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Vol. 17 No. 6 CHINESE JOURNAL OF CHEMEIRY 1999
Primary structure of p-momorcharin , a ribosome-inactivating protein from the seeds
Momordica Charantia Linn ( Cucurbitaceae ) of
Abstrsct The complete amino acid sequence of ~momorcharh, a ribosome-inactivating p t e i n from the
seeds of Morn& &rrantia Linn ( Cucurbiteceae) has been determined. 'Ihis has been done by the sequence analysis of pepides obtained by enymatic digestion with win, chynmtq-psin and S . weus V8 protease, as well as by chemical cleavage with BNPSskatole. The protein consists of 249 amino acid residues containing one
asparape - linked sugar p u p attached to the site of Asn 5 1 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of hichosanthin and other r i h i n a c t i v % pmteins &om different species of plants shows a signircant homology with each other.
Regarding the similarity of their biological proprties, an active domain of thesz proteins has been predicted hen:.
Keymwds Momorcharin, ribommeinactivating protein (RIP), primary struchm, sequence comparison
Introduction
The chemistry of trichosanthin has been extensively studied because of its special pharmacological properties. ' This protein was isolated from the Chinese traditional drug Tianhuafen, the root tuber of Tri- chosanthes Kirilowii Maxim (Cucurbitaceae) . Trichosanthin induces abortion in labomtory animals and is effective for both mid-term abortion3 and the termination of early pregnancy4 in clinical trials in China. Its therapeutic value has been reported in patients with ectopic pregnancy, hydatidiform mole, invasive mole and choriocarcinoma. Very recently, trichosanthin has been found a RE' with RNA N-glycosidase activi-
Received November 17, 1998; accepted May 24, 1W. Roject supported by G m t s from the High Technolagy Development P m p m of China and from the National Natural Science Foundation of China (No. 29272074)
"he present address: Kovler Viral onodogv Lab, The Llniuersity of Chamgo ,910 E 58th Stmet Chrcago, IL 60637, U . S . A . E d : [email protected]
YE et d. Pnmary structure of p-momochar;l 659
ty6 and an anti-human immunod&ciency virus (anti-HW) agent.7 It inhibits HW-1 in vitro infection and replication and has been applied in clinical trials in the United States of America.
a- and p-momorcharin (a- and PMMC) are two glycoproteins isolated from the seeds of Mm& charantia (Bitter melon) and shown to have abortifacient a c t i ~ i t y . ~ ' ~ They appear to correspond to the previously reported proteins of Fraction I and 11, respectively, lo although the molecular weights and amino acid compositions of both a- and PMMC detedned in this study differ greatly hm the published data. Subsequent studies found that a- and PMMC share a great deal of the properties of trichosanthin. They have a similar molecular weight (29, OOO Da) , a strong basic PI value (9.10-9.40) , a similar confor- mation as shown by circular dichmism,lland they are also ribosome-inactivating proteins (RIP) with RNA N-glycosidase activity. 12*L3PMMC was also shown a very effective, nontmic inhibitor of human immun- odeficiency vim ( HW-1) . l4
Now, the amino acid sequence of a-MMC detemined by cDNA cloning as well as by sequencing of enzymatic peptides derived from the purified protein had been reported respectively, although there is a big diffenmx between these two reported results. In our previous paper, we r e p o d the crystal struc- ture of QMMC to an extent of 3. OA. l7 We also presented the complete amino acid sequence analysis of k MMC as well as the structure of its carbohydrate moiety on the 3rd SineFrench Symposium on Biologically Active Natural products in 1993.1&'b'19 In this paper, we describe the experimental details l e d q to the elucidation of the complete amino acid sequence of QMMC. In addition, a biological active domain of @ MMC is also proposed.
*
Materials and methods
TPCK-trypsin, TkK-chynmtrypin , and S . aureus V8 protease wem purchased from Sigma Chemi- cal Co., and DABITC h m F'lh Chemical Co., BNPS-skatole was synthesized accordmg to the reported procedurea in our laboratory. Au reagents used were of analytical grade and treated LuxoTding to the de- mands of protein sequencing. PMMC was prepared fmm the extract of the seeds of Momonlica CharMtia as previously ~ p o r t e d ' ~ and its homogeneity was controlled by SDSPAGE.
