Btochrmrco et Brophywa Acta 1088 (1991) 311-314 51 1991 Ekevier Science Publishers B.V. 01674781/91/SO3.50 ADONIS 0167478191000920
BBAEXP 90208
BBA Report - Short Sequence-Paper
Cloning of the cDNA of a-momorcharin: a ribosome inactivating protein Walter K.K. Ho’, S.C. Liu ‘, P.C. Shaw ‘, H.W. Yeung ‘, T.B. Ng ’ and W.Y. Chan ’ ’
The Biotechnology Laboratory and Deparrment o/ Btochemtstry, Chrnese Unrrwsrty of Hong Kong, Sham. .V. T IHwxg Kong) and ’ Oklahama Medrcal Research Fmmdatron. Oklahoma Ctty. OK flJ S.A.) (Received 23 October 1990)
Key words: cDNA: Momorcharin;
Ribowme mrccitia:mg protem. Tnchosanthm
Using a hgtll cDNA library constructed from the seeds of the bitter melon (Momordiea clu~ru&z~, we have obtained a full length cDNA containing the entire seqoence of a-momorcharin by immunowree Ring. I iw length of this cDNA is 1044 basepain long and it consists of an open reading frame coding for a polypeptide of 286 amino acids. The first 23 residues of this polypeptide probably code for a signal sequenuz. ?he N-termirrsl sequence of the deduced protein is exactly identical to that determined by peptide sequencing. The seqwnce identity between a-momorcharin and other ribosome inactivating proteins, such as trichosanthii and ricin A chain, is high, i.e., 3443%. Examination of the predicted secondary struchue of I-momorcharin and trichosanthin indicates that these proteins have regions of high strwtt4 similarities and this may account for the common biofqical activities that tbey share, viz, abwtificant, inununosuppressive, antitumor and inhibi*ion of HIV-l. The seeds of the bitter melon (Momordica churuntiu) are used in the Orient as a source of medicinal ingredients. In recent years, a number of biologically active proteins have been isolated from these seeds and most of them have been demonstrated to have ribosome inactivating property [1.2]. Our group has purified and characterized two basic proteins, a- and p-momorcharin (aMMC, /3MMC), from these seeds 13) and have demonstrated that they possess biological activities which can account for some of the medicinal value of the bitter melon. These activities include the induction of mid-term abortion [4,5], inhibition of tumor growth (Leung, K.N., unpublished results) and suppression of immune response 16). Whether these eiiects are directly related to the ribosome inactivating property is stilt uncertain. Recently, McGrath et al. [7] have shown that trichosanthin (TCS). a protein isolated from the root tubers of Trichosanthes kirilowii, can effectively inhibit
Abbreviations: MMC, momorcharin; TCS. crichosamhin: RIP. r&osome inactivating pro~tin; HIV, human imrmuwxehciency virus: IPTG, isopropyl-&~thiogalactopyranoside; X-gal. 5-hromo-khloro3-indolyl-/&~galactoside. Correspondence: W.KK. Ho. Department of Biochenkistry, Chmar University of Hong Kong. Shatin, N.T.. Hong Kong
the replication of type I human immunodeficien;y virus (HIV-l) in acuteely infected lymphoblastoid cells or chronically infected macrophages. As ICS, MMC and other ribosome inactivating proteins (RIPS: e.g., ticin A chain) are structurally related, it is not unexpected that they all have been subsequently shown to inhibit the propagation of HIV-l [g]. In spite of this generalization, the dose of these proteins required for inhibition of protein synthesis is significantly higher than that for inhibiting HIV-1 propagation ]7]. Thus, there may be other structural features of these proteins which are responsible for the inactivation of this type of wr(s,. in order to gain a better understanding on the structurefunction relationship of TCS and aMMC. we have cloned their cDNAs and deduced their amino acid sequences. This report deals with the results on aMMC. The amino acid sequence of the N-terminal residues of aMMC has been determined [9]. Using this information, we have attempted to design oligonucleotide probes for hybridization screening. but the codons for the N-terminal sequence were too degenera eti -Id a large number of oligonucleotides had to L * synthesized to cover all the permutations. On the other hand, aMMC is a very immunogenic protein and we were able to raise a number of high titer antisera against it. Therefore, we constructed a cDNA library from mRNA isolated from fresh seeds of the bitter melon (lO,ll] in the expression vector. Xgtll, for immunoscrcening (12.13).
