Molecular and Biochemical Parasitology, 54 (1992) 125 128 (i'; 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00
125
MOLBIO 01797
Short Communication
Sequence of the gene for a Trypanosoma cruzi protein antigenic
during the chronic phase of human Chagas disease A l e j a n d r o Buschiazzo, Oscar E. C a m p e t e l l a , R o b e r t o A. M a c i n a , S u s a n a Salceda, A l b e r t o C.C. F r a s c h a n d Daniel O. Sanchez lnstituto de Investigaciones Bioquimicas Fundaci6n Carnpomar, Buenos Aires, Argentina (Received 14 May 1992; accepted 15 May 1992)
Key words: Trypanosoma cruzi; Antigen; D N A .sequence: Gene cloning
DNA sequencing studies showed two major characteristics of Trypanosoma cruzi trypomastigote antigens. Firstly, a large number of them contain repeated amino acid motifs (see ref. 1 for a review) and secondly, analyzing the non-repeated region of several molecules it became clear that they belong to a major family of surface antigens [2-6]. Members of this family have an amino acid sequence homology ranging from 20% to 80% and contain copies of the sequence Ser-X-Asp°XGly-X-Thr-Trp, which is conserved among bacterial neuraminidases [7]. Proteins belonging to this major antigen family are expressed in the trypomastigote stage of the parasite, have a size of around 85 kDa or 130-200 kDa and are located on the parasite surface or shed into the medium. A member of this antigen family is SAPA, a shed-acute-phase-antigen homologous to the T. cruzi neuraminidase [5] and able to mediate neuraminidase and transsialidase activity [8]. One of the genes that we have previously Correspondence address." Alejandro Buschiazzo, Instituto de Investigaciones Bioquimicas Fundacion Campomar, Antonio Machado 151, 1405 Buenos Aires, Argentina.
Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M92049.
identified is that coding for antigen number 2 (TcCA-2, ref. 9, 10). This protein is a major antigen during the chronic phase of the human Chagas disease [11]. It is trypomastigotespecific, and immunofluorescence experiments performed with living parasites support that it is located at the parasite surface [10]. Subsequent failure to detect TcCA-I by immunoelectron microscopy [12] is probably due to its unstability to several fixation procedures, like glutaraldehyde [12], formaldehyde, ethanol or methanol/acetic acid (unpublished observations). In this paper the sequence of TcCA-2 is presented. A DNA clone encoding TcCA-2 gene was identified by screening a genomic library constructed with DNA of the CA-1/65 clone of T. cruzi with a DNA probe containing the 3' end of the gene [10]. It was sequenced using the dideoxinucleotide chain termination procedure [13]. After 4 upstream stop codons there is an open reading frame of approximately 4000 bp (see Fig. 1). An in-frame methionine is present 72 bp after the last stop codon at the 5' end. There are two putative trans-splicing sites, 68 and 73 bp upstream of the ATG, conforming to the consensus sequence for trypanosomes [14,15]: UUUCU---44 bp (84% Py)---AG. The gene contains, starting from the 5' end, a non repeated region of 645 bp, then approximately
126
t TGCGCCRTRG G A C R A T ~ G GCCGT~AT TGTAATGTTT GCTTGRGGCA'CAATTICTAC CTTTTTITTT 121 TTTTRTTTTT GTTTTTCTGT TGTTGBTTGT TGTGOGGTCCCGTGRTTTAR TRARTRTGGC CTTGCGTCCC f l R L R P 241 TGGRCTTGCA 8GIITRCRGG RGCTCTGCCR GCRGGTGRGA CTGCTGCGCG CCRRTGTRCR RCGCCTTOCR G L R S L Q E L C O Q U R L L R A H U O R L R 361 TCRCCTGCRC CTOCCGTTGC RTRCTRATTT TCRTGTTGOC ATGCRGGflGR RGRAACCGGC RCCRGGAAGT H L H L P L H T H F H U G ~ O E K K P R P G S 481 AGACT GCACR CGTGGOTCGC ACAGGCTGCC CGROflRGGRA CGCCCCRCAC ACflTGATGTB CGCGGTGGGG 0 C T R G S H R L P E K E R P T R M M Y R U G 601 RTRCRRTGRA RGRGRRRAGR TRGRRGRRRR RRRRGRTRC~ RCRTCGTRCR CRCCCAGCRR CCCCTCCCRR Y It E R E K I E E K K O T T S Y T P S H P S O 721 CCRRCRRRTT RRRCCTCRTR