Enzymatic cleavage
The protein P M M C was digested with TPCK-trypsin as follows: the protein was denatured by dis- solving in 8.0 M urea and incubating for 10 min at 55 T , and then diluted with 0.2 M NI&HC@ b d e r
added in two portions over a 24 h period at 37T ( final trypsidprotein ratio of 1 : 25) . TLCK-chy- motrypsin digestion of PMMC was done by a procedure similar to that described above for trypsin diges- tion, except that the digestion was done for 1 h at a final enzyme/substrate ratio of 1 :50. TPCK-trypsin digestion of maleyl-QMMC (enzyme/substmte ratio of 1 : 50) was done in 0.2 M N-methylmoIphorine ac- etate buffer (pH 8.1 ) , at 379: for 24 h and S. m u s V8 protease digestion of succinyl-QMMC (en-
andwatertoafinalconcentdonof2.0M ureaandO.1 M W H C Q (pH8.1) . TPCK-tryp' mwas
660 &nese Journal of Chemistry Vol. 17 No. 6 1999
zyme/substrate ratio of 1 :U) was performed in 0.1 M mHC@ buffer (pH 8. 1) , at 37°C for 45 h.
BNPS-skatole cleavage
BNPSskatole cleavage at TIP of PMMC was done accoding to the pmedure reported.21 p-MMC was dlssolved in 80% fomic acid containing Tyr (5.0 mg/mL), and equal amount of BNF'S-skatole dis- solved in glacial acetic acid was then added. The mixture was allowed to react for 24 h at mom tempera- ture in the dark. After two-fold dilution with water, it was extracted with benzene until the extracts were colorless. The mixture of peptides in the aqueous layer was then lyophilized.
Peptide pwfiatwn
Mixtures of peptides derived firm various enzymatic digestions or chemical cleavage were separated by reverse-phase hgh performance liquid chmmatography (Rp-HPLC) on columns of Spherisorb S5 ODs2 (10 x 250 mm), Vydac C-18 (30 nm, 4.6 x 250 mm) or Delta Pak C-4(30 nm, 4.6 x 150 mm) using an acetonitrile d e n t in 0.1 % aqueous trifluoroacetic acid, and effluents were monitored by absorption at W 230 nm. In some cases the mixture of peptides derived from the -tic digestion of maleyl-/%MMC was prechtographed on a column of Sephadex 625 in 5% acetic acid. The purification of peptides from chemical degradation of PMMC was also performed by chromatography on a column of Sephadex G 50.
. h n o acld compositwn and sequence d y s i s
Protein ( 1 . 0 nmol) or peptide( 5.0 nmol) were hydrolyzed in 5.7 N HCl (containing 0.02% phe- nol) at 1109: for 24 h in evacuated sealed tubes. ?he amino acids were analyzed on a LKB4400 amino acid analyzer. The amino acid composition of /3-MMC was also checked by analyzing on a Beckman 6300 amino acid analyzer. Trichosanthin was used as a reference for calibration.
Tryptophan was measured photometrically.p Amino acid sequences of peptides were done by the 4- N , N'-dimethylaminoazobenzene4isothiocyanate/phenylisothiocyanate ( DAl3WPIK ) double cou- pling methodzl and, for large peptides, d y s e d with an applied biosystem model 470A gas-phase. protein sequencer.
The C-terminal sequence of PMMC was determined by Dr Y u - w E. PANG, Hoi5nann-h Roche INC. in U . S . A .
Molecular might determination ofpeptides by W I - T O F - M S
The MALDI-TOF-MS determination of the molecular weight was carried out on the b t o s Kompact MALDI 1 instrument by Mr. Lei Zeng, Shimadzu company in Beijing.
YE e t d . Pnmary structure of p-momocharin 66 1
Resultsand discussion
PWLficatwn and sequence analysis of t w t k peptides fiom gMMC
The try-ptic digestion of p-MMC in the buffer system of 0.1 M m H C @ ( p H 8.1, 2.0 M urea) was separated into a soluble fraction (Ts fraction) and precipitate( Tp fraction) via centdugation. The Ts fraction was first separated by RP-HPLC on a Spherisorb s5 ODs2 column as depicted in Fig. 1. "he res- olution of Ts fraction gave a number of peaks, each of which was further purified by mhmmatography on a Vydac C- 18 column using the same solvent system. Rechmmatography of the peaks revealed that most of them were not pure and could be further separated into two or more peaks as indicated in Fig. 1 (peaks yielding two or more peptides upon mhmmatography were assigned two or more numbers). A total of 23 peptides were obtained from the Ts fraction. On the other hand, the Tp fraction (Hicult to dissolve in normal buffers) was dissolved in the mixed solvent of 20% formic acid, 20% acetonitde and water, and then purified by Rp-HF'K on a Vydac C-18 column (for pmtein and large peptide) . As shown in Fig. 2, the Tp fraction gave 16 peptides, of which three were new (Tp-11, Tp-14, Tp-16) and were not found or detected in the Ts &tion. The amino acid compositions of m t of the contributive tryptic peptides were listed in Table 1. Ihe amino acid sequence of these tryptic peptides were also listed in Table 2.
