Molecular and Biochemical Parasttology, 25 (1990) 153-154 Elsevier
153
MOLBIO 01391
Short C o m m u n i c a t i o n
The epitope of a protective monoclonal antibody occurs in a region of microheterogeneity in Plasmodium chabaudi A n d r e w M. L e w and Dianne J. B e c k Veterinary Research Institute, Attwood, Victoria, Australia (Received 19 April 1990; accepted 15 May 1990)
Key words: Merozoite surface; Plasmodium chabaudi; Microheterogeneity; DNA sequence
The precursor to the major merozoite surface antigens in Plasmodium chabaudi is a 250-kDa protein and is known by several acronyms, including PMMSA and MSA1 [1,2]. We have previously reported the sequence of 1.4 kb (clone 3101) of the MSAI gene from P. chabaudi adami DS [3]. A protective monoclonal antibody, 5C10/66, identified a core epitope sequence of ETTET within this clone. We now report the sequence of the homologous region of the MSA1 genes from P. chabaudi adami DK and P. chabaudi chabaudi CB. All 3 strains are cloned lines from different isolates and were originally obtained from D. Walliker, Edinburgh. The oligonucleotides were phosphorylated by T4 kinase and then used to amplify a 1.4-kb fragment from genomic DNA by the polymerase chain reaction [4]. This was cloned into PUC 19 and double-stranded dideoxy sequencing was performed in both directions. Fig. 1 shows the alignment of the predicted protein sequence of this segment of MSA 1 from P. c. adami DS, P. c. adami DK and P. c. chabaudi CB. The nucleotide sequence is available in GenBank. There is a single Correspondence address: Andrew Lew, Veterinary Research Institute, 475-485 Mickleham Road, Attwood 3049, Victoria, Australia. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession numbers M34047 and M34255.
nucleotide difference between DS and DK which results in a change of hydrophilic Thr to a hydrophobic lie. It should be noted that the previously published DS sequence has several errors [3]. These errors occur at residues 128, 218, 293, 300, 305, 309, 370, 373 and 387 of that published sequence. The amino acid homology [6] between DS and DK, P. c. chabaudi CB, Plasmodium yoelii YM [5] and Plasmodium falciparum Fc27 [6] is 99.8%, 77%, 53% and 25%, respectively. This is in accord with the relatedness of these plasmodium species. There are two notable aspects of the single amino acid change between DS and DK. Firstly, it resides in the core epitope region of the protective monoclonal antibody mentioned above. Secondly all MSA 1 genes of P.falciparum have been shown to be derived from two primordial alleles [6,7]. Within regions that show low homology between these two alleles (< 20%) as described by Tanabe et al. [7], there are occasionally, albeit rarely, single amino acid changes in the MSA1 genes of strains of P.falciparum even though they were derived from the same primordial allele. Such a region of microheterogeneity between P.falciparum Fc27 and P. falciparum Camp is shown in Fig. 2. This region aligns with the microheterogeneity described between P. c. adami DS and DK. The difference between Fc27 and Camp in this region is similar to the different between DS and DK in that the charged Glu is changed to the hydropho-
0166-6851/90/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
154 2 DS DK CB
DRLAKFIPKI DRLAKFIPKI ERLAKFIPKI i01 DS PTAEEPQPAT DK PTAEEPQPAT CB PTEQEAQPAA 201 DS VKAGVIEPEP DK VKAGVIEPEP CB VKAGVIDPEP 301 D$ APAQEATTET DK APAQEATTET CB AQAPEAATET 401 DS AESEDEMFVI) DK AESEDEMFVD CB AESEDEMFVD
