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

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• 作者：Keisuke Ito ; Taishi Sugawara ; Ayako Koizumi ; Ken-ichiro Nakajima ; Akiko Shimizu-Ibuka ; Mitsunori Shiroishi ; Hidetsugu Asada ; Takami Yurugi-Kobayashi ; Tatsuro Shimamura ; Tomiko Asakura ; Katsuyoshi Masu
• 关键词：Miraculin ; Sweet protein ; Taste ; Yeast expression system ; N-glycan ; Steric hindrance
• 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
• 出版年：2010
• 出版时间：September 2010
• 年：2010
• 卷：1800
• 期：9
• 页码：986-992
• 全文大小：1255 K

文摘

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.