THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264,No. Issue of April 25, pp.

66554659,1989
0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A.

Complete Amino Acid Sequence and Structure Characterizationof the

Taste-modifying Protein,Miraculin*
(Received for publication, July 21,1988)

Sarroch TheerasilpS, Hiromu Hitotsuya, Shigeo NakajoQ, Kazuyasu NakayaQ, Yasuharu NakamuraQ,
and Yoshie Kuriharall
From the Department of Chemistry, Faculty of Education, Yokohama National University, Yokohamu 240, Japan and the
§Laboratory of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo 152Japan

The taste-modifying protein, miraculin, has the un- with 191 amino acid residues. The calculated molecular weight
usual property of modifying sour into tastesweet taste. of miraculin based on the amino acid sequence and the car-
The complete amino acid sequenceof miraculin puri- bohydrate content is 24,600.
fied from miracle fruits by a newly developed method
(Theerasilp,S., and Kurihara,Y. (1988) J. Biol. Chern. EXPERIMENTALPROCEDURES
263, 11636-11639) was determined by an automatic Materials-Miracle fruits (R. dulcifica) were obtained from plants
Edman degradation method. Miraculin was a single grown in the green house of Yokohama National University. Mira-
polypeptide with 191 amino acid residues. The calcu- culin was purified from pulps of the fruits free from seeds and skins
lated molecular weight based on the amino acid se- as described in the previous paper (5).
quence and thecarbohydratecontent (13.9%) was The sources of proteases are as follows.Achrornobacterlyticus
24,600. Asn-42 and Asn-186 were linked N-glycosid- protease I (lysyl endopeptidase), Wako Pure Chemicals Industries,
ically tocarbohydratechains.Highhomology was Ltd.; l-chloro-3-tosylamido-7-amino-2-heptanone-chymotrypsin and
found between the amino acid sequences of miraculincarboxypeptidase A, Sigma; Staphylococcusaurew V8 protease, Miles
and soybean trypsin inhibitor. Laboratories, Inc.; ~-l-tosylamido-2-phenylethyl chloromethyl ke-
tone-trypsin, Worthington.
All other chemicals used were of analytical grade.
Preparation of S-Carboxyamidomethylted Miraculin-Seven mil-
ligrams of the purified miraculin were dissolved in 5 mi of 0.4 M Tris
Richadella dulcifica aisnative shrubof tropical West Africa. buffer, pH 8.2,containing 6 M guanidine hydrochloride, 2 mM EDTA,
It yields red berries which have an unusual property in mod- and 60 mM dithiothreitol. The solution was incubated at 37 “C under
of nitrogen gas for 24 h. Iodoacetamide, 0.2 g, was added
ifying sour taste into sweet taste. For example, lemons elicit atmosphere
to thesolution, mixed, let stand atroom temperature for 10 min, and
sweet taste afterchewing pulps of the berries. Because of this then placed in an ice bath for 60 min. The obtained S-carboxyami-
unusual property, the berry has been called “miracle fruit.” domethylated miraculin was desalted by using a Sephadex G-25
Kurihara and Beidler (1)first isolated the active principle column (1.6X 5 cm) equilibrated with 50 mM EDTA.
of miracle fruit and showed that it is a basic glycoprotein. Enzymatic Cleavage-Lysyl endopeptidase digestion of S-carbox-
Brouwer et al. (2), Giroux and Henkin (3), and Kurihara and yamidomethylated miraculin was performed in 50 mM ammonium
Terasaki (4) also isolated the active principle, and Brouwer bicarbonate buffer, pH 8.0,containing 2 M urea and 2 mM EDTA at
37 “C for 20 h. The protein concentration was 1 mg/ml, and the
et al. (2) named it “miraculin.” The miraculin samples isolated enzyme:substrate ratio was 1:lOO (w/w). The reaction was terminated
in all these studies were not completely pure, and hence any by addition of HCI to give a final pH 2.0. There was no insoluble
attempt to determine miraculin’s primary structure has not material formed after digestion. The solution was injected into a
been made. Recently Theerasilp and Kurihara(5) established HPLC toisolate the peptides.
a new method to obtain miraculin in very pure form. They Chymotrypsin digestion of the modified miraculin was performed
showed that it is a basic glycoprotein having a molecular under the same conditions as those for lysyl endopeptidase digestion
except that the digestion time was 90 min. The reaction was also
weight of about 28,000 as estimated by SDS-PAGE,’ while terminated in the same manner. There was no insoluble material
molecular weights reported in previous papers (1-4)ranged formed, and the solution was injected into a HPLC to isolate the
from 40,000 to 48,000. Miraculin contained asmuch as 13.9% peptides.
of carbohydrate (5). S-Carboxyamidomethylated miraculin was digested by S. aureus
In the present study, we have determined the complete V8 protease in 50 mM ammonium bicarbonate buffer, pH 7.8,con-
amino acid sequence of miraculin. It is a single polypeptide taining 2 M urea, and 2 mM EDTA at 37 “C for 3 h. The protein
concentration was 1mg/ml, and theenzyme:substrate ratio was 1:30,
* This work was supported by grants from the Ministry of Educa- w/w. Precipitates formed after digestion. Solid urea was added to the
tion of Japan, Mitajiri Chemical Industry Co., and Mishima Science reaction mixture until the solution became clear. The solution was
Foundation. The costs of publication of this article were defrayed in injected in a HPLC toisolate the peptides.
part by the payment of page charges. This article must therefore be Peptide Isolation-The peptides in hydrolysates of the enzymatic
hereby marked “advertisement” in accordance with 18U.S.C. Section digestion were separated by HPLC (Tosoh PC 8000) with a TSK-
1734 solely to indicate this fact. ODs-IZOT column (0.46X 25 cm) (Tosoh). The peptides were eluted
$Present address: Dept. of Chemistry, Faculty of Science and from the column by a lineargradient of acetonitrile containing0.05%
Technology, Sri-Ayudhya United Colleges at Pranakornsri-Ayudhya, trifluoroacetic acid a t a flow rate of 1 ml/min. The eluted peptides
Ayudhya 13000, Thailand. were monitored by measuring absorption at 210 nm, and each peak
ll To whom all correspondence should be addressed Dept. of Chem- was collected manually.
istry, Faculty of Education, Yokohama National University, Toki- Amino Acid Analysis-Amino acid compositions of the peptides
wadai, Hodogaya-ku, Yokohama, 240 Japan. were determined by a Waters Picotag system (6).The peptide was
The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate- hydrolyzed by HCl vapor at 110 “C for 22 h. The obtained amino
polyacrylamide gel electrophoresis; HPLC, high performance liquid acids were converted to phenylthiocyanate derivatives, and their
chromatography; LEP, lysine endopeptidase; Ch, chymotrypsin. contents were analyzed by HPLC using a TSK-ODs-80TM column