DDMIEKEKQK MEQEPVATGE SEQVTPSSGA GTSTQTAQTT QTP...pAAp APVKETTETT EKATQETQAA DTTTPEG .... TTPTEQEAA DDMIEKEKQK MEQEPVATGE SEQVTPSSGA GTSTQTAQTT QTP.,.pAAp APVKETTEIT EKATQETQAA DTTTPEG .... TTPTEQEAA DNMIEKEKQK MEQEHVATGE SEQ..ASSAS GTGSSTETTS QTAPAVPAAP APAEKAKEGT ESTEETPAAS KPAEGAASTG ATTPTEQEAA PETPAEVPA. .TPAApAAPA APAAPAKPVM TKLYYLEKLK KFLAFSYSCH KYVLLQNSTI NKDALSKYAL TPEEDKIRTL KRCSELDVLL PETPAEVPA. .TPAAPAAPA APAAPAKPVM TKLYYLEKLK KFLAFSYSCH KYVLLQNSTI NKDALSKYAL TPEEDKIRTL KRCSELDVLL PETPAEVPAP TTPAAPATPA APAAPAKPVM TKLYYLEKLK KFLAFSYSCH KYVLLQNSTI NKDALSKYAL TPEEDKIRTL KRCSELDVLL EPVAPTPAVP APETAPETVP ETPAQEATQE ATQEATQPES AIQNNMPTMY SLYENVVDGL QNIYTELYEK EMMYHIYNLK DKNPAVKALL EPVAPTPAVP APETAPETVP ETPAQEATQE ATQEATQPES AIQNNMPTMY SLYENVVDGL QNIYTELYEK EMMYHIYNLK DKNPAVKALL ..VAPTPAVP APETAPETAP ETAPETPAQE APQ...QPES AIQNNMPTMY SLYENVVDGL QNIYTELYEK EMMYHIYNLK DKNPAVKALL T T P A E P T P E T Q E G A S T N , . , T S E T S T . E G T P A P E A P S T E V P A S P P A T P A A P S A S S P .... A P A Q P A P A Q P V T S Q P V S G E S T N V E G S T Q V K T T P A E P T P E T Q E G A S T N . . . T S E T S T . E G T P A P E A P S T E V P A S P P A T P A A P S A S S P .... A P A Q P A P A Q P V T S Q P V S G E $ T N V E G S T Q V K TTPAESASTE P T P K A P T A T P TSETVTQEGT . . . . . . . . . . . . . . . . TPAA PKAQEGASSS APAQPAPAKP APAQTVTGQS TNVEGSTQVR DFEVDNFYKS YLQQVDGNNT QFIDFIKSKK ELINALTPEK VNQLYLDIAH LKELSEHYYN RYYKYKLKLE RLYQKHEQIE AANQKVKEI DFEVDNFYKS YLQQVDGNNT QFIDFIKSKK ELINALTPEK VNQLYLDIAH LKELSEHYYN RYYKYKLKLE RLYQKHEQIE AANQKVKEI DFEVDNFYKS YLQQVDGNNT QFIDFIKSKK ELINALTPEK VNQLYLDIAH LKELSEHYYN RYYKYKLKLE RLYQKHEQIE AANQKVKEI
Fig. 1. The predicted protein sequence of the middle porhon of the MSAI genes from P c adamt DS, P c adamt DK and P. c chabaudi CB. Dots represent gaps to maximlse homology. Pl F c 2 7
V
T
E
E
T
E
V
T
E
sity in the 45-kilodalton merozoite surface antigen of Plasmodium falciparum. Mol. Biochem. Parasitol. 39,
PI C A M P
*
"
*
*
a
~
E
*
Pea DS
*
K
*
T
*
*
T
*
Pea DK
*
K
*
T
~
*
1
Fig. 2. The region of mmroheterogeneity between P c. adaml DS and DK corresponds to a region of microheterogeneity between P. falciparum Fc27 and CAMP. The first Val residue corresponds to position 734 and 758, respectively, for the two P. falctparum strains. b i c Val. R e g i o n s o f m i c r o h e t e r o g e n e i t y are pres u m a b l y s e l e c t e d for to e v a d e p r o t e c t i v e i m m u n e responses. T h e fact that both in P. f a l c i p a r u m and P. c h a b a u d i there is a h o m o l o g o u s r e g i o n o f mic r o h e t e r o g e n e i t y and that the e p i t o p e o f a p r o t e c tive m o n o c l o n a l a n t i b o d y a g a i n s t P. c h a b a u d i lies in this r e g i o n w o u l d indicate that this r e g i o n is i n d e e d an i m p o r t a n t one.
References 1 Holder, A.A. (1988) The precursor to major merozoite surface antigens: structure and role in immunity. Prog. Allergy 41, 72-97. 2 Smythe, J.A., Peterson, M.G., Coppel, R.L., Saul, A., Kemp, D.J. and Anders, R.F (1990) Structural diver-
227-234. 3 Lew, A.M., Langford, C.J., Anders, R.F., Kemp, D.J., Saul, A., Fardoulys, C. and Sheppard, M. (1989) A protective monoclonal antibody recognises a linear epitope m the precursor of the major merozolte antigens of Plasmodium chabaudt adamt. Proc. Natl. Acad. Sci. USA 86, 3768-3772. 4 Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.H., Higuchi, R., Horn, G.T., Mullus, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. 5 Lewis, A.P. (1989) Cloning and analysis of the gene encoding the 230 kilodalton merozoite surface antigen of Plasmodium voelii. Mol. Biochem. Parasitol. 36, 271-282. 6 Peterson, M.G., Coppel, R.L., Mclntyre, P., Langford, C.J., Woodrow, G., Brown, G.V., Anders, R.F. and Kemp, D.J. (1988) Variation in the precursor to the major merozolte surface antigens of Plasmodium falciparum. Mol. Biochem. Parasitol. 27, 291-302. 7 Tanabe, K., MacKay, M., Goman, M. and Scalfe, J.G. (1987) Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodiumfalciparum. J. Mol. Biol. 195, 273-287.
