Role of the connecting peptide in insulin biosynthesis.
In single-chain insulins (SCIs), the C terminus of the insulin B-chain is contiguous with the N terminus of the A-chain, connected by a short bioengineered linker sequence. SCIs have been proposed to offer potential benefit for gene therapy of diabetes (Lee, H. C., Kim, S. J., Kim, K. S., Shin, H. C., and Yoon, J. W. (2000) Nature 408, 483-488) yet relatively little is known about their folding, intracellular transport, or secretion from mammalian cells. Because SCIs can be considered as mutant proinsulin (with selective shortening of the 35-amino acid connecting peptide that normally includes two sets of flanking dibasic residues), they offer insights into understanding the role of the connecting peptide in insulin biosynthesis. Herein we have explored the relationship of the linker sequence to SCI biosynthesis, folding, and intracellular transport in transiently transfected HEK293 or Chinese hamster ovary cells or in stably transfected AtT20 cells. Despite previous reports that direct linkage of B- and A-chains produces a structure isomorphous with authentic two-chain insulin, we find that constructs with short linkers tend to be synthesized at lower levels, with a significant fraction of molecules exhibiting improper disulfide bonding. Nevertheless, disulfide-mispaired isoforms from a number of different SCI constructs are secreted. While this suggests that a novel folded state goes unrecognized by secretory pathway quality control, we find that misfolded SCIs are detected at higher levels in Chinese hamster ovary cells with artificially activated unfolded protein response mediated by inducible overexpression of active ATF-6. Such a maneuver allows analysis of more seriously misfolded mutants with further foreshortening of the linker sequence or loss (by mutation) of the insulin interchain disulfide bonds.
Liu
Ming
M
Diabetes Research and Training Center and the Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Ramos-Castañeda
Jose
J
Arvan
Peter
P
D000818:Animals; D002460:Cell Line; D004220:Disulfides; D015195:Drug Design; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D021122:Protein Subunits; D013329:Structure-Activity Relationship; D014162:Transfection
D004220:Disulfides; D007328:Insulin; D010455:Peptides; D021122:Protein Subunits