Formation of Disulfide Bonds in Proinsulin is Dependent on Both the Substrate Chemical Form and the Conditions Used for Purification and Folding
Authors: Mackin Robert B 
Published: June 2021

Misfolding of proinsulin (PI), the biosynthetic precursor to insulin, is believed to be the underlying cause of some forms of both type 1 and type 2 diabetes mellitus. It has been proposed that the ER stress response plays a role in recognizing and responding to PI misfolding in the cell. My laboratory is developing an in vitro model system to examine the roles of PI, protein disulfide isomerase (PDI) and the redox co‐factor glutathione in the folding reaction.

Both PI and PDI were expressed as N‐terminal poly‐His‐tagged proteins in bacteria. The PI fusion protein was recovered from inclusion bodies and the poly‐His tag was removed by cyanogen bromide cleavage. Liberated PI was recovered by rotary evaporation and subjected to further chemical treatment. PDI was purified from the soluble fraction of bacteria using a combination of IMAC and gel filtration chromatography.

Two forms of PI were studied: sulfonated and reduced (PI‐SO4 and PI‐red, respectively). Both versions of PI were subjected to gel filtration chromatography at pH 7.2. PI‐SO4 eluted within the included volume of the column while PI‐red eluted at the void volume, suggesting that the latter version had undergone significant aggregation. Subsequent analysis using non‐reducing SDS‐PAGE indicated that both forms of PI migrated through the gel as monomers. Reaction of PI‐red with iodoacetic acid prior to non‐reducing SDS‐PAGE resulted in a mobility shift, reinforcing the hypothesis that the aggregation event was not due to the formation of incorrect disulfide cross‐links between PI molecules.

Folding of both versions of PI (25 μM) was tested in the presence of reduced and oxidized glutathione (GSH/GSSG, 3 mM of each), PDI (5 μM) or a combination of GSH/GSSG and PDI. Previous studies had established that samples of PI‐red taken directly from the reduction reaction (performed in the presence of either 6 M guanidine or 8 M urea); obtained following buffer exchange without chaotropic agents at pH 10.6; or obtained after folding in the presence of GSH/GSSG at pH 10.6; could all be analyzed using RP‐HPLC. However, PI‐red that had been subjected to chromatography between pH 7 and 8 in the absence of chaotropic agents could not be analyzed using this method as no PI‐red peak could be detected and the HPLC column quickly became clogged. As an alternative, native PAGE was investigated as an alternative analytical method. Results from this study indicated that folding of PI‐SO4 at pH 7.2 can occur in the presence of either GSH/GSSG or a combination of GSH/GSSG and PDI, but not in the presence of PDI alone. In contrast, it was found that PI‐red does not enter the native PAGE gels under any of the conditions tested. This suggests that the non‐disulfide bonded, aggregated form of PI‐red could not be properly folded at pH 7.2 using concentrations of PI and GSH/GSSG similar to those that resulted in proper folding at pH 10.6.

In conclusion, these results suggest that folding of PI in islet beta cells must take place under tightly controlled conditions and that mimicking these conditions in an in vitro assay will require further understanding of the biochemistry of the PI molecule.

Support or Funding Information

Supported by the Creighton University Health Science Strategic Investment Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.