153
MOLBIO 01391
Short C o m m u n i c a t i o n
The epitope of a protective monoclonal antibody occurs in a region of microheterogeneity in Plasmodium chabaudi A n d r e w M. L e w and Dianne J. B e c k Veterinary Research Institute, Attwood, Victoria, Australia (Received 19 April 1990; accepted 15 May 1990)
Key words: Merozoite surface; Plasmodium chabaudi; Microheterogeneity; DNA sequence
The precursor to the major merozoite surface antigens in Plasmodium chabaudi is a 250-kDa protein and is known by several acronyms, including PMMSA and MSA1 [1,2]. We have previously reported the sequence of 1.4 kb (clone 3101) of the MSAI gene from P. chabaudi adami DS [3]. A protective monoclonal antibody, 5C10/66, identified a core epitope sequence of ETTET within this clone. We now report the sequence of the homologous region of the MSA1 genes from P. chabaudi adami DK and P. chabaudi chabaudi CB. All 3 strains are cloned lines from different isolates and were originally obtained from D. Walliker, Edinburgh. The oligonucleotides were phosphorylated by T4 kinase and then used to amplify a 1.4-kb fragment from genomic DNA by the polymerase chain reaction [4]. This was cloned into PUC 19 and double-stranded dideoxy sequencing was performed in both directions. Fig. 1 shows the alignment of the predicted protein sequence of this segment of MSA 1 from P. c. adami DS, P. c. adami DK and P. c. chabaudi CB. The nucleotide sequence is available in GenBank. There is a single Correspondence address: Andrew Lew, Veterinary Research Institute, 475-485 Mickleham Road, Attwood 3049, Victoria, Australia. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession numbers M34047 and M34255.
nucleotide difference between DS and DK which results in a change of hydrophilic Thr to a hydrophobic lie. It should be noted that the previously published DS sequence has several errors [3]. These errors occur at residues 128, 218, 293, 300, 305, 309, 370, 373 and 387 of that published sequence. The amino acid homology [6] between DS and DK, P. c. chabaudi CB, Plasmodium yoelii YM [5] and Plasmodium falciparum Fc27 [6] is 99.8%, 77%, 53% and 25%, respectively. This is in accord with the relatedness of these plasmodium species. There are two notable aspects of the single amino acid change between DS and DK. Firstly, it resides in the core epitope region of the protective monoclonal antibody mentioned above. Secondly all MSA 1 genes of P.falciparum have been shown to be derived from two primordial alleles [6,7]. Within regions that show low homology between these two alleles (< 20%) as described by Tanabe et al. [7], there are occasionally, albeit rarely, single amino acid changes in the MSA1 genes of strains of P.falciparum even though they were derived from the same primordial allele. Such a region of microheterogeneity between P.falciparum Fc27 and P. falciparum Camp is shown in Fig. 2. This region aligns with the microheterogeneity described between P. c. adami DS and DK. The difference between Fc27 and Camp in this region is similar to the different between DS and DK in that the charged Glu is changed to the hydropho-
0166-6851/90/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
154 2 DS DK CB
DRLAKFIPKI DRLAKFIPKI ERLAKFIPKI i01 DS PTAEEPQPAT DK PTAEEPQPAT CB PTEQEAQPAA 201 DS VKAGVIEPEP DK VKAGVIEPEP CB VKAGVIDPEP 301 D$ APAQEATTET DK APAQEATTET CB AQAPEAATET 401 DS AESEDEMFVI) DK AESEDEMFVD CB AESEDEMFVD