6655

This is an Open Access article under the CC BY license.

6656 Amino Acid Sequence of Miraculin
(0.46 X 15 cm) (Tosoh). Phenylthiocyanate-amino acid derivatives Carboxyl-terminal amino acid sequence was determined by using
were eluted from the column by a linear gradient from solution A carboxypeptidase A as described by Ambler (8). Miraculin, 200 pg,
(3% acetonitrile in 50 mM phosphate buffer of pH 7.0 containing 0.1 was dissolved in 0.9 ml of 0.1 M N-ethylmorpholine acetate buffer,
M sodium perchlorate) to a mixture of solution A and 40% acetonitrile pH 8.0. Carboxypeptidase A, 1pg, was added, and thereaction mixture
(1:4, v/v) for 20 min. The elution was carried out atcolumn temper- was incubated at room temperature. Aliquots were taken at 15, 30,
ature of 40 "C and at a flow rate of 1 ml/min. The eluted phenylthio- 60, and 120 min, and the proteins were precipitated by addition of
cyanate-amino acid derivatives were monitored by measuring absorp- trichloroacetic acid to give a final concentration of 10%. The precip-
tion at 254 nm. itates wereremovedby centrifugation, andthesupernatant was
Sequence Analysisof Protein and Peptides-Amino-terminal amino subjected to analysis of the released amino acids by a Waters Picotag
acid sequence analysis by automatic Edman degradation was per- system as described above.
formed with an Applied Biosystem Protein Sequencer (model 470A). Detection of Carbohydrates-Carbohydrates in peptides were de-
Phenylthiohydantoin amino acid derivatives were analyzed by HPLC tected by orcinol-sulfuric acid reaction (9). In addition, the carbohy-
using a TSK-ODs-12OT column, 0.46 X 25 cm, as described by drate-containingpeptides (LEP-6 andLEP-12) were subjected to acid
Tsunasawa et al. (7) or by an Applied Biosystem on-line HPLC hydrolysis and the presence of carbohydrates in the hydrolysate was
system. confirmed by HPLC on an ISA-O7/S2504 column (5).