312
P
E
E
mmc118
E
nunc41
I
x
P
I
I
780
bp
I E
1044
bp
I I
II
e .
.
.
Fig. 1. Alignment of various aMMC cDNA clone:. The relationships of the cDNA scquenw of clones MMCll. 41 and 118 are as indicated. The lowet part of the figure shows the sequencmg strategy. P. Pcrl; E, f&RI; X. Xholl.
Altogether. six positive clones were isolated in the first round of screening. Their insert size ranged from about 0.65 kb up to 1.27 kb. Upon rescreening by SDS-PAGE and Western blotting using an affinity purified ‘251-labeIed-anti-aMMC antibody, three clones, MMCI 1 (1.27 kb). MMC41 (0.78 kb) and MMC42 (0.78 kb), gave strong positive signals. By restriction mapping, clones MMC41 and 42 were identical. To facilitate sequencing, the inserts from MMCll and MMC41 were subcloned into pUCl8. The nucleotide sequence of MMCll can be divided into two fragments of 670 bp and 600 bp long by EcoRI digestion (Fig. 1). The longer fragment has a sequence identical to that of MMC41. Examination of the MMC41 sequence reveal-d :Zsi i, has a poly(A) tail and a polyadenylation signal, AATAAA, 31 bp upstream. These features were not found in the 670 bp fragment of MMCll. In order tc deteimine the identity of the nucleotide sequences of these -tones, a homology search in the Genebank Database was performed. The short fragment (600 bp) of MMCll was found to have some homology with the SS ribosomal RNA. No significant homology with sequences in the database was found to associate with the common sequence of MMCll and MMC41. 5’ upstream from the polyadenylation signal of the common sequence, several termination codons were noticed. Starting with one of these codons located at 123 bp 5’ to the AATAAA signal, we were able to back translate an uninterrupted polypeptide all the way to the 5’ end at the cloning site. Since the length of this peptide is only 186 amino acids and its nucleotide sequence does not encode an initiation codon, the common sequence of MMCll and MMC41 probably only contained a partial cDNA. The N-terminal sequence of TCS and aMMC have been shown to be highly homoiogous to each other. Since the primary structure of TCS is known, we compared it with the translated sequence
of our partial cDNA. They showed an identity of greater th.m 60%. The alignment of the two amino acid squences indicated that the partial cDNA ( M M C 4 1 ) probab!y codes for only two-thirds of the entire aMMC sequence. To obtain the full sequence of the uMMC cDNA, we prepared a 120 bp fsrI/&-oR1 fragment from the MMC41 insert (Fig. 1) and used it to reprobe the library. Altogether, over 250000 plaques were screened and only one positive clone carrying an insert of greater than 1 kb was obtained. This clone. abbreviated as MMC118, was subcloned into pUCl8 and its sequence determined. As presented in Fig. 2, this sequence contains an open reading frame encoding for a polypeptide of 286 amino acids long and the nucleotide sequence starting from base 322 to 1044 is identical to the cDNA
Fig. 2. The cDNA and its deduced xnino acid sequence of clone MMCI 18. Special features as described in the text are indicated in bold type. The mature oMMC sequence starts at Asp-24 and ends at Tyr-286. The potentia! gl>cosylatlon site is at Asn-250 (Asn-227 of the mature protein). The signal peptide is between Met-l IO Gly-23. The underlined amino acid sequence is identical to that determined by peptide sequencing of the natural protein.
313 insert found in the common fragment of MMC11 and MMC41. The deduced amino acid sequence of MMC118 contains a stretch of residues, 23 amino acids downstream from the initial methionine, identical to amino acid 1 to 45 at the N-terminal of a M M C as determined by peptide sequencing. The 23 a m i , o acids in front of the N-terminal sequence probably code for a signal peptide which is cleaved off from the mature protein. The high content of hydrophobic amino acids in this region further supports its role as a signal peptide. According to the deduced amino acid sequence of clone M M C l l S , the mature a M M C has altogether 263 residues and a calculated molecular mass of 29092 Da. Its estimated isc~ electric point is 9.5 which is slightly higher than that determined empirically ( p i 8.5 to 9; Ref. 14). There is one potential glycosylation site at amino acid residue 227 and the presence of carbohydrate may account for the discrepancy between the estimated molecular mass and that obtained from empirical determination (29 vs. 31 kDa). The amino acid composition of the deduced a M M C is almost exactly the same as that determined by amino acid analysis of the pure protein (result not shown).
