Processing and secretion of human carboxypeptidase E by C6 glioma cells.
Carboxypeptidase E (CPE) catalyses the removal of C-terminal basic amino acids and is implicated in the processing of peptides and hormones in secretory vesicles. The enzyme has been isolated in multiple forms from both soluble and membrane-bound compartments, and has been demonstrated to be co-secreted with peptides from pancreatic and adrenal cells. To address the question regarding the origin of the multiple forms of the enzyme, we have transfected a construct containing the cDNA for human CPE under the control of the murine-sarcoma-virus enhancer and metallothionein promoter into the C6 rat glioma cell line, which itself has extremely low levels of CPE expression. Within transfectants, multiple forms of the enzyme have been detected by Western blotting, and their sizes are comparable with that in pituitary. Fractionation of the intracellular forms of CPE with Triton X-114 at various pH values indicates that the membrane-bound, but not the soluble, form is amphipathic; this difference probably arises from post-translational modification of the enzyme. Interestingly, the secreted enzyme is found to have the amphipathic characteristics of the membrane-bound form. By modification of the cDNA sequence, enzyme lacking N-terminal and C-terminal domains have been expressed: deletion of the 'pro' region of CPE, leading to loss of the signal cleavage site, gives a more hydrophobic but active enzyme which is not efficiently secreted from the cell; deletion of a C-terminal region gives a secreted form of the enzyme which no longer exhibits amphipathic behaviour. Our data indicate that a single mRNA species can give rise to the multiple forms of CPE enzyme that have been identified and that the C6 cells are able to carry out the intracellular processing events to generate these forms.
Manser
E
E
Molecular Neurobiology Laboratory, National University of Singapore.
Fernandez
D
D
Lim
L
L
D000595:Amino Acid Sequence; D000818:Animals; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D015870:Gene Expression; D005910:Glioma / Q000473:pathology; D006801:Humans; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection; D014407:Tumor Cells, Cultured
D002268:Carboxypeptidases; D043423:Carboxypeptidase H