Amino Acid Sequence of Sweet-Taste-Suppressing Peptide (Gurmarin) from the Leaves of Gymnema Sylvestre

J. Biochem.111,109-112 (1992) Amino Acid Sequence of Sweet-Taste-Suppressing Peptide (Gurmarin) from the Leaves of Gymnema sylvestre KaekoKamei,* Ryo Takano,* Akiko Miyasaka,** Toshiaki Imoto,* * and Saburo Hara*, 1*Department of Chemistry and M T aterial Technology, Faculty of Engineering and Design, Kyoto Institute of echnology, Matsugasaki, Sakyo-ku, Kyoto, Kyoto 606; and **Department of Physiology, Tottori University School of Medicine, Yonago, Tottori 683 Received for publication, June 26, 1991 The complete amino acid sequence of a sweet-taste-suppressing peptide, gurmarin, from the leaves of Gymnema sylvestre was determined by the Edman analysis of peptides derived from digests obtained with Staphylococcus aureus V8 protease , pyroglutamyl aminopeptidase, and lysyl endopeptidase. Gurmarin consists of 35 amino acid residues with an amino-terminal pyroglutamyl residue and has the molecular weight of 4,209. Gurmarin has no significant homology with other known proteins. It has long been known that chewing a piece of the leaf of aspects of the sweet taste receptor protein. This paper Gymnema sylvestre, used as a folkloric remedy for diabetes reports the amino acid sequence of gurmarin from G. mellitus in India, causes complete loss of sweet taste sylvestre. sensation (1). The active substance which suppresses sweet taste was first extracted as a mixture of acidic compounds, MATERIALS AND METHODS and was named gymnemic acid (2). The extent of anti sweetness effect of gymnemic acid varies greatly from Materials•\Gurmarin was purified from the leaves of G. species to species even in mammals (3). No significant sylvestre according to the procedure of Imoto et al. (6). effect was observed in pig, rabbit, or rat (4, 5). Lysyl endopeptidase, S. aureus V8 protease, Wakosil-PTH Recently, a new substance (gurmarin) which suppresses column (4.6 x 250 mm), PTH-amino acids mobile phase, strongly the sweet taste responses in the rat, was isolated and 4M methanesulfonic acid containing 0.2% 3 (2-amino from the hot water extract of the leaves of G. sylvestre (6). ethyl)indole were from Wako Pure Chemical Industries. The inhibitory effect of gurmarin was highly specific to the Pyroglutamyl aminopeptidase from Boehringer Mannheim sweet taste responses to sucrose, glucose, glycine, and GmbH and YMC-packed column R-ODS-5 (4.6 x 250 mm) saccharin, so that the responses to the salty taste of NaCl, from YMC Inc. were used. the sour taste of HCl, and the bitter taste of quinine were Amino Acid Analysis-Samples were hydrolyzed in hardly affected. The inhibitory effect was detectable even sealed, evacuated test tubes with 50,u 1 of 4M methanesul at the low concentration of 1.2 x 10-6 M. The effect of fonic acid containing 0.2% 3-(2-aminoethyl)indole for 24 h gurmarin was reversible, but complete recovery of the at 110•Ž (11). Amino acid analyses were carried out with an suppressed responses required at least 3 h (6). This sug amino acid analyzer (Hitachi Model 835). gests that the binding of gurmarin to sweet taste receptor of Sequence Analysis-Manual Edman degradation was rat is quite strong and specific. carried out as described by Iwanaga et al. (12). Automated It is interesting that gurmarin showed no effect or only a Edman degradation was performed with a gas phase se very weak effect on the sweet taste sensation in humans. quencer (Shimadzu PSQ-1). PTH-derivatives of amino This is in contrast to the case of gymnemic acid which does acids were identified with a Wakosil-PTH column (4.6 x not affect the sucrose response of the rat, but completely 250 mm) equilibrated and eluted with PTH-amino acids suppresses the sweet taste sensation in man. mobile phase at a flow rate of 1 ml/min at 41•Ž. PTH Besides gymnemic acid, some other materials that amino acids were detected at 269 nm. reduce sweet taste recognition are known. They include Determination of Free SH Groups-The number of free ziziphin from Ziziphus jujuba (7, 8), hodulcin from Hovenia SH groups was determined by measuring the reaction with dulcis (9), and escin from Aesculus hippocastanum (10). Ellman's reagent, DTNB. Gurmarin (20-40 nmol) was All of them, as well as gymnemic acid, are derivatives of dissolved in 1.25 ml of 0.1M Tris-HCl buffer (pH 8.0) triterpene glycosides. Gurmarin is the first peptide found to containing 6M guanidine-HCl and 0.01M EDTA. To the show a strong and specific inhibitory effect on mammalian gurmarin solution was added 50,u 1 of a freshly prepared sweet taste responses. DTNB solution (0.01M in 0.05M sodium phosphate buffer, The study of gurmarin structure might reveal some pH 7.0), and the change of absorbance