The Mechanism of Fibril Formation of a Non-inhibitory Serpin Ovalbumin Revealed by the Identification of Amyloidogenic Core Regions

Naoki Tanaka,Yumi Morimoto,Yurika Noguchi,Tomoko Tada,Tomonori Waku,Shigeru Kunugi,Takashi Morii,Yin-Fai Lee,Takashi Konno,Nobuyuki Takahashi

doi:10.1074/jbc.m110.176396

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.

Highlights

Zation of the serpin family of serine protease inhibitors provide well defined structural models of neurodegenerative diseases
The fluorescence intensity of thioflavin T bound to OVA fibril is very low at 80 °C, and the kinetic measurement of OVA fibril formation at elevated temperature is difficult
We examined the effect of the peptide fragments of the reactive center loop 338GREVVGSAEAGVDA351, 346EAGVDA351, and R339T variant 338GTEVVGSAEAGVDA351 on the heat-induced fibril formation of OVA (Table 1), but the fibril morphology as monitored by Transmission Electron Microscope (TEM) was unaffected by these peptides

Summary

Amyloidogenic Regions of Ovalbumin

OVA have been characterized in detail using disulfide rearrangement analysis (18 –22). OVA possesses a metastable conformation like other serpins, and the intermolecular partial loop insertion model has not been completely ruled out as the mechanism for heat-induced fibril formation [37]. These results suggest that the heat-induced OVA fibril is formed by a unique mechanism. Detailed characterization of the core of ␤2-microgloblin (␤2m) amyloid fibrils at single-residue resolution has been performed by the hydrogen/deuterium exchange of amide protons combined with NMR analysis [39] This method is limited to proteins whose three-dimensional structure in solution can be solved by NMR and, not suitable for OVA, which has a higher molecular weight than ␤2m and whose NMR solution structure is unsolved. By identifying the core regions for heat-induced aggregation of SH-OVA, we discuss the molecular mechanism for the fibril formation of OVA in the light of the aggregation of serpins

EXPERIMENTAL PROCEDURES

RESULTS

Amyloidogenic core regions hB

DISCUSSION