Document Type
Article
Disciplines
Life Sciences | Medicine and Health Sciences
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.
Recommended Citation
Carlson, Kristina; Mains, Richard E.; and Eipper, Betty A., "Optimizing Production of Fc-amidated Peptides by Chinese Hamster Ovary Cells" (2015). UCHC Articles - Research. 286.
https://digitalcommons.lib.uconn.edu/uchcres_articles/286
Comments
Originally published in :
BMC Biotechnology 2015, 15:95 doi:10.1186/s12896-015-0210-4
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1472-6750/15/95
PMID:26475607
PMCID:PMC4609047