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


CONCLUSIONS
The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.
Ito
Keisuke
K
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Sugawara
Taishi
T
Koizumi
Ayako
A
Nakajima
Ken-Ichiro
K
Shimizu-Ibuka
Akiko
A
Shiroishi
Mitsunori
M
Asada
Hidetsugu
H
Yurugi-Kobayashi
Takami
T
Shimamura
Tatsuro
T
Asakura
Tomiko
T
Masuda
Katsuyoshi
K
Ishiguro
Masaji
M
Misaka
Takumi
T
Iwata
So
S
Kobayashi
Takuya
T
Abe
Keiko
K
D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D011134:Polysaccharides / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013649:Taste / Q000187:drug effects* / Q000502:physiology
D006023:Glycoproteins; D011134:Polysaccharides; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C008533:miraculin protein, Synsepalum dulcificum; C431926:taste receptors, type 1