Abstract
BackgroundAmidation 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.MethodsTo 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.ResultsPAM 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.ConclusionsPeptide 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.
Highlights
Amidation of the carboxyl terminal of many peptides is essential for full biological potency, often increasing receptor binding and stability
When expressed in endocrine cells, the peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL) catalytic cores are efficiently packaged in secretory granules; when expressed in Chinese hamster ovary (CHO) cells, both active enzymes are secreted into the medium
Since we wanted to use the same tag for each targeting vector, enhanced green fluorescent protein (EGFP) was inserted into the linker region that separates PHM from PAL in PAM1; 55 amino acids of the linker region were replaced by EGFP, with no deleterious effect on either enzymatic activity [22]
Summary
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. 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 active site of PHM contains two copper residues, each of which is essential for activity. 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]. Mice bearing a mutation in the Atp7a gene display similar symptoms and survive for less than two weeks after birth. Chelation of copper in vitro or in vivo leads to a reduced ability to produce amidated peptides [9]
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