17 I
0 10 20 30 40 50
Time (min)
Flg. 1 High performance liquid chrwnatography of the peptide mixhm (super clear 6actior1, Ts &tion) derived fmm tryptic digestion of PMhiC. Column: Spherisorb s5 ODs (10 x 250 mm). Eluents: buffer A , 0.1% TFA in water; bder B, 0.05 % WA, 20% water in acetonitrile. Gradient: 0-40-50-55-60 min , 10-70- 100- 100-10 B%. Flow rate: 2.0 mvmin. The hh W&S monitored at 230 ~IIII, 0.1 ALJFS.
PwifiCatwn and sequence analysis of chymotrptic peptides of PMMC
a-Chymotryptic digestion of p-MMC was also separated into two fractions, the supematant and the precipitate fraction (Cs and Cp fraction) by centrifugation. Purification of Cs fraction by RF-HPLC gave a much more complicated resolution profile than that of Ts fraction (as indicated in Fig.3). Each of the- se+ was further p d i e d with a Vydac C-18 column and almost all of them gave two or more peaks.
662 Chinese Journal of Chemistry Vol. 17 No. 6 1999
Table 1 Amino acid composition of tTyptic peptides and of the selected S. aureus V8 protease peptide
from PMMC
Ts-lob Ts-3c TPII-14 TPIII-17 Ts-6b TPD-12 Ts-15 .enin0 acid
ASP 1.60(3)" 2.23(2) 1.11(1) 1.43(1) 2.10(2) 'Ihr 1.65(2) 1.57(2) 0.98(1) 0.97(1) 2.36(3) Ser 1.17(1) 2.53(3) 1.40(2) 0.%(1) 0.%(1) Glu 1.79(2) 1.56(2) 1.98(2) 2.02(2) Pro 0,74(1) 2.83(2) 3.03(2) 1.90(2) 0.82(1)
Ala 1.67(2) 0.91(1) 1.11(1) 1.03(1) 1.83(2) GlY 0.97(1) 1.24(1)
Val 1.12(1) l . M ( l ) 0.99(1) Ile 1.92(2) l.Os(1) 1.06(1) Leu 1.21(1) 2.30(2) 1.34(1) l.PQ(2) Tyr i . i i ( 1 ) 1.50(2) 1.60(2) 1.38(1) 0.80(1) 1.55(2)
His 0.97(1) h e 1.26(1) 1.67(2) 1.03(1)
LYS l.Oo(1) l.Oo(1) 1.05(1) 1.35( 1) 1.88(2)
Total 12 4 15 14 7 15 19 position 1-12 13-16 31-45 7a9 i 85-91 85-99 10@118
Arg 0.87(1) 0.91(1)
pept No. T1 T2 Ts+m l9+Tlo T-10 T10+T11 T12
'PI- 1 1 Tp16 TPIII-9 Ts-3a Tp14 Ts-9a Ts-6a AmuK,
Acid
ASP 1 .%(2) 2.71(3) 2.81(3) 0.89(1) Ihr 2.55(3) 2.43(4) 1.41( 1) SeT 2.54(4) 2.24(3) Glu 2.19(2) 3.87(4) 1.17(1) 2.01(2) 2.40(3)
GlY 1.15(1) 1.15(1) 1 .63( 2) Ala 2.02(2) 5.12(7) l.OO(1) 2.51(3) 0.%(1)
Ile 1.67(2) 2.65(3) 0.93(1) 1.37(2) 0.92(1) Leu 2.08(2) 4.54(7) 2.41(3) 1.04(1)
Phe 1.55(1) 1.07(1) 1.14(1) l.OO(1) 1.12(1) His 0.95( 1) 0.95( 1) LYS 2.Oo(2) 1.21(1) O.PQ(1) l.Oo(1) 1.87(2) 1.01(1)
Pro (-1 (1) 1.74(2) (-)(1)
Val 1.46(1) 0.!38(1) 1.10(1)
Tyr 1.57(2) 1.92(2) 0.99(1) 0.94(1)
Arg 0.85(1) l.Oo(1) 0.89(1) 1 .Oo( 1) Trp (-)(1)
