Abstract:
Adiponectin is a circulating protein that is assembled and modified post-translationally in the endoplasmic reticulum (ER). The ER chaperone ERp44 plays an important role in adiponectin assembly and interacts with adiponectin by forming a mixed-disulphide linkage with the conserved cysteine in the adiponectin N-terminal domain. Under ER stress conditions, as occurs in obesity, ERp44 is up-regulated, inhibiting the release of adiponectin and is therefore an area of considerable interest. This research investigates the mechanism of adiponectin-binding by ERp44, using biophysical methods. For this purpose, adiponectin-derived peptides of various lengths either side of a critical cysteine residue in the N-terminal domain have been utilised to study complexation with ERp44. Mutants of ERp44 were produced by expression of a plasmid vector in an E. coli system. The mutations included alteration of residues within the CRFS motif and hydrophobic patch on the A domain, as well as the C160 residue in the B domain of ERp44 as these regions are conserved and have been implicated in ERp44 substrate binding. After over-expression in E. coli ERp44 mutants were purified using standard chromatographic techniques such as immobilised metal affinity chromatography (IMAC), ion exchange and size-exclusion chromatography. Binding studies were then conducted using synthetic adiponectin-mimetic peptides. These studies included tryptophan quenching to assess complexation in the native protein and non-reducing SDS-PAGE to assess complexation requiring disulphide bond formation. Complexation was verified by mass spectroscopy. Our results support the involvement of ERp44 C29 in complexation, however suggest that C160 may also be involved. The peptide mimetics were shown to behave in a manner expected of ERp44 substrates. ERp44 dimers were observed, and their formation of which appears to involve both disulphide and non-covalent interactions. This dimerisation was increased by the W28A mutation. Thus, an attempt was made to crystallise W28A ERp44, however this was unsuccessful. This research improves our understanding of the way in which adiponectin is bound by ERp44 and gives insight into the mechanism of retainment. Furthermore it demonstrated in-vitro formation of ERp44 dimers and identified C29 to be stabilising and W28 to be moderating, in dimerisation. P a g e | II Keywords: Adiponectin, ERp44, chaperones, obesity, diabetes, long peptide, short peptide, W28A, WT, C29S, C160S, complexation, dimerisation.