Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line, CHO.
Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain-C-peptide-A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitute pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of 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 against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels 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.
Hunt
S M
SM
Department of Biotechnology, University of New South Wales, Kensington, Australia.
Tait
A S
AS
Gray
P P
PP
Sleigh
M J
MJ
D000818:Animals; D016466:CHO Cells / Q000737:chemistry / Q000187:drug effects / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007118:Immunoassay; D007328:Insulin / Q000737:chemistry* / Q000235:genetics*; D016296:Mutagenesis / Q000502:physiology; D010957:Plasmids; D011384:Proinsulin / Q000737:chemistry* / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection
D017077:Culture Media, Conditioned; D007328:Insulin; D012333:RNA, Messenger; D011384:Proinsulin