Total 21 41 6 4 24 11 2 position 100-120 121-161 162-167 168-171 172-195 196-206 207-208 pept No. TI2 + T13 T14 + Tl5 T16 + T17 T18 T19 T-20 T21
YE et d. primary structure of pmOm0char;l 663
Amino acid
TPI-5 Ts-lob Ts-1 sv-23-3 sv-53
Asp 1.98( 2) 0.93( 1) 1.56(2) 3.23(3)
Ser 0.89( 1) 0.93( 1) 0.80(1) Glu 1.24(1) 1.15(1) 1.16(1) 2.30(2) Ro . 1 .76( 2) (-)(1)
?hr 1.23( 1) 0.99(1) 1.03(1)
GlY 1.13( 1) Ala 1 .72( 2) 1.25(2) 2.43 (3) Val 1.07(1) 2.14( 2) Xle 0.98( 1) 0.92(1) 1.82(2) Leu 1.17( 1) 3.00(3) 2.30(2) Tyr 0.94(1) 0.86(1) Phe l.oo(1) 1.41( 1) His 1.16(1) LYS 1.27( 1) 0.94(1) 1.90(2) Arg 0.90(1) 1.15(1) 1.00(1) 1.20(1)
Total 12 6 7 8 23
pept' No. 'I22 T25 126 T15-Tl6-Tl7 T18-Tl9a. "Values in parentheses indicate residues deduced firm sequence.
167- 189 position 209-220 237-242 243-249 159-166
60 50 40 30 20 10
Time (min)
a. 2 High pedomce liquid chmmsmgqhy of the peptide mixture (precipitate fraction, Tp fraction) derived from tryptic digestion of PMMC. column: Vydac c N ( 4 . 6 ~ 2.50 mm). Eluents: bd€er A: 0.1% TFA in water, M e r B: 0.05% TFA, 20% water in acetonitrile. Gradient: 0-3047-50-60-65 min, 0-30-80.100- 100-0 B% . Flow rate: 1 .O mVmin. The absorbance was monitored at 230 nm, 0.20 AUFS.
664 Chinese Journal of Chemistry Vol. 17 No. 6 1999
Table 2 Amino acid sequence analysis of tqptic peptides from PMMC
seguence Peptide
peak Position
T~yptic peptide No.
Ts-lob Ts3c TPIII- 18 TPII-17' TPH- 14 T p l l TPm-17 Ts-6b Ts-2 wn-12' Ts- 15 wi-11' Tp16'
Ts-Sa Ts-5b wm-9 Ts-3a Tp14' T p l Ts6s 771-5 * Tsl6a Tp3 * Ts-3b T s l l b
1-12 13-16 13-22 23-38 3145 47- 78-91 85-91 97-99 85-99 101-118 100-120
121-161 162-163 164-167 162-167 168-171 172- 195 1%% 207-208 209-218 207-218 221-232 233-236 237-242
TI T2 n+T3 T4+T5 n+Tfi T7 'I9 + TI0 TI 0 T11 T10+T11 TI 2 T12 + T13
T14 + TI5 T16 .
T17 T16 + TI7 T18 T19 m 721 'I22 T21+l22 m T24 125
T s l * ASTADEN 243-249 'I26
* Indicates peptides also analysed with ABI 470A d l p p h e s e sequencer. Ts and Tp r e p e n t the t3rptic peptides obtained fmn the soluble and precipitate fraction respectively. PI, PIl and TPID are p u p s of contributory arginine containing peptides finm hyptic digest of maleyl-PMMC after prelirmnary fractionation on a Sephadex 625 column. Low-
ercase assignment denote less than full confidence. x: no assignment.