TCRCRRRTRR TRRRCRARRR RCRRCRCTTG RRRRRRCTRR RRCRGRRCAR K P H ' T N H K 0 K T T L E K T K T E O o o 841 TGORCRGGCI GCTGCR RR ~ ~ G T G R C R G 0 KRA G 0 R 2q68 ACAGGCCGCC GCA~GTGRCfi RflCCRTCGCT ATTTGGRCRO G~CGCTGCA~ GTGRCRRRCC RCCRCCfTTT R|O 0 K P $ L F IG (~ R R Rl G 0 K e P P F 3088 A~G6TGRCR RR CCRTCRCCRT TTGGRCAOGC C G C T G C ~ G I GRCRRRCCflT CRCCRTTTGG RCRGGCCGCT P s e r ° ~ R r r~G 0 K P S P r G q R R I0 o K 3200 RTCRCCRTTT OC GRC CI~ RG GG G R CTGC RRA ~ GTGRO G CRR RTCRK GCP C S CC TGORCp BIT~ 0 oAGGCCGC B TGCAR ? R GTA G0 BCB G K S P F GCR~OTGRCR RflCCATCGCT RTTTGGRCBG GCCOCTGCA~O GTGflCRRACC RCCflCCRITT R ~G 0 K P S L F Gt . ql R R R| G 0 K P P P F 3~48 oro.c... cc~qTcRCCRT TTGOACROGC COCTGCOIGGT GRCflARCCRC CRCCRTTTG6 RCRGGCTGCT ~GG 0 K P S P F G 9 A fl R1 G 0 K P P F F G ~ ~ R 3568 TC~BAGCRCT G'GTTTGCGfl RTGCGCCTGG TGTTGCCCAG GTGTCGTTTG GAflRGCCRTC GACGRCGTTT R S T U F R H A P G U A Q J S F G K P S I I F 3688 tGGTTCTATT TTRCAGARTG TTCATGCT~C ATTGCCTGCC GGARSCGCGT TGTCTCA;AT TGGARGTGCC L 0 ~ U H R T L P R 0 5 A L S H I G S A G S 38138 AAGTGGTCCG GCAATIGGTG GTOGTqfiACT ACCTGCRTTG 5RGOGflFGTG GGTTTGGTCA'GGCTTTTAGT S G P R I G G G K L P A L E G S G F G Q R F S 3928 AGGTGRGGGC RCCTITGGTR CT3TOGOAGC CCRTGGGRCG GGTTCCGCCA CCCCGITITC RARGRCCAGT T F G T U G f l H G T G S R T P F S K T S G E G 4048 TCTCCGCRRT TTR~GRGTGT TTTTGGTCCT CRTTGRTRTG CGGTTTTTtT TTRRTCTGR~ TTTITGCTTG L B H L R U ~ k Y L I 0 M R F F F ~ l ~ F C L 4168 TTGCTTOCTG RRfiGCCRTTT GTTTTTTGGT TGTTGTTGTT GTTGTTTTTG GCTCAAGGAP TABCATTTGG 4288 GRTGT RCCOR GTCTTTTqGR TCTTCTCAGG C R C C T G A C T G flTGCqCCGGC TOTGRCTGCA TCTGCGCTOC 4408 Ri ;CRCRGRC CRACAAGGCG TGTCGTRGGG R C I G C R T C T C TTTTTGTTGR ACCCAARTTT TCGCGCGGTG 4520 ~ 3 G C ~ R R G CTTGGGCRGG CCC~T'GRTG qCCTtGCRBT ATTGCCGT
0
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GTCRTCTIIG U i F TATRCCCCCC Y T P CCCflRCGCCC P i4 A
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0
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OTGCTGRGTC GCTCCTTTCR CRCTGCRRRR GGCTCflCTGC G fl E S L L S 14 C K R L T R ATTflTCCCAA CfiGRRRRATG CCTTTGTRCR RITCflACCflA H ¥ P N R ~ ff P L Y H S T i~ flRRRflRGCTT TflRTCCTTGT QCGIGflCRRRT TGRflTRTRFIA Q K S F H P C T 0 K L H I 14
A~R~CfiGCGC"C'R-T'r'T'G))-C'R'G'G];'C'Gi~"T'O'C'R'[GOTGnCRmnC CRtCGCTRTT K
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CCflTCRCCOT TIGGflCRGGCCGCCGCfl~OTGRCRRGCCRT c R c c e r r r e e P s P F 0 ~ R ~ R 10 O K P S P F G GGRCRGGCCG C R G C ~ G T G R CRRRCCRTCG CTATTTGGRC RGGCCGCIGC i
GCR~°T G f l0C R0R IzRRCCRTCGCTp S L RTTTGGRCflGF ° q GCCGCTGCRIGR R RO / .G, GTGflGRRRCCIz P CCflTCRCCRT P S P GGRCRGGCCG G ~ A GCR~GTGRCR
R| O 0 GCAACCflCRT R T T S TTTGGTTCrC F G S GCTTTIGGCA A F O H ACCACATTTC T T F Q TGfiATGRT~A * GCATGTCGGR RGGCRRTTGC GAAAATCIGT
TTfiORCflGGC COCTOCRIGGI OflCRflRCCRC CRCCRTTI00 F 0 ~ R ~ A ~G 0 K P P P F fi CTGC ~ G T G R CRRRCCATCA CCRTTTOGRC RGGCCGCRGC A R| G 0 K P S P F G ~ A 8 G~CCRTCRCC RTTTGGRCRG GGRRC,~G~GT TTORTGCOTC
R P 0
P
CCTTTGCTGC F R R G CTGGRATGCR P G R X ATCfiTGCflTC H R S T ARTCTTCTTC S S S ~ TCAATTflATT
F G O ~_..T...U
TGGTGGTGGG G G F G TTCTGGCGGT $ G G RRCTGTTTTA V L G H TTTTTCCRGT S $ 1 A TGRRGTIGTT
F 0
fi
$
TTTGGRGCAC CRRGTGCTTT R P S B F GCGTTTGGTG GRGCPGGTGT A F G G R G U 00RARTTTTG GTCflGRRRGR F G O K E RTTGCGGGAA RCIRCRRGGC G H V K A T;GGGGRTGT TTGTGCRRCT
TGRRTTGGGT GRACTGGAAT TPRGGGRRGT TGAGGCRCGT CGTCRRGCTG 3CATGGTAAC GGGRCRCfiTT GfiRGCATGTC GTTGTTGRTC
TGAflGRGGGG ARRGGTTTTT CCTCRTRTCG
Fig. 1. Nucleotide and deduced amino acid sequence of the TcCA-2 gene. Four upstrcam codons are underlined. Arrows indicate predicted trans-splicing cleavage sites. The repeat units of 12 amino acids are boxed. The shadowed box represent approximately 57 repeat units as estimated by Hhal and Pstl digestion analysis (sec text). The hatched lines denole degenerate repeat units.
