Neuronal cell lines expressing PC5, but not PC1 or PC2, process Pro-CCK into glycine-extended CCK 12 and 22.
Endocrine tumor cells in culture and in vitro cleavage assays have shown that PC1 and PC2 are capable of processing pro-CCK into smaller, intermediate and final, bioactive forms. Similar studies have shown that PC5 has the ability to process a number of propeptides. Here, we use GT1-7 (mouse hypothalamic) and SK-N-MC and SK-N-SH (human neuroblastoma) tumor cell lines to study the ability of PC5 to process pro-CCK. RT-PCR and Western blot analysis showed that the cells express PC5 mRNA and protein, but not PC1 or PC2. They were engineered to stably overexpress CCK and cell media was analyzed for pro-CCK expression and cleavage of the prohormone. Radioimmunoassays showed that pro-CCK was expressed, but no amidated CCK was detected. Lack of production of amidated CCK may be due to the lack of the appropriate carboxypeptidase and amidating enzymes. Production of glycine-extended CCK processing products was evaluated by treatment of media with carboxypeptidase B followed by analysis with a CCK Gly RIA. Glycine-extended forms of the peptide were found in the media. The predominant forms co-eluted with CCK 12 Gly and CCK 22 Gly on gel filtration chromatography. The results demonstrate that these cell lines which express PC5 and not PC1 or PC2 have the ability to process pro-CCK into intermediate, glycine-extended forms more closely resembling pro-CCK products in intestine than in brain.
Cain
B M
BM
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA 02111, USA. bcain@opal.tufts.edu
Vishnuvardhan
D
D
Beinfeld
M C
MC
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis*; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002766:Cholecystokinin / Q000378:metabolism*; D002850:Chromatography, Gel; D005822:Genetic Vectors; D005998:Glycine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D045702:Proprotein Convertase 5; D043484:Proprotein Convertases; D011498:Protein Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011863:Radioimmunoassay; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000096:biosynthesis*; D013381:Subtilisins / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D011498:Protein Precursors; D012333:RNA, Messenger; C437845:cholecystokinin 12 C-terminal fragment; D002766:Cholecystokinin; C044926:cholecystokinin 22 C-terminal fragment; D045702:Proprotein Convertase 5; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C473805:propeptide convertase 5, mouse; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases; D005998:Glycine