Comparison of the amino acid sequence of a M M C with that of TCS and other RIPs indicates that there is a I:igh degree of homology among them. With the optimal alignment of these sequences, the percentage identity between a M M C and TCS, ricin A chain, and abrin A chain is 63, 35 and 34, respectively (Fig. 3). In contrast to TCS, a M M C is a glycoprotein. The potential glycosylation site in a M M C is located at residue 227 with the sequence NVT. At the same position, TCS has a sequence N V D instead. Examination of the corresponding nucleotides shows that this change is due to an alteration of two bases. As both TCS and otMMC have similar activities in inhibiting protein synthesis and HIV-1 replication, glycosylation at this particular site does not appear to be essential. Production of a recombinant otMMC in Escherichia coil without glycosylation, would allow us to test this hypothesis directly. In this study, we have initially isolated a c D N A clone. M M C I I , which hao a partial a M M C c D N A fused with an unknown sequence. Whether this is a phenomenon in the genomic D N A or an error arising from e D N A cloning is uncertain. We believe the latter possibility is more likely because no true reading frame could be detected in this clone. The fact that expression
t
Alpha MHC TCS Abrln A Rlcln A
2O
DVS[~LS~PI~'Y~GP~KDL~NA~P FR EKV. Y N ~ . . L P QDRP i . . . . . IFPKQYPI
..
DVS~LSFArr SSFY~VIF~S,L~KMLr'NERKL. YDB~ . . gs KIFIST ~ fir s qs ~ q F Zl~-^q~l~~LFo o L. I . . DII ~ q P DPT INEp'TAE_AITVC~_~'SlFq~Aq~RFJTTG^DV~HDLi~L~NRV
40 Alpha
Abrtn Ricin
HHC TCS A A
Alpha
HHC
60
80
SLPGSq~YAL I ~L~YAD. ET I SV AI~VT~V~IH(~Y~G DTS~ ~FN EAS AT~A[ TLQ ERN~YI TV~Lp~$. DTESI EVGI p V T q A M V V ~ T Q S ~ ~LRDAPSS.~l GLPINQ~F ILVI~S~HAE. LSVTLA~L~_~T_~AIYIVVG[~IqA~N$A~._~FHPDNQE12~J
tO0
120
140
TCo Abrln A Rictn A
AK.. YV~IKDAHRKVTLPV~G~I~ R~QTA~GKI~IENF~L ~ PA~DSA~FTL FYY SD.. YH~TGTDQH. SiP FY~I~GI~qER~HQS~QqI~I~t~I~TH(~qS • • FFR
Alpha HHC TCS Abrtn A RlcXn A
....
.....
....
NANSA.~SAL, VLF~ST S[EAA~YK~IIEQQIGK RVDKTF . . . . .
EAI TH LI~TDVQNRYTFAF~ R ~ E Q
L~G NL~EN[IIEL~hG [ ~E EALI~SALYYY
160 S. G G N D ~ E £ ~ R T L
Abrln Rlcin
HHC TCS A A
Alpha HMC TCS Abrin A Rlctn A
LA
I.~LA I
IV Ii2~IV/~EAA~FR~SNRVRVS IQTGTAFQ...!t~DAAM
qTGGTQLPTLLA~SFI IC~=_,.~qIS~AAI~FQyI~EGEHRTRIRYN...RRSA~PSV 200
Alpha
180 'ti
220
G~SKQI~L AQGNNOI ~ TP IV~VD~KGNRVQITNVTSKVVTSN IQ ~qSKQI ~ I ASTNNGQII~ESPVV~I NAQNQRVT I TNVDAGVVTSNIA N~G . V ~ . SV. QDT~PNQVT~TNI RNEPVIVDSLSH. PTVAVLA P~STA I~E. S. NQGA~ASPIQ~QRRNGSKFSVYDVS.. I L I P I I A
~S~D ~
240 263 L N T R N I A E G D N G D V $TTH. G FS SY L N R N N M A A H D D D V P ~ T Q S FGCGSYA I
LFVCNPPN
VYRCAPPPSSQF
Fig. 3. Optimalali~mentofaMMC sequence with [ho~ of other RIPs. Positions where the amino acids of all of the four RIPs are idemw.a~ are boxed.
