title: Probing the production of amidated peptides following genetic and dietary copper manipulations.
abstract: Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.
authors.affiliation: Department of Chemistry, Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
authors.forename: Ping
authors.initials: P
authors.lastname: Yin
authors.forename: Danielle
authors.initials: D
authors.lastname: Bousquet-Moore
authors.forename: Suresh P
authors.initials: SP
authors.lastname: Annangudi
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Southey
authors.forename: Richard E
authors.initials: RE
authors.lastname: Mains
authors.forename: Betty A
authors.initials: BA
authors.lastname: Eipper
authors.forename: Jonathan V
authors.initials: JV
authors.lastname: Sweedler
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000737:chemistry / Q000378:metabolism; D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001127:Arginine Vasopressin / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid; D003300:Copper / Q000494:pharmacology*; D004032:Diet; D005998:Glycine / Q000378:metabolism; D057895:Haploinsufficiency / Q000187:drug effects / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism; D011333:Pro-Opiomelanocortin / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014020:Tissue Extracts
chemical_list: D000577:Amides; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D010455:Peptides; D014020:Tissue Extracts; D001127:Arginine Vasopressin; D011333:Pro-Opiomelanocortin; D003300:Copper; C064114:pro-opiomelanocortin joining peptide(79-108); D000324:Adrenocorticotropic Hormone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D005998:Glycine