DDMIEKEKQK MEQEPVATGE SEQVTPSSGA GTSTQTAQTT QTP...pAAp APVKETTETT EKATQETQAA DTTTPEG .... TTPTEQEAA DDMIEKEKQK MEQEPVATGE SEQVTPSSGA GTSTQTAQTT QTP.,.pAAp APVKETTEIT EKATQETQAA DTTTPEG .... TTPTEQEAA DNMIEKEKQK MEQEHVATGE SEQ..ASSAS GTGSSTETTS QTAPAVPAAP APAEKAKEGT ESTEETPAAS KPAEGAASTG ATTPTEQEAA PETPAEVPA. .TPAApAAPA APAAPAKPVM TKLYYLEKLK KFLAFSYSCH KYVLLQNSTI NKDALSKYAL TPEEDKIRTL KRCSELDVLL PETPAEVPA. .TPAAPAAPA APAAPAKPVM TKLYYLEKLK KFLAFSYSCH KYVLLQNSTI NKDALSKYAL TPEEDKIRTL KRCSELDVLL PETPAEVPAP TTPAAPATPA APAAPAKPVM TKLYYLEKLK KFLAFSYSCH KYVLLQNSTI NKDALSKYAL TPEEDKIRTL KRCSELDVLL EPVAPTPAVP APETAPETVP ETPAQEATQE ATQEATQPES AIQNNMPTMY SLYENVVDGL QNIYTELYEK EMMYHIYNLK DKNPAVKALL EPVAPTPAVP APETAPETVP ETPAQEATQE ATQEATQPES AIQNNMPTMY SLYENVVDGL QNIYTELYEK EMMYHIYNLK DKNPAVKALL ..VAPTPAVP APETAPETAP ETAPETPAQE APQ...QPES AIQNNMPTMY SLYENVVDGL QNIYTELYEK EMMYHIYNLK DKNPAVKALL T T P A E P T P E T Q E G A S T N , . , T S E T S T . E G T P A P E A P S T E V P A S P P A T P A A P S A S S P .... A P A Q P A P A Q P V T S Q P V S G E S T N V E G S T Q V K T T P A E P T P E T Q E G A S T N . . . T S E T S T . E G T P A P E A P S T E V P A S P P A T P A A P S A S S P .... A P A Q P A P A Q P V T S Q P V S G E $ T N V E G S T Q V K TTPAESASTE P T P K A P T A T P TSETVTQEGT . . . . . . . . . . . . . . . . TPAA PKAQEGASSS APAQPAPAKP APAQTVTGQS TNVEGSTQVR DFEVDNFYKS YLQQVDGNNT QFIDFIKSKK ELINALTPEK VNQLYLDIAH LKELSEHYYN RYYKYKLKLE RLYQKHEQIE AANQKVKEI DFEVDNFYKS YLQQVDGNNT QFIDFIKSKK ELINALTPEK VNQLYLDIAH LKELSEHYYN RYYKYKLKLE RLYQKHEQIE AANQKVKEI DFEVDNFYKS YLQQVDGNNT QFIDFIKSKK ELINALTPEK VNQLYLDIAH LKELSEHYYN RYYKYKLKLE RLYQKHEQIE AANQKVKEI
Fig. 1. The predicted protein sequence of the middle porhon of the MSAI genes from P c adamt DS, P c adamt DK and P. c chabaudi CB. Dots represent gaps to maximlse homology. Pl F c 2 7
V
T
E
E
T
E
V
T
E
sity in the 45-kilodalton merozoite surface antigen of Plasmodium falciparum. Mol. Biochem. Parasitol. 39,
PI C A M P
*
"
*
*
a
~
E
*
Pea DS
*
K
*
T
*
*
T
*
Pea DK
*
K
*
T
~
*
1
Fig. 2. The region of mmroheterogeneity between P c. adaml DS and DK corresponds to a region of microheterogeneity between P. falciparum Fc27 and CAMP. The first Val residue corresponds to position 734 and 758, respectively, for the two P. falctparum strains. b i c Val. R e g i o n s o f m i c r o h e t e r o g e n e i t y are pres u m a b l y s e l e c t e d for to e v a d e p r o t e c t i v e i m m u n e responses. T h e fact that both in P. f a l c i p a r u m and P. c h a b a u d i there is a h o m o l o g o u s r e g i o n o f mic r o h e t e r o g e n e i t y and that the e p i t o p e o f a p r o t e c tive m o n o c l o n a l a n t i b o d y a g a i n s t P. c h a b a u d i lies in this r e g i o n w o u l d indicate that this r e g i o n is i n d e e d an i m p o r t a n t one.
References 1 Holder, A.A. (1988) The precursor to major merozoite surface antigens: structure and role in immunity. Prog. Allergy 41, 72-97. 2 Smythe, J.A., Peterson, M.G., Coppel, R.L., Saul, A., Kemp, D.J. and Anders, R.F (1990) Structural diver-
227-234. 3 Lew, A.M., Langford, C.J., Anders, R.F., Kemp, D.J., Saul, A., Fardoulys, C. and Sheppard, M. (1989) A protective monoclonal antibody recognises a linear epitope m the precursor of the major merozolte antigens of Plasmodium chabaudt adamt. Proc. Natl. Acad. Sci. USA 86, 3768-3772. 4 Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.H., Higuchi, R., Horn, G.T., Mullus, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. 5 Lewis, A.P. (1989) Cloning and analysis of the gene encoding the 230 kilodalton merozoite surface antigen of Plasmodium voelii. Mol. Biochem. Parasitol. 36, 271-282. 6 Peterson, M.G., Coppel, R.L., Mclntyre, P., Langford, C.J., Woodrow, G., Brown, G.V., Anders, R.F. and Kemp, D.J. (1988) Variation in the precursor to the major merozolte surface antigens of Plasmodium falciparum. Mol. Biochem. Parasitol. 27, 291-302. 7 Tanabe, K., MacKay, M., Goman, M. and Scalfe, J.G. (1987) Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodiumfalciparum. J. Mol. Biol. 195, 273-287.
