title: Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin.
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.
  
  
  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.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ito
authors.forename: Taishi
authors.initials: T
authors.lastname: Sugawara
authors.forename: Ayako
authors.initials: A
authors.lastname: Koizumi
authors.forename: Ken-Ichiro
authors.initials: K
authors.lastname: Nakajima
authors.forename: Akiko
authors.initials: A
authors.lastname: Shimizu-Ibuka
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Shiroishi
authors.forename: Hidetsugu
authors.initials: H
authors.lastname: Asada
authors.forename: Takami
authors.initials: T
authors.lastname: Yurugi-Kobayashi
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Shimamura
authors.forename: Tomiko
authors.initials: T
authors.lastname: Asakura
authors.forename: Katsuyoshi
authors.initials: K
authors.lastname: Masuda
authors.forename: Masaji
authors.initials: M
authors.lastname: Ishiguro
authors.forename: Takumi
authors.initials: T
authors.lastname: Misaka
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: Takuya
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Keiko
authors.initials: K
authors.lastname: Abe
mesh_terms: 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
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C008533:miraculin protein, Synsepalum dulcificum; C431926:taste receptors, type 1