I RESULTS
Amino acid sequence from the amino terminus of miraculin
to the46th amino acid residue has been determined with the
whole moleculeof S-carboxyamidomethylatedmiraculin (Fig.
3). Aspartic acid is the amino-terminal amino acid.
Several peptides were obtained by digestion of the modified
miraculin with lysyl endopeptidase (Fig. 1).The peptides were
""
well separated on HPLC except LEP-3 which contained two
peptides, LEP-3A and LEP-3B. These peptides were sepa-
rated from each other upon rechromatography on the same
column but using a narrow gradient of acetonitrile and a
longer running time.

A
The sequences of minor peaks (LEP-11,LEP-13, and LEP-
14) from the amino-terminal to at least 10 residues toward
their carboxyl terminus were identical with that of LEP-12.
Therefore these minor peaks seem to be either apart of LEP-
0 IO 20 30 40 50 60 12 peptide or LEP-12 peptide with some extending amino
R e t e n t i o n T i m( m i " . )
acid sequences from its carboxyl terminus. Similarly, peptides
FIG. 1. HPLC separation of peptides obtained by lysine en- from the minor peaks, LEP-16, LEP-17,and LEP-19, had the
dopeptidase digestion of S-carboxyamidomethylatedmiracu- same sequence as LEP-18 at least 10 residues from their
lin. Separation was carried on a reverse phase column, TSK-ODS- amino terminus toward the carboxyl terminus. Amino acid
120T (0.46 X 25 cm). The peptides were eluted by a linear gradient
of acetonitrile containing 0.05% trifluoroacetic acid from 10 to 60% sequences and amino acid compositions of all major peaks
at a flow rate of 1 ml/min for 1h. The eluted peptides were monitored were determined as shown in Fig. 3 and Table I, respectively.
by absorption at 210 nm. The modified miraculin was cleavedby lysyl endopeptidase

TABLEI
Amino acid compositionsof miraculin and peptides obtained from digestion of S-carboxyamidomethyluted miraculin
with lysyl endopeptidase
Amino LEP-lb LEP-2 LEP-3A LEP-3B
LEP-4
LEP-6 LEPS LEP-9
LEP-10 LEP-12 LEP-15
LEP-18
peptides obtained
(170-179)
(159-169)
(1-14)
(145-156)
(134-144)
(180-191)
(170-187) (170-179)
(57-72) (159-169)
(105-120)
(15-56) (1-14)
(145-156)
(121-133) (134-144)
(73-104) (180-191)
(170-187)
(57-72)
(105-120)
(15-56)
(121-133) peptides obtained
(73-104)
residuesjmolecule'
Asx(D/N) 0.9(1) 1.9(2) 3.4(4) 0.6(1) 1.7(2) 2.7(3) 0.9(1) 2.6(3) 0.9(1) 4.7(5) 20.1(21)
Glx(E/Q) 1.0(1) 1.2(1) 1.8(2) 0.7(1) 2.1(2)
0.8(1) peptides obtained 1.0(1) 1.2(1) 1.4(1) 11.8(12)
CYS(C) 0.8(1) 2.6(3) 0.8(1) 0.9(1) 0.9(1) 6.5(7)
Ser(S) 0.8(1) 0.9(1) 0.9(1) 0.9(1) 0.9(1) 0.8(1) 0.9(1) 1.8(2) 4.5(5) 12.8(13)
GMG) 0.9(1) 1.8(2) 1.1(1) 1.1(1) 1.9(2) 1.4(1) 3.0(3) 4.3(5) 2.1(2) 15.6(17)
His(H) 0.9(1) 1.2(1) 1.8(2)
Thr(T) 0.8(1) 1.4(1) 1.6(2) 6.4(7) 0.9(1) 2.8(3) 14.6(15)
Ala(A) 0.9(1) 1.3(1) 0.6(1) 1.9(2) 1.1(1) 1.3(1) 1.3(1) 6.2(6)
Pro(P) 2.1(2) 1.0(1) 0.5(1) 0.9(1) 2.9(3) 4.4(4) 2.2(2) 1.2(1) 13.9(14)
2.7(3) 2.8(3) 0.9(1) 4.2(4) 2.7(3) 10.8(11)
Tyr(Y) 0.8(1) 0.7(1) 1.2(1) 1.5(2) 1.7(2) 7.3(7)
Val(V) 1.0(1) 0.9(1) 2.1(2) 1.1(1) 1.2(1) 2.2(2) 7.4(6) 3.7(4) 21.3(19)
Met(M) 1.2(1) 0.9(1)
Ile(1) 2.3(2) 1.0(1) 1.5(1) 1.4(1) 0.8(1) 1.1(1) 1.3(1) 8.4(8)
Leu(L) 2.2(2) 0.9(1) 0.9(1) 2.4(2) 0.9(1) 2.6(3) 2.0(2) 9.5(10)
Phe(F) 0.9(1) 2.1(2) 3.6(4) 2.7(3) 1.9(2) 1.2(1) 1.7(1) 1.0(1) 2.1(2) 14.5(14)
Lys(K) 1.3(1) 1.4(1) 0.9(1) 1.0(1) 1.2(1) 1.1(1) 2.1(2) 1.4(1) 1.1(1) 1.1(1) 0.9(1) 1.2(1) 12.4(12)
TdW) ND(1)"
ND(1) ND(2)
Total (10) (11) (14) (12) (11) (12) (18) (16) (16) (42) (13) (32) (191)
a One-letter symbols are given in parentheses.
* Name of peptides and thesequence position (in parentheses).
e Determined by amino acid analysis or from the sequence (in parentheses).
ND, not determined.
 Amino Acid Sequence of Miraculin 6657