From the Cs fraction, a total of 34 chymotryptic peptides were obtained. 'Ihe Cp fraction, dissolved in a solvent of 20% formic acid, 20% acetonitrile and water, was sepamted on a Vydac C-18 column (Fig. 4 ) . It gave 43 peaks, of which one was new ( C p 4 ) . The amino acid compositions of those contributive peptides were given in Table 3 , and the amino acid sequences of these peptides were listed in Table 4. Sequence analysis of the a-chymotryptic peptides thus identified 245 residues comprising 98.4% of p- MMC ,
YE et al. Primary StluCillIe of p-momochar;l 665
Table 3 Amino acid composition of chymotrypzic peptides from PMMC
Thr 1.73(3) 0.77(1) 0.89(1) l.Oo(1)
Glu 1.36(1) Pro 1.01(1)
GlY
ser 0.56(1) 1.92(3) 0.99(1)
Ah 1.80(2) 1.17( 1) Val l.Oo(1) 1.61(2)
Leu l.Oo(1) 1.37(2) 1.88(3) Ile l.Oo(1) l.Oo(1) l.Oo(1) l.Oo(1)
Tyr Phe 1.21(1) 1 .70( 2) 0.99(1)
0.78(1) 1.02(1) l.Oo(1) l.Oo(1)
His LYS 0.73(1) 0.83(1) Arg 1.78(2)
Total 4 10 7 6 9 8 4 position 1-4 5- 14 15-21 33-38 39-47 4-55 71-74 Pep No. c1 Q c3 (15 c6 CI c10
Cs-16a Cs-lOa G l 8 a Csdb Cs-13a G15a G17a Amino
acid
ASP 1.02(1) 2.06(2) 1.24(1) nu Ser Glu Pro
GlY Ala Val ne
Leu
Tyr Phe His LYS
0.98(1) 1.43(2) 0.95(1) 0.81(1)
2.35( 2) 2.54(2) 0.98(1)
1.11(1) 1 .Oo(1)
l.Oo(1) 1.06(1) 1.04(1)
0.89(1) 1.02(1) 1.06(1) 0.88(1) 1.09(1)
1.90(2) 1.55(2)
2.61(2) 1.13(1)
1.42(1) 1.80(2) 2.27(3)
1.12(1)
1.23(1) 1.83(2) 1.20(1) 1.67(2)
l.Oo(l) 0.95(1) 1.54(2)
1.66(2) (-)(1)
2.14(2) 1.13( I )
1.85(2) l.Oo(1) 0.88( 1) O.%( 1) 1.07(1) l.Oo(1) 0.89(1)
1.54(2) 1.32(1)
0.93( 1) 1.49(2)
l.Oo(1) 0.84(1) Arg 1.66(2) l.Oo(1) Total 8 9 10 12 12 12 10
position 75-82 83-91 %lo5 16117 139-150 151-162 163-172
Pep No. c11 c12 C14 C15 C17 C18 C19
666 Chinese Journal of Chemistry Vol. 17 No. 6 1999
continued
Cs-12b Cs8b G l 9 b G16b cs-3 Amino acid
ASP 2.60(3) 0.95 ( 1 ) 2.28(2) 3.38(4) 2.14(3)
Cs-20
Thr 0.90(1) 0.95( 1 ) 1.08(1) 0.94(1) Ser 0.68(1) 0.65(1) 0.%(1) 1.51(2)
pro (-)(I) ( * 1 0 )
Ah 1.89(2) 1.16(1) 1.66(2) Val 1.09(1) 0.89( 1) 3.02(4)
Leu 1.83(2) 1.#(1) 1.43(1) 1.81(2) l.Oo(1)
TYr Phe 1.15(1) 1.04(1) 0.92(1) 0.90(1)
Glu 2.19(2) 1.26(1) 2.16(2) 1.30(1) 1.32(1) 1.13( 1)
GlY 1.90(2) 1.48(1) 1.14(1)
Ile 1.59(2) l.OO(1) 1.25(1) 0.98(1)
His LYS l.Oo(1) 0.?2(1) 0.47(1) 0.79(1) 1 .82( 2) Arg 1.69(2) l.oo(1)
Trp (-)(1) Total 18 5 9 14 17 11 position 173-190 194-198 199-207 208-221 222-238 239-249
pep No. c20 C2l c22 c23 a4 c25
Values in parentheses indicate residues deduced fmm sequence.
Table 4 Sequence analysis of selected chymotryptic peptides and S. aureus V8 protease peptides from PMMC
Peptide
peak Sequence GYmoVPtic Pasition
peptide No.
