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 journalArticlepeer-review

13 Citations (Scopus)

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 2010

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

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