title: The mechanism of fibril formation of a non-inhibitory serpin ovalbumin revealed by the identification of amyloidogenic core regions.
abstract: Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys(73) and Cys(120) in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4-5B are highly β-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to β-strands, thereby forming sequential intermolecular linkages.
authors.affiliation: Department of Biomolecular Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan. tanaka@kit.ac.jp
authors.forename: Naoki
authors.initials: N
authors.lastname: Tanaka
authors.forename: Yumi
authors.initials: Y
authors.lastname: Morimoto
authors.forename: Yurika
authors.initials: Y
authors.lastname: Noguchi
authors.forename: Tomoko
authors.initials: T
authors.lastname: Tada
authors.forename: Tomonori
authors.initials: T
authors.lastname: Waku
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kunugi
authors.forename: Takashi
authors.initials: T
authors.lastname: Morii
authors.forename: Yin-Fai
authors.initials: YF
authors.lastname: Lee
authors.forename: Takashi
authors.initials: T
authors.lastname: Konno
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takahashi
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D002645:Chickens; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010047:Ovalbumin / Q000737:chemistry* / Q000235:genetics; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
chemical_list: D000682:Amyloid; D004220:Disulfides; D010455:Peptides; D010047:Ovalbumin; D003545:Cysteine