High affinity binding of hydrophobic and autoantigenic regions of proinsulin to the 70 kDa chaperone DnaK
- Equal contributors
1 German Diabetes Centre, Leibniz Institute at Heinrich Heine University Düsseldorf, Institute of Clinical Diabetology, Auf'm Hennekamp 65, D-40225 Düsseldorf, Germany
2 Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland
3 Applied Genomics Research Group, McClay Research Center for Pharmaceutical Sciences, Queen's University, Belfast BT9 7BL, UK
4 Current Address: Research Group Immunobiology, Heinrich-Heine-University Düsseldorf, D-40225 Düsseldorf, Germany
BMC Biochemistry 2010, 11:44 doi:10.1186/1471-2091-11-44Published: 8 November 2010
Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. Complexes of peptides with chaperones induce peptide-directed immunity. Here we analyzed the interaction of (pre)proinsulin with the best characterized chaperone of the hsp70 family, bacterial DnaK.
Of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to DnaK was found for the signal peptide and one further region in each proinsulin domain (A- and B-chain, C-peptide). Among the latter, peptides covering most of the B-chain region B11-23 exhibited strongest binding, which was in the range of known high-affinity DnaK ligands, dissociation equilibrium constant (K'd) of 2.2 ± 0.4 μM. The B-chain region B11-23 is located at the interface between two insulin molecules and not accessible in insulin oligomers. Indeed, native insulin oligomers showed very low DnaK affinity (K'd 67.8 ± 20.8 μM) whereas a proinsulin molecule modified to prevent oligomerization showed good binding affinity (K'd 11.3 ± 7.8 μM).
Intact insulin only weakly interacts with the hsp70 chaperone DnaK whereas monomeric proinsulin and peptides from 3 distinct proinsulin regions show substantial chaperone binding. Strongest binding was seen for the B-chain peptide B 11-23. Interestingly, peptide B11-23 represents a dominant autoantigen in type 1 diabetes.