G l o b DVNF G 8 C DLSTATAKTY
1-4 c1 5-14 c2
C15b TKFIEDF 15-21 0 CPIII- 18 RATlpFsHKw 22-32 CA Gs-l7b D P U Y 33-38 c5 G l 2 a SnSDSRFW 39-47 c6 Gila' HI" LEY 48-55 c7 CP40' SEW3WU" LTSYAYETISVAIDVINVY 39-74 C6+ 7 + 8 + 9 + 10
cs-7 W A Y 71-74 c10 C+16a RTRDVSYF 75-82 c11 Cs-lOa r n P E A Y 83-91 c12 G18a KGTFWTLPY %lo5 C14 Cs-6b xmJ4J4H 106-117 C15
WAX
YE et d. Primary structure of p-momocharin 667 ~~~~ ~ ~
continued
Cp38 mNIx 118-? C16 Cs-13a YYNAQSAPSALL 139-150 C17
Cs-17a KYIERHVAKY 163-172 C19 Cs-l5a WOTISEAARF 151-162 C18
cs-20 V A T N F K P ” Q W 173-190 c20 Cs-19c W Q W 191 - 198 (221
Cs-8b UQNQCGKF 199-207 a2 Cs-19b - m m 208-221 a Cs-16b QvnvvDsDWKGNWL 222-238 m Cs-3 LN!3RAsrADm 239249 (25 sv-26 VTlWYVVAYRlRD 66-78 ClCLC11 sv-23-3 AARFKYIE 159166 T15-T16-T17 sv-53 RHVAKYVAX 167-? T18-Tl9
Glycopeptides which carbohydrate attached residue Asn was d & d h the amino acid composition analysis. Indicates peptides also analyzed with ABI 470A model gaqhaee sequencer. x: no assignment.
60 50 40 30 20 10 0
Time (min)
Flg. 3 High performance liquid chromatography of the peptide mixture (super clear fraction, Cs hction) derived fmm chymotrypsin digestion of PMMC. column: Spherisorf, s5 ODs (10 x whnm). Eluents: Mer A: 0 .1% “FA in water; buffer B: 0.05% TFA, 20% water in acetonitrile. Gradient: 0-5-50-55-58-62 min,
0-15-7CLlOO-lWO B% . Flow rate: 2.0 mVmin.’lhe &O&IKX ~ 8 s monitored at 230 IIIII, 0.30 Am.
668 Chinese J o d of Chemistry Vol. 17 No. 6 1999
41 I
60 50 40 30 20 0
Time (min)
Fig. 4 High performance liquid ciuwmtop$y of the peplide mixture (precipitate fraction, Cp fraction) derived fromchymobypsindigestionof~MMC. Column: V y d a c C - 1 8 ( 4 . 6 ~ 2 5 O m m ) . Eluents: W e r A : 0.1% "FA in water, M e r B: 0.05% TFA, 20% water in acetonitrile. Gradient: 0-S58-61 min, 0-1001WO B% . Flow rate: 1.0 U r n i n . The hrbance was monitored at 230 m, 0.25 AUFS.
Isolation of arginine-wniuining tryptic peptides and S . aweus V 8 protease peptides j b m succinylated p- MMC
Some of the alignments of the tryptic or chymotryptic peptides obtained above were also confirmed by analysis of tryptic peptides from rnaleyl-p-MMC or peptides derived from S. w e u s V8 protease digest of succinyl-FMMC. This was also served to double check the peptide sequence which had been analysed before. Armno acid composition of some new arginine-containing tryptic peptides contributed greatly to the completion of amino acid sequence of PMMC were also included in Table 1. These include TPn-14, TPIII-17, TPII-12, TPI-11, WID-9, and TPI-5. Meanwhile, the sequence of S . aluell~ V8 pmtease peptide SV-26, SV-23-3, and SV-53 (Table 4) confirmed the linkage of C10-C11 , T15-Tl6-Tl7, and T18-Tl9, respectively.
