TY - JOUR
T1 - Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin
AU - Ito, Keisuke
AU - Sugawara, Taishi
AU - Koizumi, Ayako
AU - Nakajima, Ken ichiro
AU - Shimizu-Ibuka, Akiko
AU - Shiroishi, Mitsunori
AU - Asada, Hidetsugu
AU - Yurugi-Kobayashi, Takami
AU - Shimamura, Tatsuro
AU - Asakura, Tomiko
AU - Masuda, Katsuyoshi
AU - Ishiguro, Masaji
AU - Misaka, Takumi
AU - Iwata, So
AU - Kobayashi, Takuya
AU - Abe, Keiko
N1 - Funding Information:
This study was supported in part by the ERATO Iwata Human Receptor Crystallography Project (JST) (to S.I.), the Targeted Proteins Research Program (to S.I. and T.M.), a Grant-in-Aid for Scientific Research (B) ( 20370035 ) (to T.K.), a grant from the Research and Development Program for New Bio-Industry Initiatives (to K.A.), Grants-in-Aid for Scientific Research (S) (to K.A.), Grants-in-Aid for JSPS Fellows (to A.K.) and Grants-in-Aid for JSPS Fellows (to K.I.) in Japan.
PY - 2010/9
Y1 - 2010/9
N2 - Background: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. Methods: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. Results: As a result, a 2. mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. Conclusions: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. General significance: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.
AB - Background: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. Methods: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. Results: As a result, a 2. mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. Conclusions: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. General significance: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.
KW - Miraculin
KW - N-glycan
KW - Steric hindrance
KW - Sweet protein
KW - Taste
KW - Yeast expression system
UR - https://www.scopus.com/pages/publications/77955281666
UR - https://www.scopus.com/pages/publications/77955281666#tab=citedBy
U2 - 10.1016/j.bbagen.2010.06.003
DO - 10.1016/j.bbagen.2010.06.003
M3 - Article
C2 - 20542090
AN - SCOPUS:77955281666
SN - 0304-4165
VL - 1800
SP - 986
EP - 992
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
IS - 9
ER -