TY - JOUR
T1 - Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP)
AU - Krampert, Monika
AU - Bernhagen, Jürgen
AU - Schmucker, Jürgen
AU - Horn, Anita
AU - Schmauder, Anke
AU - Brunner, Herwig
AU - Voelter, Wolfgang
AU - Kapurniotu, Aphrodite
N1 - Funding Information:
We are grateful to G. Geiger for assistance with the expression of ProIAPP and mutProIAPP. We thank A. Kazantzis and A. Buck for HPLC purifications and the SPPS of mutProIAPP. We thank G. Tovar, S. Stoeva, M. Klärer, and K. Laib for MS measurements. We thank M. Duszenko and G. Dußling for assistance with the EM. This work was supported by a grant from The University of Tübingen to A.K. and an institutional grant from the Fraunhofer IGB to J.B.
PY - 2000
Y1 - 2000
N2 - Background: Pancreatic amyloid has been associated with type II diabetes. The major constituent of pancreatic amyloid is the 37-residue peptide islet amyloid polypeptide (IAPP). IAPP is expressed as a 67-residue pro-peptide called ProIAPP which is processed to IAPP following stimulation. While the molecular events underlying IAPP amyloid formation in vitro have been studied, 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 purification of ProIAPP and a processing site mutant, mutProIAPP, as thioredoxin (Trx) fusion proteins. Conformation and amyloidogenicity of cleaved ProIAPP and mutProIAPP and the fusion proteins were assessed by circular dichroism, electron microscopy and Congo red staining. We find that ProIAPP and mutProIAPP exhibit strong self-association potentials and are capable of forming amyloid. However, the conformational transitions of ProIAPP and mutProIAPP during aging and amyloidogenesis are distinct from the random coil-to-β-sheet transition of IAPP. Both proteins are found to be less amyloidogenic than IAPP and besides fibrils a number of non-fibrillar but ordered aggregates form during aging of ProIAPP. ProIAPP aggregates are cytotoxic on pancreatic cells but less cytotoxic than IAPP while mutProIAPP aggregates essentially 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.
AB - Background: Pancreatic amyloid has been associated with type II diabetes. The major constituent of pancreatic amyloid is the 37-residue peptide islet amyloid polypeptide (IAPP). IAPP is expressed as a 67-residue pro-peptide called ProIAPP which is processed to IAPP following stimulation. While the molecular events underlying IAPP amyloid formation in vitro have been studied, 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 purification of ProIAPP and a processing site mutant, mutProIAPP, as thioredoxin (Trx) fusion proteins. Conformation and amyloidogenicity of cleaved ProIAPP and mutProIAPP and the fusion proteins were assessed by circular dichroism, electron microscopy and Congo red staining. We find that ProIAPP and mutProIAPP exhibit strong self-association potentials and are capable of forming amyloid. However, the conformational transitions of ProIAPP and mutProIAPP during aging and amyloidogenesis are distinct from the random coil-to-β-sheet transition of IAPP. Both proteins are found to be less amyloidogenic than IAPP and besides fibrils a number of non-fibrillar but ordered aggregates form during aging of ProIAPP. ProIAPP aggregates are cytotoxic on pancreatic cells but less cytotoxic than IAPP while mutProIAPP aggregates essentially 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.
KW - Aggregation
KW - Amyloidogenicity
KW - Cytotoxicity
KW - Islet amyloid polypeptide
KW - Pro-islet amyloid polypeptide
UR - http://www.scopus.com/inward/record.url?scp=0033758248&partnerID=8YFLogxK
U2 - 10.1016/S1074-5521(00)00034-X
DO - 10.1016/S1074-5521(00)00034-X
M3 - Article
C2 - 11094339
AN - SCOPUS:0033758248
SN - 1074-5521
VL - 7
SP - 855
EP - 871
JO - Chemistry and Biology
JF - Chemistry and Biology
IS - 11
ER -