at 412 nm was recorded (13). Reduction and S-Carboxymethylation•\Gurmarin was 1 To whom correspondence should be addressed. dissolved in 0.5M Tris-HCl buffer (pH 8.0) containing 6M Abbreviations: DTNB, 5,5•L-dithiobis(2-nitrobenzoic acid); PTH, guanidine-HCl and 2mM EDTA, and then reduced with phenylthiohydantoin; RCm-, reduced and S-carboxymethylated; 2-mercaptoethanol (100 times the molar amount of half TFA, trifluoroacetic acid. Vol. 111, No. 1, 1992 109 110 K. Kamei et al. cystines) at 50•Ž under nitrogen (14). After 4 h, an amount presented in Table I. Thr, Ser, Ala, Met, Phe, and Arg of iodoacetic acid equimolar to 2-mercaptoethanol was residues were not present. added. The reaction mixture was left for 10 min at room On the manual Edman degradation up to 5th step of temperature, and then gel-filtered through a column of intact gurmarin, no PTH-derivative of any amino acid was Sephadex G-25 equilibrated with 50mM ammonium detected, indicating that the amino terminus of gurmarin is bicarbonate. blocked. Enzymatic Cleavage•\Reduced and S-carboxymethylat S. aureus V8 Protease Peptides•\The digest of RCm ed (RCm-) gurmarin (13.6nmol) in 10mM ammonium gurmarin with S. aureus V8 protease was separated by bicarbonate was digested with S. aureus V8 protease at HPLC to yield three peptides, V1 to V3 (Fig. 1S). The 37•Ž for 4 h. The molar ratio of enzyme to substrate was 1: amino acid compositions of the obtained peptides are listed 100. V8 protease peptideV1 (12.1nmol) was dissolved in in Table IS. The sum of the amino acid compositions of the 50mM ammonium bicarbonate and 0.01 unit of pyrogluta three peptides coincided with the total residues of RCm myl aminopeptidase was added. The digestion was per gurmarin. The hydrolysates of the peaks without numbers formed at 30•Ž for 26 h. in Fig. 1S contained no or very little amino acid, so they RCm-gurmarin (12.7nmol) in 10mM Tris-HCl buffer were not used for sequencing. Because V2 lacked glutamic (pH 9.0) was digested with lysyl endopeptidase at 37•Ž for acid, V2 was considered to be the carboxyl-terminal 4 h. The molar ratio of enzyme to substrate was 1:400. peptide. The amino acid sequences of V2 and V3 were Separation of Peptides•\Peptides obtained by enzymatic completely determined by the gas phase sequencer (Fig. digestion were separated by HPLC on a YMC-packed 4S). Sequence analysis of V1 yielded no PTH amino acid up column R-ODS-5 (4.6 x 250 mm) with an 80 min linear to 3 cycles of Edman degradation, indicating that Vl is the gradient of 0-80% acetonitrile in 0.1% TFA at a flow rate of amino-terminal peptide. 1 ml/min. Peptides were monitored at 220 nm. Vl (12.1nmol) was digested further with pyroglutamyl Nomenclature of the Peptides•\Peptides are designated aminopeptidase. The digest was separated by HPLC on a by a serial number prefixed by a letter. The letters indicate column of YMC-packed column R-ODS-5 (Fig. 2S). The the type of digestion: L, lysyl endopeptidase; P, pyrogluta peaks without numbers in Fig. 2S were not peptides as myl aminopeptidase; V, S. aureus V8 protease. The judged from the result of amino acid analyses. On the amino numbers in the peptide designation correspond to the order acid analysis, 12nmol of glutamic acid derived from 12.1 of their elution in HPLC. nmol of V1 was detected in V1-P1 fraction (Fig. 2S). The Mass Spectrometry•\FAB-MS measurements were car amino acid composition of V1-P2 was one glutamic acid less ried out on a JEOL HX110A/110A tandem mass spectro than that of V1 (Table IIS). These results indicated the meter. The accelerating voltage was 10 kV. Ions were amino-terminal residue of VI is pyroglutamic acid. Be produced with xenon using a JEOL FAB gun at 6 kV. The cause the amino-terminal amino acid residue newly formed resolution was about 1,000. All spectra were measured in by digestion with pyroglutamyl aminopeptidase was a the positive ion mode and recorded on a JEOL DA-5000 glutaminyl residue and because it was converted partially data system. The matrix used was either glycerol or a 1 : 1 to pyroglutamic acid under acidic HPLC conditions, it was mixture of glycerol/thioglycerol. considered that the peak of V1-P2 in Fig. 2S contained two components. The amino acid sequence of V1-P2 was RESULTS AND DISCUSSION determined as shown in Fig. 4S. Lysyl Endopeptidase Peptides-To align the S. aureus Amino Acid Composition and Amino Terminal Sequence V8 protease peptides, RCm-gurmarin was digested with of Gurmarin•\The amino acid composition of gurmarin is lysyl endopeptidase. The digest was separated by HPLC to yield seven peptides, Ll to L7 (Fig. 3S). Their amino acid compositions are presented in Table HIS. The Ll fraction in Fig.

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