314 o f the C - t e r m i n a l t w o - t h i r d s o f the a M M C c D N A is e n o u g h to p r o d u c e a positive signal o n W e s t e r n blot s u g g e s t s that the m a j o r a n t i g e n i c d e t e r m i n a n t s of a M M C m a y be a s s o c i a t e d with this region. A s there are i n d i c a t i o n s that t h e f u n c t i o n a l region o f a M M C d o e s n o t overlap with its a n t i g e n i c d e t e r m i n a n t ( s ) ( Y e u n g , u n p u b l i s h e d results), it is attractive to s p e c u l a t e that the N - t e r m i n a l p o r t i o n o f this m o l e c u l e m a y p l a y a m o r e i m p o r t a n t role in d i c t a t i n g biological activities. W e t h a n k Ms. W . H . T s a n g a n d Mr. T . K . M a n for their excellent technical a s s i s t a n c e a n d t h e C r o u c h e r F o u n d a t i o n of H o n g K o n g for d o n a t i n g the facilities of the B i o t e c h n o l o g y L a b o r a t o r y . T h i s r e s e a r c h was s u p p o r t e d by a g r a n t f r o m t h e U n i v e r s i t y a n d P o l y t e c h n i c Grants Committee. Hong Kong Government.
References 1 Stripe, F. and Barbieri. L. (1986) FEBS Left. 195. 1-8+ 2 Yeung, H.W,, Li. W.W.. Feng. Z.. Barbieri, L. and Stirpe, F. (1988) Int. J. Pept. Prof. Res. 31,265-268. 3 Yeung, H.W. Li+ W.W., Law. LK.. Chan+ W.Y, and Ng. T.B. (1986) Int. J. Pept. Prot. Res. 28, 518-524.
4 Law. LK., Tam, P.P.L. and Yeung, H.W. (1983) J. Reprod. Fert. 69, 597 604. 5 Chan, W.Y., Tam, PP+L. and Yeung, H.W+ (1984) Contraception 29, 91-100. 6 Leung, S.O., Yeung. H.W. and Leung, K.N. (1987) lmmunopharmacotogy 13, 159-171. 7 Mc(~rath. M.S.. Hwang, K M., Caldwell, S.E., Gaston, I.. Luk, K.C., Wu, P., Ng, V.L., Crowe, S., Daniels, J., Marsh. J. Deinhart, T., Lekas, P.V., Vennari. J.C.. Yeung, HW. and Lifson, J.D.(1989) Prec. Natl. Acad. Sci. USA 86, 2844-2848. 8 Lifson. J.D., McGrath, M.S.. Yeung, H.W. and Hwang, K. (1988) International Patent W088/0912. 9 Yeung. H.W., Ng, TB., Li, W.W. and Cheung. W.K. (1987) Planta Medica 53. 164-166. l0 Gulber, U. and Hoffman, B.J. (1983) Gene 25. 263-269. tl Cathala. G., Savouret, J.F.. Mendez. B., West, B.L., Karin, M., Martial, J+A, and Baxter. ,I.D. (1983) DNA 2, 329-335. 12 Huynh. T.V.. Young. R.A. and Davis, R.W. (1985) in DNA Cloning - A Practical Approach (GIover, D.. ed.), Vol. !, pp. 49-78. IRL Press, Oxford. 13 Young, R.A. and Davis, R.W. 0985) in Genetic Engineering Principles and Methods (Setlow, J. and Hollanender. A., eds.), Vol. 7, pp+ 29 41, Plenum Press, New York. 14 Kubota, S., Yeung, H.W. and Yang. J.T. (1986) Biechim Biophys. Acta 871, 101-106.
