 1997b;Czyzyk et al, 2005). PAM missense alleles have been associated with metabolic disorders (Huyghe et al, 2013;Steinthorsdottir et al, 2014).…”

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

“…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).…”

“…When AP-1 Levels Are Reduced: 18-kDa Fragment-NH 2 -Peptide amidation is an essential post-translational modification; neither flies nor mice lacking PAM can complete development (16,46). Copper is absolutely essential for PAM function.…”

“…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.…”