Optimizing production of Fc-amidated peptides by Chinese hamster ovary cells

Carlson et al. BMC Biotechnology (2015) 15:95 DOI 10.1186/s12896-015-0210-4 RESEARCH ARTICLE Open Access Optimizing production of Fc-amidated peptides by Chinese hamster ovary cells Kristina Carlson1, Steven C. Pomerantz2, Omid Vafa2, Michael Naso2, William Strohl2, Richard E. Mains1 and Betty A. Eipper1,3* Abstract Background: Amidation of the carboxyl terminal of many peptides is essential for full biological potency, often increasing receptor binding and stability. The single enzyme responsible for this reaction is peptidylglycine α-amidating monooxygenase (PAM: EC 1.14.17.3), a copper- and ascorbate-dependent Type I membrane protein. Methods: To make large amounts of high molecular weight amidated product, Chinese hamster ovary (CHO) cells were engineered to express exogenous PAM. To vary access of the enzyme to its substrate, exogenous PAM was targeted to the endoplasmic reticulum, trans-Golgi network, endosomes and lysosomes or to the lumen of the secretory pathway. Results: PAM was equally active when targeted to each intracellular location and assayed in homogenates. Immunocytochemical analyses of CHO cells and a pituitary cell line demonstrated that targeting of exogenous PAM was partially successful. PAM substrates generated by expressing peptidylglycine substrates (glucagon-like peptide 1-Gly, peptide YY-Gly and neuromedin U-Gly) fused to the C-terminus of immunoglobulin Fc in CHO cell lines producing targeted PAM. The extent of amidation of the Fc-peptides was determined by mass spectrometry and amidation-specific enzyme immunoassays. Amidation was inhibited by copper chelation, but was not enhanced by the addition of additional copper or ascorbate. Conclusions: Peptide amidation was increased over endogenous levels by exogenous PAM, and targeting PAM to the endoplasmic reticulum or trans-Golgi network increased peptide amidation compared to endogenous CHO PAM. Keywords: CHO cell, Glucagon-like peptide 1, Peptide YY, Neuromedin U, Mass spectrometry Background Amidated peptides act as hormones, neuromodulators and autocrine growth factors [1, 2]. Each amidated peptide is synthesized from a peptidylglycine precursor through the actions of peptidylglycine α-amidating monooxygenase (PAM: EC 1.14.17.3). In mice engineered to lack the Pam gene, peptide amidating activity is not detectable and embryos die at mid-gestation [3]. PAM contains two catalytic domains, peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL). The enzymes act sequentially, first converting the peptidylglycine substrate into a short-lived peptidyl-α* Correspondence: 1 Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3401, USA 3 Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT 06030, USA Full list of author information is available at the end of the article hydroxyglycine intermediate and then cleaving the C-N bond to produce the amidated peptide and glyoxylate [2]. The active site of PHM contains two copper residues, each of which is essential for activity. Copper is not tightly bound to PHM and no other metal can substitute for it. ATP7A, a P-type ATPase, transports the copper it receives from cytosolic copper-binding chaperones into the lumen of the secretory pathway, where the copper is available to PHM [2, 4, 5]. Mutations in human ATP7A cause Menkes Disease, a lethal disorder characterized by copper deficiency [6, 7]. Mice bearing a mutation in the Atp7a gene display similar symptoms and survive for less than two weeks after birth. Among the many deficits observed in these mice is the inability to produce normal levels of amidated peptides [8]. Chelation of copper in vitro or in vivo also leads to a reduced ability to produce amidated peptides [9]. © 2015 Carlson et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Carlson et al. BMC Biotechnology (2015) 15:95 The reaction catalyzed by PHM is still not fully understood, but requires two single electron transfer steps. Ascorbic acid (vitamin C) is present at high levels in the secretory pathway and is generally the source of the reducing equivalents needed to support peptide amidation [2]. In the absence of ascorbic acid in cell culture systems, peptide amidation fails to occur and other single electron donors or reducing agents (e.g. NADH, NADPH, dithiothreitol, dopamine), cannot fully substitute for ascorbate [10]. Previous studies of the production of amidated peptides in cell lines have had mixed results. Using transfected CHO and COS7 cells, Takahashi et al. [11] found very efficient amidation of salmon calcitonin (C-terminal Pro-NH2), while Hayashi et al. [12] reported that amidation of gastrin (C-terminal Phe-NH2) was efficient in CHO cells but not in COS7 cells. These results are puzzling, since peptides terminating with –Phe-Gly are far better substrates for PAM that peptides terminating with –Pro-Gly, using test tube assays and purified enzyme [1]. Johansen et al. [13] showed that amidation of NPY (C-terminal Tyr-NH2), another excellent PAM substrate, only proceeded to 50–80 % completion in different CHO cell lines. Work using neuroendocrine lines which express prohormone convertases along with PAM consistently always shows complete amidation after transfection of preprohormone precursor cDNAs [14–18]. Thus, it is difficult to predict which peptide precursors will be efficiently amidated in which cell lines, especially if the goal is to achieve essentially 100 % amidation without extraneous or unwanted endoproteolytic cleavages. In an attempt to prolong the half-lives of amidated peptides, we engineered CHO cells to produce Fcpeptidylglycine fusion proteins in the absence and presence of exogenous PAM; both soluble and integral membrane forms of PAM were tested for their ability to support Fc-peptidylglycine fusion protein amidation in CHO cells [19]. The extent of amidation observed varied from 25 to 90 % for different Fc- peptidylglycine substrates, but the expression of exogenous PAM always increased the amidation of Fc- peptidylglycine substrates [19]. The extent of amidation never reached 100 %, which would be essential for many pharmacotherapeutic applications. It is clear that PAM activity is rate-limiting for peptide amidation in CHO cells, since increasing PAM increased amidation [19], while decreasing PAM low (...truncated)