PROCESSING OF MUTATED HUMAN PROINSULIN TO MATURE INSULIN IN THE NONENDOCRINE CELL-LINE, CHO

Heterologous genes encoding proproteins, including proinsulin, general ly produce mature protein when expressed in endocrine cells while unpr ocessed or partially processed protein is produced in non-endocrine ce lls. Proproteins, which are normally processed in the regulated pathwa y restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the cons titutive pathway, the principal protein processing pathway in non-endo crine cells. Human proinsulin consists of B-Chain-C-peptide-A-Chain an d cleavage at the B/C and C/A junctions is required for processing. Th e B/C, but not the C/A junction, is recognised and cleaved in the cons titutive pathway. We expressed a human proinsulin and a mutated proins ulin gene with an engineered furin recognition sequence at the C/A jun ction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency o f the mutant proinsulin was 56% relative to 0.7% for native proinsulin . However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized agai nst mRNA levels, were 18-fold lower in the mutant proinsulin-expressin g cells. As a result, there was only a marginal increase in absolute l evels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.