TY - JOUR
T1 - Transition of ovalbumin to thermostable structure entails conformational changes involving the reactive center loop
AU - Shinohara, Hiroshi
AU - Horiuchi, Masahisa
AU - Sato, Mamoru
AU - Kurisaki, Junichi
AU - Kusakabe, Takahiro
AU - Koga, Katsumi
AU - Minami, Yuji
AU - Aoki, Takayoshi
AU - Kato, Ikunosin
AU - Sugimoto, Yasushi
N1 - Funding Information:
We are very grateful to Mr. Y. Arai and Mr. K. Morizane for ardent technical assistance. We thank Dr. K. Inoue for his valuable suggestion and kind help with X-ray scattering experiments, which were performed under the approval of the Program Advisory Committee of the SPring-8 (Proposal No. 2002B0223-NL2-np). Special thanks are given to Professor K. Watanabe of Saga University for expert advice with GdmCl denaturation procedures of proteins. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, and Culture of Japan.
PY - 2007/1
Y1 - 2007/1
N2 - Ovalbumin is a serpin without inhibitory activity against proteases. During embryonic development, ovalbumin in the native (N) form undergoes changes and takes a heat-stable form, which was previously named HS-ovalbumin. It has been known that N-ovalbumin is artificially converted to another thermostable form called S-ovalbumin by heating at an alkaline pH. Here, we characterized further the three ovalbumin forms, N, HS, and S. The epitope of the monoclonal antibody 2B3/2H11, which recognizes N- and HS-ovalbumin but not S-ovalbumin, was found to reside in the region Glu-Val-Val-Gly-Ala-Ser-Glu-Ala-Gly-Val-Asp-Ala-Ala-Ser-Val-Ser-Glu-Glu-Phe-Arg, which corresponds to 340-359 of amino acid residues and is contained in the reactive center loop (RCL). Removal of RCL by elastase or subtilisin mitigated binding of the antibody. Dephosphorylation experiments indicated that the phosphorylated Ser-344 residue located on RCL is crucial for the epitope recognition. We suggest that the shift to the heat-stable form of ovalbumin accompanies a movement of RCL.
AB - Ovalbumin is a serpin without inhibitory activity against proteases. During embryonic development, ovalbumin in the native (N) form undergoes changes and takes a heat-stable form, which was previously named HS-ovalbumin. It has been known that N-ovalbumin is artificially converted to another thermostable form called S-ovalbumin by heating at an alkaline pH. Here, we characterized further the three ovalbumin forms, N, HS, and S. The epitope of the monoclonal antibody 2B3/2H11, which recognizes N- and HS-ovalbumin but not S-ovalbumin, was found to reside in the region Glu-Val-Val-Gly-Ala-Ser-Glu-Ala-Gly-Val-Asp-Ala-Ala-Ser-Val-Ser-Glu-Glu-Phe-Arg, which corresponds to 340-359 of amino acid residues and is contained in the reactive center loop (RCL). Removal of RCL by elastase or subtilisin mitigated binding of the antibody. Dephosphorylation experiments indicated that the phosphorylated Ser-344 residue located on RCL is crucial for the epitope recognition. We suggest that the shift to the heat-stable form of ovalbumin accompanies a movement of RCL.
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U2 - 10.1016/j.bbagen.2006.06.019
DO - 10.1016/j.bbagen.2006.06.019
M3 - Article
C2 - 16987608
AN - SCOPUS:33845440895
SN - 0304-4165
VL - 1770
SP - 5
EP - 11
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
IS - 1
ER -