Proinsulin lacking the A7-B7 disulfide bond, Ins2Akita, tends to aggregate due to the exposed hydrophobic surface.
A single mutation (C96Y) in the Ins2 gene, which disrupts the A7-B7 disulfide bond, causes the diabetic phenotype in Akita mice. We biochemically analyzed the conformation of wild-type and Akita mutant recombinant proinsulins. Gel filtration chromatography and dynamic light scattering revealed that the apparent size of the mutant proinsulin molecules was significantly larger than that of wild-type proinsulin, even in the absence of intermolecular disulfide bonds. Titration with a hydrophobic probe, 1-anilinonaphthalene-8-sulfonate, demonstrated that the mutant proinsulin was more hydrophobic than the wild type. In addition, circular dichroism studies revealed that the conformation of the mutant proinsulin was less stable than the wild type, which is consistent with the observation that hydrophobic residues are exposed on the surface of the proinsulin molecules. Studies with antiserum against the C-peptide of proinsulin indicated that the mutant proinsulin had an immunoreactivity that was at least one-tenth weaker than wild-type proinsulin, suggesting that the C-peptide of mutant proinsulin is buried inside the aggregate of the proinsulin molecule. These findings indicate that increased hydrophobicity of mutant proinsulin facilitates aggregate formation, providing a clue to the dominant negative effect in the Akita mouse.
Yoshinaga
Takeo
T
Department of Health and Environmental Sciences, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan.
Nakatome
Keisuke
K
Nozaki
Jun-ichi
J
Naitoh
Motoko
M
Hoseki
Jun
J
Kubota
Hiroshi
H
Nagata
Kazuhiro
K
Koizumi
Akio
A
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D002942:Circular Dichroism; D003921:Diabetes Mellitus, Experimental / Q000235:genetics; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D005799:Genes, Dominant; D057927:Hydrophobic and Hydrophilic Interactions; D008027:Light; D051379:Mice; D008822:Mice, Transgenic; D008968:Molecular Conformation; D009154:Mutation; D010316:Particle Size; D011384:Proinsulin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012542:Scattering, Radiation
D000817:Anilino Naphthalenesulfonates; D004220:Disulfides; D011994:Recombinant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D011384:Proinsulin