Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations

et al. (2011) Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations. PLoS ONE 6(12): e28679. doi:10.1371/journal.pone.0028679 Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations Ping Yin 0 Danielle Bousquet-Moore 0 Suresh P. Annangudi 0 Bruce R. Southey 0 Richard E. Mains 0 Betty A. Eipper 0 Jonathan V. Sweedler 0 Paul Proost, University of Leuven, Rega Institute, Belgium 0 1 Department of Chemistry, Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America, 2 Department of Neuroscience, University of Connecticut Health Center , Farmington, Connecticut , United States of America Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine a-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 (hydroxyglycineextended peptide) by the copper-dependent peptidylglycine-a-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-a-hydroxyglycine a-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 c-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+/2) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-a-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+/2 mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM+/2 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. - Funding: This work was supported by the National Science Foundation by Award No. CHE-05-26692, the National Institute on Drug Abuse by Award No. P30DA018310 (JVS), and the National Institute of Diabetes and Digestive and Kidney Diseases by Award No. DK32949 (BAE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Neuropeptide production typically involves endoproteolytic cleavage of prohormones by prohormone convertases, followed by exoproteolytic cleavage by carboxypeptidase E. As the prohormone and its cleaved products traverse the secretory pathway, additional post-translational modifications occur. One common modification is C-terminal a-amidation, a reaction catalyzed only by peptidylglycine a-amidating monooxygenase (PAM) [1,2]. The C-terminal amide group often increases the affinity of the peptide for its receptor, extends its half-life, and is essential for biological activity [3]. Eliminating the PAM gene prevents amidated peptide synthesis and is lethal in Drosophila [4] and mouse [1]. PAM heterozygous (PAM+/2) mice, with half the PAM activity of wild-type (WT) mice, survive to adulthood and reproduce [3]. However, PAM+/2 mice are unable to maintain body temperature in a cold room and show increased anxiety-like behavior [5,6]. In vertebrates, PAM is a Type I integral membrane protein; its luminal catalytic domains and unstructured cytosolic domain are highly conserved. A c-secretase-mediated cleavage within the PAM transmembrane domain generates a cytosolic fragment that accumulates in the nucleus and is thought to affect gene expression [5,7,8,9]. PAM has two enzymatic domains, peptidylglycine-a-hydroxylating monooxygenase (PHM) and peptidyl-a-hydroxyglycine aamidating lyase (PAL). PHM converts glycine-extended peptides into hydroxyglycine-extended peptides by using molecular oxygen to hydroxylate the a-carbon of the C-terminal glycine in a copperand ascorbate-dependent reaction. The hydroxyglycine-extended peptide is then converted into the corresponding amidated peptide and glyoxylate by PAL [10,11]. Although several glycine-extended peptides (e.g., TRH-Gly [5,12,13] and gastrin-Gly [14,15]) have been detected using radioimmunoassays, they have not been studied systematically. Hydroxyglycine-extended peptides have not been detected in vivo, suggesting that the product of the monooxygenase reaction might be passed directly to the lyase. In addition, PAM is amongst the small number of enzymes that require copper for their catalytic function [16,17,18,19], and behavioral deficits similar to those observed in PAM+/2 mice were observed in WT mice kept on a copper-deficient diet [5]. Whether the deficits observed in PAM+/2 and copper-deficient mice reflect altered levels of amidation or changes in the regulatory processes responsive to PAM and copper is not yet clear. Liquid chromatography (LC)-mass spectrometry (MS) provides both qualitative and quantitative information and enables sensitive and accurate determination of neuropeptide forms [20,21,22, 23,24]. Here we used two LC-MS based neuropeptidomics approaches to evaluate the effects of PAM haploinsufficiency and dietary copper deficiency on the levels of several glycine- and hydroxyglycine-extended peptides as well as amidated peptides in the mouse pituitary. We applied a standard isotope labeling approach with LC-MS analysis to perform relative quantitation for amidated peptides in PAM+/2 mice maintained on a normal diet compared to WT mice. We also found that endogenous intermediates were present at much lower levels than their amidated counterparts, with the large dynamic range of peptide concentrations making their identification and quantitation challenging. In this case, following LC separation, we employed both matrixassisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS and electrospray ionization (ESI) ion-trap (IT) MS to identify the intermediate peptides. After identification, the levels of glycineand hydroxyglycine-extended peptides were compared to those of amidated products using MS-based peak intensities, and differences were correlated to genetic and/or dietary manipulations. The standard labeling approach requires multiple sample processing steps and is well suited for assays of peptides at higher and similar concentrations; however, these additional steps can cause peptides alre (...truncated)