Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP)

Background: Pancreatic amyloid has been associated with type II diabetes. T he major constituent of pancreatic amyloid is the 37-residue peptide islet amyloid polypeptide (IAPP). IAPP is expressed as a 67-residue pro-peptide c alled ProIAPP which is processed to IAPP following stimulation. While the m olecular events underlying IAPP amyloid formation in vitro have been studie d, little is known about the role of ProIAPP in the formation of pancreatic amyloid. This has been due in part to the limited availability of purified ProIAPP for conformational and biochemical studies. Results: We present a method for efficient recombinant expression and purif ication of ProIAPP and a processing site mutant, mutProIAPP, as thioredoxin (Trx) fusion proteins. Conformation and amyloidogenicity of cleaved ProIAP P and mutProIAPP and the fusion proteins were assessed by circular dichrois m, electron microscopy and Congo red staining. We find that ProIAPP and mut ProlAPP exhibit strong self-association potentials and are capable of formi ng amyloid. However, the conformational transitions of ProIAPP and mutProIA PP during aging and amyloidogenesis are distinct from the random coil-to-P- sheet transition of IAPP. Both proteins are found to be less amyloidogenic than IAPP and besides fibrils a number of non-fibrillar but ordered aggrega tes form during aging of ProIAPP. ProIAPP aggregates are cytotoxic on pancr eatic cells but less cytotoxic than IAPP while mutProIAPP aggregates essent ially lack cytotoxicity. The Trx fusion proteins are neither amyloidogenic nor cytotoxic. Conclusions: Our studies suggest that ProIAPP has typical properties of an amyloidogenic polypeptide but also indicate that the pro-region suppresses the amyloidogenic and cytotoxic potentials of IAPP.