YE et al. primary structure of pmomocharin 669
Isolation and partid amino acid sequenoes of BNPS-skatolefragmats of PMMC
Based on the sequence analysis of tryptic and chymotryptic peptides of P-MMC, a preliminarily amino acid sequence of PMMC was proposed. However, as shown in Fig. 8, the overlaps between pep- tides of T15-Tl6, T18-Tl9, T2O-T21, T22-T23 and T24-T25 remain to be further strengthened. Consid- ering no Met or Cys but one Trp residue contained in PMMC, BNPS-skatole cleavage at TIP in P-MMC was p e r f o d . Following BNPS-skatole cleavage of PMMC, the reaction mixture was fractionated on Sephadex G50 ( Fig. 5 ) . Since cleavage at tryptophan was incomplete, the major peak fraction of Fig. 5 contained QMMC which had notbeen cleavaged. SDS-PAGE analysis of the fraction compondmg to the shoulder of the main peak (solid bar in Fig.5) indicate the presence of a predominant component of the large fragment having the N-terminal sequence of QMMC. Peak IN, although its absorbance at 280 nm was very weak, was corresponding to the small C-terminal fragment (Fs) resulting from chemical degm- dation of P-MMC at Trp and presented just one band or one peak in SDS-PAGE and RP-HPLC analysis respectively (data not shown) . The small C-terminal hapen t (Fs) can also be directly purified fmm the degradation mivture using RP-HPLC (Fig. 6) . The amino acid composition of Fs was included in Table 5 . Sequencing of Fs up to the 46th step by protein sequencer demonstmted the linkage of T19-T20-"21-"22- "23, and the amino acid composition analysis of Fs agreed very well to the sequence we had propced . The molecular weight of the Fs was determined to be 6443 by IMALDI-TOF-MS (Fig. 7) , while the cal- culated value is 6442.9 (Table 5) .
The c-terminal amino acid of pMMc
The C-terminal amino acid of P-MMC was determined to be -E-N on the C-terminal amino acid se- quencer.
Characterization of &MMC
The protein consists of 249 amino acid residues and has a calculated molecular weight of 28453 Da, without addition of the carbohydrate, as given in Table 5 . The amino acid composition of the protein, de- rived fmm the sequence, agrees well with that found by amino acid analysis (Table 5 ) .
In P-MMC , h n i n e , alanine, and leucine a . ~ the most abundant residues, and a number of basic amino acids, lysine, arginine and histidine (31 residues totally) compared with 22 acidic residues, as- partic acid and glutamic acid, account for the basic character of PMMC: its PI value of 9 . 3 is slightly higher than that of a - m o m o r c ~ n (PI: 9.1 ) .
The amino acid sequence of PMMC includes one potential asparagine-linked glywsylation site with the sequence of Asn-h-Thr (position 51 to 53) . 'Ihe oligosaccharide chain containing one or two amino acids at its reducing terminal has been obtained from enzymatic digestion of PMMC. Recent studies showed that it consists of six monosaccharides (2 glumsamines, 2 mannoses, 1 fucose and 1 xylose) and has a mass of 1,009 Da. The p r e m result of the oligosaccharide structure analysis by NMR had been presented on the 3rd Sino-French Symposium on the Chemistry of Natural Products, and the detailed result will be described elsewhere.
Chinese Joumal of Chemistry Vol. 17 No. 6 1999 -
670
TaMe 5 Amino acid cornpition of PMMC and the Fs fiagment
ARliIlO
acid Fs
fragment
ASP Thr Ser Glu Ro Gly Ala
Val Ile
L&U
Tyr Phe His
28.9( 29) 21.8(22) 16.7( 19) 24.3( 19) 9.6( 10) 8.6(7)
23.1(23) 16.2( 16) 19.1(18)
25.3(25) 16.0( 15) 16.3(14) 4.1(3)
9.5(10)
3.2(3) 4.6(5) 7.4(6)
(-)(2) 5.1(4) 5.2(4) 5.0(5) 3.4(3) 5.9(6)
3 . 4 0 )
LYS 16.4( 16) 4.6(5) Arg 11.7( 12) 3.1(3) TrP 0.89(1)
Total 249 59 Mr(calcd. ) 28,453 Da 6442.9 Da
Measured photometrically.
OD 230 nrn 'I OD 280 nm
c 1.50
1 .oo
0.50
0 Tube number (5 rnL/per tube) Time ( x 10rnin)
Fig. 5 separation of peptide miKture derived froin F&. 6 Hi& performan~e liguid c w h y of pep- BNPSskatole dejpdahon of PMMC on tide mixture derived fnnn BNPSskatole degrada- Sephadex G50. Equilibrated and eluted tion of PMMC. Column: Delta pak GI (4.6 x with 5 % HOAC (containing 8 M urea). 150 mm). Buffer A: 0.1% "FA in water; B: Column: 2 x 150 cm. Flow rate: 5.0 mV 0.05% "FA, 20% water in acetonitrile. Flow 30 min. rate: 1 . 0 mVmin. 'Ihe atmrbance was moni-
tored at 230 nm, 0.3 AUFS.