BBAEXP 90208
BBA Report - Short Sequence-Paper
Cloning of the cDNA of a-momorcharin: a ribosome inactivating protein Walter K.K. Ho’, S.C. Liu ‘, P.C. Shaw ‘, H.W. Yeung ‘, T.B. Ng ’ and W.Y. Chan ’ ’
The Biotechnology Laboratory and Deparrment o/ Btochemtstry, Chrnese Unrrwsrty of Hong Kong, Sham. .V. T IHwxg Kong) and ’ Oklahama Medrcal Research Fmmdatron. Oklahoma Ctty. OK flJ S.A.) (Received 23 October 1990)
Key words: cDNA: Momorcharin;
Ribowme mrccitia:mg protem. Tnchosanthm
Using a hgtll cDNA library constructed from the seeds of the bitter melon (Momordiea clu~ru&z~, we have obtained a full length cDNA containing the entire seqoence of a-momorcharin by immunowree Ring. I iw length of this cDNA is 1044 basepain long and it consists of an open reading frame coding for a polypeptide of 286 amino acids. The first 23 residues of this polypeptide probably code for a signal sequenuz. ?he N-termirrsl sequence of the deduced protein is exactly identical to that determined by peptide sequencing. The seqwnce identity between a-momorcharin and other ribosome inactivating proteins, such as trichosanthii and ricin A chain, is high, i.e., 3443%. Examination of the predicted secondary struchue of I-momorcharin and trichosanthin indicates that these proteins have regions of high strwtt4 similarities and this may account for the common biofqical activities that tbey share, viz, abwtificant, inununosuppressive, antitumor and inhibi*ion of HIV-l. The seeds of the bitter melon (Momordica churuntiu) are used in the Orient as a source of medicinal ingredients. In recent years, a number of biologically active proteins have been isolated from these seeds and most of them have been demonstrated to have ribosome inactivating property [1.2]. Our group has purified and characterized two basic proteins, a- and p-momorcharin (aMMC, /3MMC), from these seeds 13) and have demonstrated that they possess biological activities which can account for some of the medicinal value of the bitter melon. These activities include the induction of mid-term abortion [4,5], inhibition of tumor growth (Leung, K.N., unpublished results) and suppression of immune response 16). Whether these eiiects are directly related to the ribosome inactivating property is stilt uncertain. Recently, McGrath et al. [7] have shown that trichosanthin (TCS). a protein isolated from the root tubers of Trichosanthes kirilowii, can effectively inhibit
Abbreviations: MMC, momorcharin; TCS. crichosamhin: RIP. r&osome inactivating pro~tin; HIV, human imrmuwxehciency virus: IPTG, isopropyl-&~thiogalactopyranoside; X-gal. 5-hromo-khloro3-indolyl-/&~galactoside. Correspondence: W.KK. Ho. Department of Biochenkistry, Chmar University of Hong Kong. Shatin, N.T.. Hong Kong
the replication of type I human immunodeficien;y virus (HIV-l) in acuteely infected lymphoblastoid cells or chronically infected macrophages. As ICS, MMC and other ribosome inactivating proteins (RIPS: e.g., ticin A chain) are structurally related, it is not unexpected that they all have been subsequently shown to inhibit the propagation of HIV-l [g]. In spite of this generalization, the dose of these proteins required for inhibition of protein synthesis is significantly higher than that for inhibiting HIV-1 propagation ]7]. Thus, there may be other structural features of these proteins which are responsible for the inactivation of this type of wr(s,. in order to gain a better understanding on the structurefunction relationship of TCS and aMMC. we have cloned their cDNAs and deduced their amino acid sequences. This report deals with the results on aMMC. The amino acid sequence of the N-terminal residues of aMMC has been determined [9]. Using this information, we have attempted to design oligonucleotide probes for hybridization screening. but the codons for the N-terminal sequence were too degenera eti -Id a large number of oligonucleotides had to L * synthesized to cover all the permutations. On the other hand, aMMC is a very immunogenic protein and we were able to raise a number of high titer antisera against it. Therefore, we constructed a cDNA library from mRNA isolated from fresh seeds of the bitter melon (lO,ll] in the expression vector. Xgtll, for immunoscrcening (12.13).
312
P
E
E
mmc118
E
nunc41
I
x
P
I
I
780
bp
I E
1044
bp
I I
II
e .
.
.
Fig. 1. Alignment of various aMMC cDNA clone:. The relationships of the cDNA scquenw of clones MMCll. 41 and 118 are as indicated. The lowet part of the figure shows the sequencmg strategy. P. Pcrl; E, f&RI; X. Xholl.
