Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin

Keisuke Ito, Taishi Sugawara, Ayako Koizumi, Ken ichiro Nakajima, Akiko Shimizu-Ibuka, Mitsunori Shiroishi, Hidetsugu Asada, Takami Yurugi-Kobayashi, Tatsuro Shimamura, Tomiko Asakura, Katsuyoshi Masuda, Masaji Ishiguro, Takumi Misaka, So Iwata, Takuya Kobayashi, Keiko Abe

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish
Pages (from-to)986-992
Number of pages7
JournalBiochimica et Biophysica Acta - General Subjects
Volume1800
Issue number9
DOIs
Publication statusPublished - Sep 1 2010

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Mannose
Yeast
Polysaccharides
Protein Sorting Signals
Recombinant Proteins
Throughput
Yeasts
Proteins
Codon
Saccharomyces cerevisiae

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Medicine(all)

Cite this

Ito, K., Sugawara, T., Koizumi, A., Nakajima, K. I., Shimizu-Ibuka, A., Shiroishi, M., ... Abe, K. (2010). Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin. Biochimica et Biophysica Acta - General Subjects, 1800(9), 986-992. https://doi.org/10.1016/j.bbagen.2010.06.003

Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin. / Ito, Keisuke; Sugawara, Taishi; Koizumi, Ayako; Nakajima, Ken ichiro; Shimizu-Ibuka, Akiko; Shiroishi, Mitsunori; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shimamura, Tatsuro; Asakura, Tomiko; Masuda, Katsuyoshi; Ishiguro, Masaji; Misaka, Takumi; Iwata, So; Kobayashi, Takuya; Abe, Keiko.

In: Biochimica et Biophysica Acta - General Subjects, Vol. 1800, No. 9, 01.09.2010, p. 986-992.

Research output: Contribution to journalArticle

Ito, K, Sugawara, T, Koizumi, A, Nakajima, KI, Shimizu-Ibuka, A, Shiroishi, M, Asada, H, Yurugi-Kobayashi, T, Shimamura, T, Asakura, T, Masuda, K, Ishiguro, M, Misaka, T, Iwata, S, Kobayashi, T & Abe, K 2010, 'Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin', Biochimica et Biophysica Acta - General Subjects, vol. 1800, no. 9, pp. 986-992. https://doi.org/10.1016/j.bbagen.2010.06.003
Ito, Keisuke ; Sugawara, Taishi ; Koizumi, Ayako ; Nakajima, Ken ichiro ; Shimizu-Ibuka, Akiko ; Shiroishi, Mitsunori ; Asada, Hidetsugu ; Yurugi-Kobayashi, Takami ; Shimamura, Tatsuro ; Asakura, Tomiko ; Masuda, Katsuyoshi ; Ishiguro, Masaji ; Misaka, Takumi ; Iwata, So ; Kobayashi, Takuya ; Abe, Keiko. / Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin. In: Biochimica et Biophysica Acta - General Subjects. 2010 ; Vol. 1800, No. 9. pp. 986-992.
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abstract = "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.",
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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

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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.

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