| Title: | Heterologous expression of human cholecystokinin in Saccharomyces cerevisiae. Evidence for a lysine-specific endopeptidase in the yeast secretory pathway |
| Author(s): | Rourke IJ; Johnsen AH; Din N; Petersen JG; Rehfeld JF; |
| Address: | "Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen O, Denmark. rourke@whi.edu.au" |
| ISSN/ISBN: | 0021-9258 (Print) 0021-9258 (Linking) |
| Abstract: | "Precursors of the human regulatory peptide cholecystokinin (CCK) have been expressed in Saccharomyces cerevisiae, and the post-translational processing of secreted CCK-related products analyzed. Recombinant plasmids expressing native human prepro-CCK and a hybrid molecule encompassing the prepro leader of the yeast alpha-mating pheromone fused to pro-CCK were examined. The latter construct resulted in considerably higher levels of pro-CCK secretion and was therefore analyzed in more detail. Two of the protein modifications essential for CCK bioactivity, C-terminal alpha-amidation and tyrosyl sulfation, were not detected in S. cerevisiae. Proteolytic cleavage of pro-CCK occurred C-terminally of three basic sites; (i) Arg105-Arg106 which, upon exposure to carboxypeptidase activity, leads to the production of glycine-extended CCK; (ii) Arg95 to produce CCK-8 related processing intermediates; and (iii) Lys81 resulting in CCK-22 related products. To elucidate which protease(s) are involved in these endoproteolytic cleavage events, pro-CCK was expressed in yeast mutants lacking various combinations of the Mkc7, Yap3, and Kex2 proteases. Only in S. cerevisiae strains deficient in Kex2 function was any of the above mentioned pro-CCK cleavages abolished, namely processing at the Arg105-Arg106 and Arg95 sites. This suggests that mammalian Kex2-like serine proteases may process pro-CCK at single arginine residues. Our data suggests that an as yet uncharacterized endopeptidase(s) in the S. cerevisiae secretory pathway is responsible for the lysine-specific cleavage of pro-CCK" |
| Keywords: | "Amino Acid Sequence Cholecystokinin/biosynthesis/chemistry/*genetics Chromatography, Gel Humans Kinetics Lysine Mass Spectrometry Metalloendopeptidases/*metabolism Molecular Sequence Data Peptide Fragments/biosynthesis/chemistry Protein Processing, Post-T;" |
| Notes: | "MedlineRourke, I J Johnsen, A H Din, N Petersen, J G Rehfeld, J F eng Research Support, Non-U.S. Gov't 1997/04/11 J Biol Chem. 1997 Apr 11; 272(15):9720-7. doi: 10.1074/jbc.272.15.9720" |