r" T
-
1 1 10 20 30
D S A P N P V L D I D G E K L R T G T N Y Y I V P V L R D H
I

I
LEP-12 LEP-31
I
1 Ch-8 LCh-15"-

31 40 50 60
G G G L T V S A T T P N G T F V C P P R V V Q T R K E V D H
- N

Ch-15--Ch-14

h
61 70 80 90
D R P L A F F P E N P K E D V V R V S T D L N I N F S A F M
-LEP-9-LEP-18
2aCh-16- LCh-9-

91 100 110 120

P C R W T S S T V S R L D K Y D E S T G Q Y F V T I C G V K

121 130 110 150

G N P G P E T I S S W F K I E E F C G S G F Y K L V F C P T
-LEP-15- L L E P - 4 - LLEP-3B-
L"Ch-6- I
-70 20 30 40 50 60 "
"""
"*
v-10
Retention Time ( m i n ) 1V-6

FIG. 2. HPLC separation of peptides 180 obtained from diges- 110 151 160
V C G S C K V K C G D V G I Y I D Q K G R R R L A L S D K P
tion of S-carboxyamidomethylated miraculin by chymotryp-
sin. Separation was carried on a reverse phase column, TSK-ODS- 1L L E P - Z - L LEP-I-
120T (0.46 X 25 cm). The peptides were eluted by a linear gradient
of acetonitrile containing0.05% trifluoroacetic acid from 20 to 90%
LEP-8 -
Ch-7- I C h - 1 2
at a flow rate of 1 ml/min for 1h. The eluted peptides were monitored
by absorption at 210 nm.
191
"""

181
"""""""""

"_
-
F A F E F N K T V Y F
atthe carboxyl-terminal site of all lysine residues. Only
peptide bonds at two positions (Lys-Pro at position 179-180 I
LEP-6