Altogether. six positive clones were isolated in the first round of screening. Their insert size ranged from about 0.65 kb up to 1.27 kb. Upon rescreening by SDS-PAGE and Western blotting using an affinity purified ‘251-labeIed-anti-aMMC antibody, three clones, MMCI 1 (1.27 kb). MMC41 (0.78 kb) and MMC42 (0.78 kb), gave strong positive signals. By restriction mapping, clones MMC41 and 42 were identical. To facilitate sequencing, the inserts from MMCll and MMC41 were subcloned into pUCl8. The nucleotide sequence of MMCll can be divided into two fragments of 670 bp and 600 bp long by EcoRI digestion (Fig. 1). The longer fragment has a sequence identical to that of MMC41. Examination of the MMC41 sequence reveal-d :Zsi i, has a poly(A) tail and a polyadenylation signal, AATAAA, 31 bp upstream. These features were not found in the 670 bp fragment of MMCll. In order tc deteimine the identity of the nucleotide sequences of these -tones, a homology search in the Genebank Database was performed. The short fragment (600 bp) of MMCll was found to have some homology with the SS ribosomal RNA. No significant homology with sequences in the database was found to associate with the common sequence of MMCll and MMC41. 5’ upstream from the polyadenylation signal of the common sequence, several termination codons were noticed. Starting with one of these codons located at 123 bp 5’ to the AATAAA signal, we were able to back translate an uninterrupted polypeptide all the way to the 5’ end at the cloning site. Since the length of this peptide is only 186 amino acids and its nucleotide sequence does not encode an initiation codon, the common sequence of MMCll and MMC41 probably only contained a partial cDNA. The N-terminal sequence of TCS and aMMC have been shown to be highly homoiogous to each other. Since the primary structure of TCS is known, we compared it with the translated sequence
of our partial cDNA. They showed an identity of greater th.m 60%. The alignment of the two amino acid squences indicated that the partial cDNA ( M M C 4 1 ) probab!y codes for only two-thirds of the entire aMMC sequence. To obtain the full sequence of the uMMC cDNA, we prepared a 120 bp fsrI/&-oR1 fragment from the MMC41 insert (Fig. 1) and used it to reprobe the library. Altogether, over 250000 plaques were screened and only one positive clone carrying an insert of greater than 1 kb was obtained. This clone. abbreviated as MMC118, was subcloned into pUCl8 and its sequence determined. As presented in Fig. 2, this sequence contains an open reading frame encoding for a polypeptide of 286 amino acids long and the nucleotide sequence starting from base 322 to 1044 is identical to the cDNA
Fig. 2. The cDNA and its deduced xnino acid sequence of clone MMCI 18. Special features as described in the text are indicated in bold type. The mature oMMC sequence starts at Asp-24 and ends at Tyr-286. The potentia! gl>cosylatlon site is at Asn-250 (Asn-227 of the mature protein). The signal peptide is between Met-l IO Gly-23. The underlined amino acid sequence is identical to that determined by peptide sequencing of the natural protein.
313 insert found in the common fragment of MMC11 and MMC41. The deduced amino acid sequence of MMC118 contains a stretch of residues, 23 amino acids downstream from the initial methionine, identical to amino acid 1 to 45 at the N-terminal of a M M C as determined by peptide sequencing. The 23 a m i , o acids in front of the N-terminal sequence probably code for a signal peptide which is cleaved off from the mature protein. The high content of hydrophobic amino acids in this region further supports its role as a signal peptide. According to the deduced amino acid sequence of clone M M C l l S , the mature a M M C has altogether 263 residues and a calculated molecular mass of 29092 Da. Its estimated isc~ electric point is 9.5 which is slightly higher than that determined empirically ( p i 8.5 to 9; Ref. 14). There is one potential glycosylation site at amino acid residue 227 and the presence of carbohydrate may account for the discrepancy between the estimated molecular mass and that obtained from empirical determination (29 vs. 31 kDa). The amino acid composition of the deduced a M M C is almost exactly the same as that determined by amino acid analysis of the pure protein (result not shown).
Comparison of the amino acid sequence of a M M C with that of TCS and other RIPs indicates that there is a I:igh degree of homology among them. With the optimal alignment of these sequences, the percentage identity between a M M C and TCS, ricin A chain, and abrin A chain is 63, 35 and 34, respectively (Fig. 3). In contrast to TCS, a M M C is a glycoprotein. The potential glycosylation site in a M M C is located at residue 227 with the sequence NVT. At the same position, TCS has a sequence N V D instead. Examination of the corresponding nucleotides shows that this change is due to an alteration of two bases. As both TCS and otMMC have similar activities in inhibiting protein synthesis and HIV-1 replication, glycosylation at this particular site does not appear to be essential. Production of a recombinant otMMC in Escherichia coil without glycosylation, would allow us to test this hypothesis directly. In this study, we have initially isolated a c D N A clone. M M C I I , which hao a partial a M M C c D N A fused with an unknown sequence. Whether this is a phenomenon in the genomic D N A or an error arising from e D N A cloning is uncertain. We believe the latter possibility is more likely because no true reading frame could be detected in this clone. The fact that expression
t
Alpha MHC TCS Abrln A Rlcln A
2O
DVS[~LS~PI~'Y~GP~KDL~NA~P FR EKV. Y N ~ . . L P QDRP i . . . . . IFPKQYPI
..
