Abstract
Part I Human regenerating gene type III (RegIII) was identified in pathognomonic lesions of Alzheimer’s disease, a disease characterized by the presence of filamentous protein aggregates. Here, we showed that, at physiological pH, RegIII forms non-Congo red-binding, proteinase K-resistant fibrillar aggregates with diameters from 6 up to as large as 68 nm. Interestingly, circular dichroism and Fourier transform infrared spectra showed that, unlike typical amyloid fibrils, which have a cross-beta-sheet structure, these aggregates have a very similar secondary structure to that of the native protein, which is composed of two □-helices and eight □-strands, as determined by NMR techniques. Surface structure analysis showed that the positively-charged and negatively-charged residues were clustered on opposite sides, and strong electrostatic interactions between molecules were therefore very likely, which was confirmed by cross-linking experiments. In addition, several hydrophobic residues were found to constitute a continuous hydrophobic surface. These results and protein aggregation prediction using the TANGO algorithm led us to synthesize peptide Thr84 to Ser116, which, very interestingly, was found to form amyloid-like fibrils with a cross-□ structure. Thus, it seems that RegIII fibrillization is initiated by protein aggregation primarily due to electrostatic interactions, followed by conformational rearrangement, especially of the exposed hydrophobic loop, which is converted into a beta-sheet structure, then the RegIII fibril with a native-like conformation grows by the stacking of this short hydrophobic loop on top of the cross-beta spine. Part II Human Regenerating (Reg) gene family encoded secreted proteins, with amino acid sequences similar to C-type lectins. Among them, RegIV is highly expressed in mucosa cells of gastrointestinal tract during pathogen infection and carcinogenesis. It has been reported that RegIV is involved in anti-inflammation, cell proliferation, and increase of apoptosis resistance. However, the exact function of RegIV is not well defined. In this study, we provide the first direct evidence by surface plasmon resonance (SPR) that RegIV binds to polymeric carbohydrates, mannan and heparin but not monosaccharides, with the binding constant in the range around μM. To elucidate the structural basis for carbohydrate binding, we tried to use NMR spectroscopy to solve the structure of RegIV protein. However, the backbone amide resonances of two segments were missing. To solve this problem, the mutant that substituted Pro63 to Ser (P63S) was generated. Circular dichroism spectra showed that the secondary and tertiary structures of RegIV-P63S are quite similar with those of RegIV protein. The carbohydrate binding ability of RegIV-P63S was almost identical to that of wild-type protein as examined by SPR. The solution structure of P63S was determined and showed that RegIV contains two alpha-helices and eight beta-strands as a typical carbohydrate-recognization domain (CRD). The binding region of RegIV with mannan was determined by chemical shift perturbations of amide resonances of RegIV in complex with mannan. We find a binding cluster on the upper lobe of RegIV which is consisted by H69, D70, K73, Q75 and H100. In addition, base on the dynamic behavior difference, we propose that the highly flexible alpha2/beta4 and beta6/beta7 loops of RegIV also have mannan-binding characteristic in vitro. Although RegIV has a rigid scaffold as CRD, the plastic loops which composed by polar residues also provide the ability to recognize the sugars with various topologies. To combine these, RegIV is a polar macromolecule for ligands binding, such as polysaccharide on pathogen cell wall, glycoprotein or receptor and providing the basis for further functional investigations.