and Lys-Thr at position 187-188) showed partial resistance 1

to thedigestion as indicated by amino acid sequences of LEP- "-J L A L
__--I
1, LEP-6,andLEP-8 (Fig. 3 and Table I). Amino acid
FIG.
compositions of all LEP-peptides were in agreement with sequence3.ofThe proof amino acid sequence of miraculin. The
the peptides isrepresented by a one-letter code. LEP and
their compositions as determined from the sequence. Mira- Ch denote peptides derived from lysyl endopeptidase and chymotryp-
culin contained 2 tryptophan residues, one in LEP-18 andthe sin digestions of S-carboxyamidomethylatedmiraculin, respectively.
other in LEP-15. Only LEP-6 had no lysine as the carboxyl- Their numbers also correspondto those in Figs. 1 and 2. V denotes
terminal amino acid. There wasno phenylthiohydantoin peptides derived from VS-protease digestion, and their numbers cor-
amino acid observed after phenylalanine. This resultsuggests respond to those in Fig. 4. Solid lines indicate theamino acid sequence
obtained from the automatic sequencing, and dashed lines indicate
that phenylalanine is the carboxyl-terminal amino acid of the sequence which was not determined by the sequencing. The urrow
miraculin. (L) indicates the sequence from the carboxyl terminus determined
LEP-6 and LEP-12 showed orcinol-sulfuric acid reaction, by carboxypeptidase A digestion.
suggesting that these peptides contain carbohydrates. In ad-
dition, the peptides were subjected to acid hydrolysis, and the obtained are shown in Fig. 2. The peptides, Ch-4 to Ch-9, Ch-
presence of carbohydrates in the hydrolysates was confirmed 12, Ch-14, Ch-16, and Ch-18 were subjected to determination
by HPLC. Amino acid residue at position 186 in LEP-6could of amino acid sequences and amino acid compositions (Fig. 3
not be identified by the automatic sequencing. It was, how- and Table 11). Amino acid compositions of the Ch-peptides
ever, identified as aspartic acid by determination of amino were in agreement with their compositions as determined
acid composition of LEP-6. Thus the amino acid a t position from the sequences. Ch-14 linked LEP-12 to LEP-9, which
186 is asparagine to which a carbohydrate chain is linked. extended the sequence from 1st to 72nd positions. Ch-16
LEP-12 was a long peptide composed of 42 amino acid resi- overlapped LEP-9 and LEP-18.Ch-18 overlapped three pep-
dues; and hence, to identify the amino acid residue to which tides, i.e. LEP-18, LEP-10, and LEP-15. Ch-7 clearly linked
a carbohydrate chain is linked, LEP-12 isolated was digested LEP-3B to LEP-2, indicating that there is a Val-Lys peptide