DVS~LSFArr SSFY~VIF~S,L~KMLr'NERKL. YDB~ . . gs KIFIST ~ fir s qs ~ q F Zl~-^q~l~~LFo o L. I . . DII ~ q P DPT INEp'TAE_AITVC~_~'SlFq~Aq~RFJTTG^DV~HDLi~L~NRV
40 Alpha
Abrtn Ricin
HHC TCS A A
Alpha
HHC
60
80
SLPGSq~YAL I ~L~YAD. ET I SV AI~VT~V~IH(~Y~G DTS~ ~FN EAS AT~A[ TLQ ERN~YI TV~Lp~$. DTESI EVGI p V T q A M V V ~ T Q S ~ ~LRDAPSS.~l GLPINQ~F ILVI~S~HAE. LSVTLA~L~_~T_~AIYIVVG[~IqA~N$A~._~FHPDNQE12~J
tO0
120
140
TCo Abrln A Rictn A
AK.. YV~IKDAHRKVTLPV~G~I~ R~QTA~GKI~IENF~L ~ PA~DSA~FTL FYY SD.. YH~TGTDQH. SiP FY~I~GI~qER~HQS~QqI~I~t~I~TH(~qS • • FFR
Alpha HHC TCS Abrtn A RlcXn A
....
.....
....
NANSA.~SAL, VLF~ST S[EAA~YK~IIEQQIGK RVDKTF . . . . .
EAI TH LI~TDVQNRYTFAF~ R ~ E Q
L~G NL~EN[IIEL~hG [ ~E EALI~SALYYY
160 S. G G N D ~ E £ ~ R T L
Abrln Rlcin
HHC TCS A A
Alpha HMC TCS Abrin A Rlctn A
LA
I.~LA I
IV Ii2~IV/~EAA~FR~SNRVRVS IQTGTAFQ...!t~DAAM
qTGGTQLPTLLA~SFI IC~=_,.~qIS~AAI~FQyI~EGEHRTRIRYN...RRSA~PSV 200
Alpha
180 'ti
220
G~SKQI~L AQGNNOI ~ TP IV~VD~KGNRVQITNVTSKVVTSN IQ ~qSKQI ~ I ASTNNGQII~ESPVV~I NAQNQRVT I TNVDAGVVTSNIA N~G . V ~ . SV. QDT~PNQVT~TNI RNEPVIVDSLSH. PTVAVLA P~STA I~E. S. NQGA~ASPIQ~QRRNGSKFSVYDVS.. I L I P I I A
~S~D ~
240 263 L N T R N I A E G D N G D V $TTH. G FS SY L N R N N M A A H D D D V P ~ T Q S FGCGSYA I
LFVCNPPN
VYRCAPPPSSQF
Fig. 3. Optimalali~mentofaMMC sequence with [ho~ of other RIPs. Positions where the amino acids of all of the four RIPs are idemw.a~ are boxed.
314 o f the C - t e r m i n a l t w o - t h i r d s o f the a M M C c D N A is e n o u g h to p r o d u c e a positive signal o n W e s t e r n blot s u g g e s t s that the m a j o r a n t i g e n i c d e t e r m i n a n t s of a M M C m a y be a s s o c i a t e d with this region. A s there are i n d i c a t i o n s that t h e f u n c t i o n a l region o f a M M C d o e s n o t overlap with its a n t i g e n i c d e t e r m i n a n t ( s ) ( Y e u n g , u n p u b l i s h e d results), it is attractive to s p e c u l a t e that the N - t e r m i n a l p o r t i o n o f this m o l e c u l e m a y p l a y a m o r e i m p o r t a n t role in d i c t a t i n g biological activities. W e t h a n k Ms. W . H . T s a n g a n d Mr. T . K . M a n for their excellent technical a s s i s t a n c e a n d t h e C r o u c h e r F o u n d a t i o n of H o n g K o n g for d o n a t i n g the facilities of the B i o t e c h n o l o g y L a b o r a t o r y . T h i s r e s e a r c h was s u p p o r t e d by a g r a n t f r o m t h e U n i v e r s i t y a n d P o l y t e c h n i c Grants Committee. Hong Kong Government.
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