Efficient amidation of C-peptide deleted NPY precursors by non-endocrine cells is affected by the presence of Lys-Arg at the C-terminus.
Post-translational processing of peptide precursors producing amidated, biologically active peptides generally occurs in specially differentiated endocrine or neural cells. However, we have previously shown that a C-peptide-deleted precursor of neuropeptide Y (NPY1-39) in which the precursor terminates in the sequence Gly-Lys-Arg was partially amidated by the non-endocrine cell line, CHO. In the present study we show that two other non-endocrine cell lines, NIH 3T3 and BHK, also possess amidating activities and that the NPY1-39 precursor was completely converted to NPY1-36 amide by the NIH 3T3 cell line. The role of the two basic residues (Lys-Arg) in the C-terminus was studied by transfection of a construct encoding a NPY precursor terminating with glycine alone. Both the CHO and NIH 3T3 cell lines, transfected with this construct, secreted a significantly smaller fraction of NPY reactive material as amidated NPY compared to the fraction of amidated NPY secreted by the cells transfected with the NPY1-39 precursor. It is concluded that the capacity to perform C-terminal amidation appears to be a universal feature of eukaryotic cells and that the carboxypeptidase E-like enzyme influences the amidation process, beyond its known ability to remove the C-terminal basic residues.
Wulff
B S
BS
Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark.
Catipovic
B
B
Okamoto
H
H
Gether
U
U
Schwartz
T W
TW
Johansen
T E
TE
D016475:3T3 Cells; D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D015152:Blotting, Northern; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D007668:Kidney; D008239:Lysine; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009478:Neuropeptide Y / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D014162:Transfection
D000577:Amides; D009478:Neuropeptide Y; D010446:Peptide Fragments; D011498:Protein Precursors; D001120:Arginine; D008239:Lysine