CHO cells synthesize amidated neuropeptide Y from a C-peptide deleted form of the precursor.
Post-translational processing of peptide precursors producing amidated, biologically active peptides is generally believed to occur only in specially differentiated endocrine or neural cells. Previously it has been shown that endoproteolytic processing of peptide precursors is very inefficient in non-endocrine cells like CHO cells. We have studied the processing of a C-peptide-deleted precursor of neuropeptide Y (NPY) in which the precursor terminates in the sequence Gly-Lys-Arg and does not require any dibasic specific endoproteolytic processing. Following transfection of CHO cells with an expression plasmid encoding this mutated NPY precursor, between 50 and 80 percent of the synthesized NPY was secreted from stable transfectants as authentic amidated NPY as assessed by both a C-terminal amide specific radioimmunoassay and by isoelectric focusing. It is concluded that amidated peptides can be produced in non-endocrine cells provided they are presented with a precursor which does not have to be endoproteolytically processed.
Johansen
T E
TE
University Department of Clinical Chemistry, Rigshospitalet, Copenhagen, Denmark.
O'Hare
M M
MM
Wulff
B S
BS
Schwartz
T W
TW
D000595:Amino Acid Sequence; D000818:Animals; D002096:C-Peptide / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009478:Neuropeptide Y / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014162:Transfection
D002096:C-Peptide; D009478:Neuropeptide Y; D011498:Protein Precursors; D004247:DNA