YE e t d . prima^^ structure of p-mmachar;n 67 1
40 :
30 :
20
10-
N O - 3
Calulated 6442.9 M + 6443
, I I . , I . . . , . - . . , . * 2000 4000 6000 8000 1 0000 12000 14000
Fig. 7 MALDI-"IF-MS of the peptide Fs fimn BNPSskatole deigestion of PMMC.
20 40
DVNFDLSTATAKTYTKFEDFRATLPFSHKVYDIPLLYSTI SDSRRF I LL . . . . . . . . , . , . > F b T 7 ,
TI T2 T3 T4 T5+l6 ---+-----,--------* c 4 c 5 C6+7+8+9+10 c',"c'---+
TI-- - r
60 80 100
NLTSY AYETISVAIDVTNVWVAY RTRDVS YFFKESPPEAYNILFKGTRK .__-
T8 T9+T I0
TLO+TI 1 b
c14 a- - C6+7+8+9+10
SV-26 ' + 120 140
ITLPYTGNYENLQTAAHK I RENIDLGLPALKSAllTLFYYNAQSAPSALL TI3 T14+T15 -- TI2
v r T12+T13
b c17 L ! ! + c - - . + C15 C16
160 180 200
VLIQTTSEAARFKYIERHVAKYVATNFKF'NLAI ISLENQWSAJSKQI FLA --- +a L A +--
A
T14+T15 T16+T17 TI8 TI9
C20 CZ1
SV-23-3 sv-53 -- 220 240
QNQGGKFRNPVDLIKPTGQRFQVTNVDSDVVKGNIK LLLNSRASTADEN ---t----------*---*-------*-
T20 T21 +T22 T23 T24 T2s T26
b b C 2 5 c22 a 3 a 4 L
~
Fs , Fs . F s . Fs , . , , . Fig. 8 Complete amino acid sequence of PMMC. Peptides obtained after cleavage with bypsin, a-chymotrypsin, S
aureus V8 pmtease and BNPSskatole are indicated by T, C, SV and F respectively. Solid m w s indicate that the sequencing p d e d unambiguously and completely. Open arrows indicate that the peptides not completely sequenced and the remaining sequence (dashed line) are from that of overlapping peptides.
67 2 Chinese Journal of Chemistry Vol. 17 No. 6 1999
Amino acid sequence of PMMC and sequence comparison
The complete amino acid sequence of p-MMC was shown in Fig. 8. The sequence of QMMC had been compared with those of trichosanthin as well as a-momorcharin.2L’ This comparison revealed a very high homogeneity between these three ribosome-inactivating proteins. Further comparison of QMMC with ricin A-chain , abrin A-chain, barley protein synthesis inhibitor, mirabilis, pokeweed antivid protein- seeds, saporin SO-6, as well as trichosanthin and a-momorcharin found seven absolutely conserved amino acid residues, they are: 14Y, 17F, 22R, 70Y, 158E, 161R, 19OW in P-MMC. Moreover, there are seventeen identical amino acid residues when the sequences of ten out of eleven RIP were compared, these are: 52L, 74Y, 107G, 109Y, 112L, 116A, 120R, 126G, 130L, 150L, 1544, 159A, 162F, 1651, 187E, 188N, 194s in QMMC. It is very clear that: (1) The proteins from Cucuhitaceae such as luffin-a and b, a- and PMMC, and trichosanthin show much higher sequence homology with each other than thm from other plant species. (2) A very conserved domain was found in the peptide segment from 109G to 205N, in h c h 15 amino acid residues revealed to be conserved in 10 RIP’S compared. It is worthwhile to note that in this region most amino acid residues are on the surface of the molecule and 120R, 158E, 161R, 187E and 19OW areclustered together around the pmposed active site cleft in the three-dimensional crystal structure of QMMC as well as trichosanthin. Therefore, we propose that the 109-205 peptide segment of P-MMC is a potential active domain and the synthesis of it with the genetic engineering method is still in progress.
AbbreViationS BNPS-skatole , 2-( 2-nitrophenyl-sulfenyl)-3-methyl-3-bromo-indolenine; DABlTC , 4- N , N-dunethylarmnoazobenzene-4’-isothi~y~e; P I E , phenylisothiocyanate; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis ; TPCK, tosyl-l-phenylalanine chloromethyl ketone.
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