with chymotrypsin, and a carbohydrate-containing peptide (position 157-158) between the two peptides. This linkage
was collected. The automatic sequencing of this peptide in- and amino acid sequences of Ch-6 and Ch-7 suggested that
dicated that itssequence is Thr-Val-Ser-Ala-Thr-Pro-X-Gly- LEP-15 links to LEP-4 which further links to LEP-3B. Ch-
Thr-Phe. The results of determination of the amino acid 12 overlapped LEP-2,LEP-1, and LEP-6. Thus the Ch-
composition was consistent with this sequence if X (position peptides overlapped LEP-peptides to give the complete amino
42) is asparticacid. Therefore it was concluded that theamino acid sequence from the amino-terminal aminoacid (Asp-1) to
acid at position 42 is asparagine to which a carbohydrate the carboxyl-terminal amino acid (Phe-191).
chain is linked. Connection between LEP-15 with LEP-4 with LEP-3B was
Attempt to overlap LEP-peptides was performed by diges- established by amino acid sequences of V-6 and V-10 peptides
tion of the modified miraculin with chymotrypsin. The results which wereobtained from digestion of the modified miraculin
6658 Amino Acid Sequence of Miraculin
TABLEI1
A m i n o acids compositions of peptides obtained from digestion S-carboxyamidomethylated
of miraculin with
chymotrypsin
Ch-8 Ch-7 Ch-6 Ch-5 Ch-9 Ch-18
Ch-12
Ch-16Ch-15Ch-14
Amino acids" Ch-4b
(95-100) (166-181) (133-143) (144-165) (1-22) (87-94)
(166-191) (46-64) (23-45) (65-86) (101-132)
residueslmolecule'
Asx(D/N) 1.9(2) 0.7(1) 3.8(5) 2.2(3) 2.3(3) 1.7(2) 4.8(5) 3.5(3)
Glx(E/Q) 1.3(1) 1.7(2) 1.0(1) 2.1(2) 2.2(2) 1.6(2) 2.9(3)
CYS(C) 0.9(1) 3234) 2.4(2)
0.8(1) 0.8(1)
Ser(S) 2.7(3) 1.1(1) 1.1(1) 0.7(1) 1.2(1)
0.9(1)
1.2(1) 2.4(2) 1.2(1) 1.3(1) 2.2(2)
GMG) 1.2(1) 3.6(3)2.4(2) 2.4(2) 1.1(1) 3.3(4) 3.8(5)
His(H) 0.8(1) 0.8(1)
Thr(T) 2.1(2) 1.2(1) 2.2(2) 1.1(1) 0.9(1) 3.2(4) 1.2(1) 2.5(3)
Ala(A) 0.9(1) 1.3(1) 1.2(1) 1.8(2) 1.1(1) 1.1(1)
Pro(P) 0.9(1) 1.3(1) 2.4(2) 2.4(2)
1.4(1) 1.1(1) 2.5(3) 2.1(2) 2.2(2) 2.4(2)
Arg(R) 2.9(3) 1.2(1) 2.4(2)
1.3(1) 2.6(3) 2.8(3) 1.2(1) 1.1(1) 1.3(1)
Tyr(Y) 1.0(1) 1.0(1) 2.0(2) 1.0(1) 1.9(2)
Val(V) 1.2(1) 4.4(4) 1.4(1) 1.4(1) 4.3(3) 3.5(3) 2.8(3) 2.1(2)
Met(M) 0.9(1)
Ile(1) 0.8(1) 0.7(1)1.3(1) 0.5(1) om) 0.9(1) 0.8(1) 1.9(2)
Leu(L) 1.7(2) 0.7(1) 1.6(2) 1.6(2) 0.9(1) 2.1(2) 1.3(1) 1.5(1)
Phe(F) 1.2(1) 1.4(1)1.5(2) 1.5(1) 3.6(4) 1.5(1) 2.8(3) 2.9(2)
Lys(K) 1.9(2) 2.5(3)1.0(1) 0.6(1) 3.3(3) 1.2(1) 1.4(1) 2.2(2)
TdW) ND(l)d NDU)
Total
(16) (6) (11) (22) (22) (26)
(8) (19) (23) (22) (32)
a One-letter symbols are given in parentheses.
* Name of peptides and thesequence position (in parentheses).
e Determined by amino acid analysis or from the sequence (in parentheses).

