A STUDY OF THE EXPRESSION, PROCESSING AND SECRETION OF CARBOXYPEPTIDASE E

Abstract

This thesis attempts to address the question of the basis of the observed heterogeneity of in vivo forms of carboxypeptidase E, a prohormone processing enzyme that may itself be subject to a number of post-translational modifications. Starting with the human cDNA sequence, expression has been achieved in bacterial and eukaryotic systems to enable a study of the processing events that convert the nascent polypeptide into the mature enzyme. In the final part, it also intends to determine if the primary amino acid sequence of CPE contains information required to sort the enzyme into vesicles for exocytosis. Following restriction mapping of the human cDNAs, a recombinant CPE fusion protein construct was expressed in E. coli. A specific 17 kDa carboxyl-terminus peptide derived by cyanogen bromide digestion of the fusion protein was purified by ion-exchange chromatography and used to generate polyclonal antibodies that recognise a protein of 53-56 kDa, identified as Carboxypeptidase E. The antibody was used to detect the protein by Western Blot and immunoflouresence. Cell lines of C6 rat glioma and AtT-10 mouse anterior pituitary tumor were transfected with the (i) full-length human CPE cDNA, (ii) the human cDNA encoding protein with 26 amino acids deleted from the carboxy terminal and (iii) cDNA encoding protein with a deletion of 16 amino acids in the 'pro' region; and the processing of these highly expressed exogenous forms of CPE were followed by western blot analysis and assayable activity. The full-length transfectants displayed expression of both soluble and membrane-bound enzyme similar to observed bovine pituitary in vivo forms. The 'pro' deletion mutant showed higher retention of activity within intracellular compartments owing to an uncleaved signal peptide, as opposed to the C-terminal deletion which expressed higher amounts of secreted activity. Two N-terminal sequences were obtained from the secreted products of the full-length Transfectants : one arising from signal peptide cleavage 27 amino-acids from the initiating methionine, and the other originating 15 amino acids downstream of the first sequence, at the carboxyl side of 5 arginine residues which could be an endopeptidase cleavage site. The different mobilities of the two secreted forms of CPE were found to be due to N-terminal processing and not to differences in glycosylation of the polypeptide core or C-terminal processing. The C-terminal deletion was found to contain sequence responsible for amphipathicity and for CPE aggregation in response to changes in pH and calcium concentration, a mechanism which, we speculate, is the means by which CPE is sorted for regulated secretion.