ND, not determined.

peptides
peptidesobtained
obtained

TY r
^""
"""""""-=-
c N
Q

peptides obtained Val

"
-*"_""_ *-

I
0.0
0 30 60 90 120
Time (min)
20 30 40 ! FIG.5. Amino acids released from the carboxyl terminusof
Retention Time ( m i n )
miraculin after digestion with carboxypeptidase A. Native
FIG.4. HPLC separation of peptides obtained from S. au- miraculin was digested by carboxypeptidase A in 0.1 M N-ethylmor-
reus Vi3 protease digestion of S-carboxyamidomethylated pholine acetate buffer of pH 8.0 at room temperature with an en-
miraculin. Separation was carried on a TSK-ODS-120T column. zyme:substrate ratio of 1:200 (w/w). 8 nmol of miraculin was used for
The peptides were eluted by a linear gradient of acetonitrile contain- the digestion. The ordinate represents moles of amino acids released/
ing 0.05% trifluoroacetic acid from 20 to 70% at a flow rate of 1ml/ mol of miraculin.
min for 1h. The eluted peptides were monitored by absorption at 210
nm. quence determined by automatic sequencing of Ch-12 and
LEP-6 peptides.
with S. aureus VS protease (Figs. 3 and 4). The complete sequence of 191 amino acid residues (Fig. 3)
Determination of the amino acid sequence from the car- is approximately in accordance with the amino acid compo-
boxyl-terminal was performed by digestion of native miraculin sition of the whole protein (TableI). The sum of the molecular
with carboxypeptidase A. The results areshown in Fig. 5. The weights of amino acid residues is 21,257. In Fig. 6, the hydro-
pathic profile (10) is plotted, which suggests that there are a
rate of release of amino acids is decreased in the order: number of hydrophobic domains in the miraculin molecule.
phenylalanine > tyrosine > valine. There was no other amino
acid released during 2 h digestion. These results indicate that DISCUSSION
the amino acid sequence from the carboxyl terminus of mir- In the present study, we have determined the complete
aculin is Phe-Tyr-Val which is in accordance with the se- sequence of 191 amino acid residues of miraculin. It is a single
 Amino Acid Sequence of Miraculin 6659
Residue I 200
40 80
160 120 residues is highly resistant to the protease digestion unless
No. dithlothreitol is applied to miraculin suggests that these half-
cystine residues form disulfide bonds.
The amino acid sequences of thaumatinand monellin,
which are sweet-tasting proteins, were already determined
r
n
+4 (12, 13). We looked for homology of amino acid sequence
between miraculin and these sweet-tastingproteins using
I E homology search program (IDEAS) developed by Kanehisa
(14), but therewas noparticular homology between miraculin
and the sweet proteins. This is consistent with the fact that
the antibody to miraculin did not exhibit cross-reactivity with
thaumatin (data not shown). However, high homology was
found between the sequences of miraculin and soybean trypsin
inhibitors A and C (Kunitz) was36.3% (the sequence of
miraculin from the amino terminusto 60th residue) and 51.1%
(the sequence of miraculin from 143rd to thecarboxyl termi-
nus). It is interesting to note that both proteins are produced
in plants and have similar molecular weights of about 20,000.
Acknowledgments-We are indebted to Professor Lloyd M.Beidler
of The Florida State University for supply of seeds of miracle fruit
and for constant encouragement. We also thank Professor Takeo Aso
FIG. 6. Hydropathic profile calculated by the method of of Yokohama National University who cultured miracle fruit. We
Kyte and Doolittle (10).The global average hydropathic index is thank Professor Noriko Takahashi of Nagoya City University for
indicated by the horizontal line. In the upper part, the location of valuable suggestions on carbohydrate chains of miraculin.
positive and negative charges along the polypeptide chain of miraculin
is represented. REFERENCES
1. Kurihara, K., and Beidler, L. M. (1968) Science 1 6 1 , 1241-1242
polypeptide chain with molecular weight of 21,257. Carbohy- 2. Brouwer, J. N., Van der Wel, H., Francke, A., and Henning, G.
drate content was as much as 13.9% (5), and hence the total J . (1968) Nature 220,373-374
calculated molecular weight is 24,600which is 8 8 % of the 3. Giroux, E. L., and Henkin, R. I. (1974) J.Agric. Food Chem. 22,
594-601
molecular weight (28,000) estimated by SDS-PAGE ( 5 ) .Ap- 4. Kurihara, Y., and Terasaki, S. (1982) Biochim.Biophys.Acta
parent molecular weight of a glycoprotein on SDS-PAGE is 719,444-449
often larger than the actual one (ll),and hence SDS-PAGE 5. Theerasilp, S., and Kurihara, Y. (1988) J. Biol. Chem. 2 6 3 ,
of miraculin seems to have given a larger molecular weight 11536-11539
than the actual one. Resistance of Lys-Thr at position 187- 6. Bidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L. (1984) J.
Chromatogr. 336,93-104
188 to lysyl endopeptidase may be due to the presence of the 7. Tsunasawa, S., Kondo, J., and Sakiyama, F. (1985) J. Biochern.
carbohydrate chain at Asn-186. Similar effect of the carbo- (Tokyo)9 7 , 701-704
hydrate chain on the proteolytic action was observed in car- 8. Ambler, R. P. (1967) Methods Enzymol. 1 1 , 155-166
boxypeptidase A digestion. 9. Francois, C., Marshall, R. D., and Neuberger, A. (1962) Biochem.
One characteristic of the miraculin structure is that the J. 83,335-341
10. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 1 5 7 , 105-132
amino-terminal half of the molecule is enriched in proline 11. Segrest, J. P., and Jackson, R. L. (1972) Methods Enzymol. 28,
residues; it contains 10 of 14 proline residues. Another char- 54-63
acteristic of miraculin structure is that 5 half-cystines of a 12. Frank, G., and Zuber, H. (1976) Hoppe-Seyler’s 2.Physiol. Chem.
total 7 half-cystines are located between positions 138 and 357,585-592
13. Iyengar, R. B., Smits, P., Van der Ouderaa, F., Van der Wel, H.,
159. That is, 2 half-cystine residues are located at positions van Brouwershaven, J., Ravestein, P., Richters, G., and Van
47 and 92, and 5 residues are at positions 138, 148, 152, 155, Wassenaar, P. D. (1976) Eur. J. Biochem. 9 6 , 193-204
and 159. The fact that the region containing 5 half-cystine 14. Kanehisa, M. (1982) Nucleic Acids Res. 10, 183-196