75 tandemly arranged 36-base-long repeats and finally, a non-repeated region of 570 bp at the 3' end. The region encoding the repeated units was not completely sequenced. The number of repeat units is based on restriction endonuclease analysis, and is known to vary between allelic forms of the gene [16]. The genomic organization of TcCA-2 has been previously studied in several parasite strains, including another clone of CA-l, number 72 [16]. Only two copies of the gene per parasite located in different chromosomes, were detected. It is possible that both copies are expressed since we have detected two RNA species, at least in RA strain [9]. We cannot tell at present whether the sequence provided belongs to a gene which is actively expressed. The expected size of the protein as deduced from the D N A sequence is around 155kDa. We had previously found that the protein
TcCA-2 has an apparent molecular weight of 85kDa in Western blots, with some reacting material remaining in the slot. This discrepancy might be due to the fact that the antigen is unstable, being quickly degraded when preparing protein extracts from parasites or even when the protein is expressed in bacterial systems (unpublished observations). New protein extracts were prepared. Parasites from the RA strain were collected by centrifugation and immediately resuspended in cracking buffer and boiled. The CA-1/72 clone used was kept at - 7 0 ~ C for one week and thawed directly in crackin~ buffer. The volumes corresponding to 2 × 10 parasites per well, were layered in a 10% SDS-PAGE and electrophoresed. The Western blot was reacted with antibodies made in rabbits against the TcCA-2 antigen expressed in a recombinant D N A clone [10]. The results in Fig. 2 show that there are two major
127
KDa
1
M
2 slot.
180 116 -
other significant homology with any published sequence, including any major surface antigen gene from T. cruzi [2-6]. Furthermore, the amino acid sequence Ser-X-Asp-X-Gly-X-ThrTrp, characteristic of bacterial neuraminidases and members of T. cruzi antigens, is not present in TcCA-2.
Acknowledgements 4/1.5-
Fig. 2. Detection of TcCA-2 protein in trypomastigote extracts. Protein extracts from RA and CA-1/72 parasites were prepared as described in the text, electrophoresed in a 10% polyacrylamide gel and transferred to a nitrocellulose filter. The blot was reacted with a rabbit antiserum raised against the carboxy terminus of TcCA-2 protein [10].
bands of around 140-160kDa in RA strain and one band of around 180kDa in CA-1/72 clone of T. cruzi. These sizes are in good agreement with the expected molecular weight deduced from the DNA sequence. In addition, there is another reacting band of around 50 kDa. If parasites are kept at -20~C or, after thawing, they are not immediately resuspended in cracking buffer and boiled, bands of smaller molecular weight are observed (results not shown). Thus, we consider likely that the size we had previously reported for TcCA-2 was not the correct one, due to degradation of this unstable protein. Hoft et al. [17] reported a partial amino acid sequence deduced from DNA sequence of a T. cruzi gene (TCR39), that includes a few repeat units and the carboxy-terminus. Comparison of TcCA-2 with TCR39 shows that both proteins contain the same repeated motif but different carboxy-termini, with several stretches of amino acids being conserved. Sequence comparison, in the GenBank (release 65) and the EMBL (release 28) data bank, showed no
We thank Carlos Ibfifiez for his help with the first sequences of TcCA-2, Drs. J.J. Cazzulo, U. Pettersson and L. A,slund for useful discussions and L. Sferco and M. Guerin for technical assistance. This work was supported by grants from the UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical diseases, the Swedish Agency for Research Co-operation with Developing Countries (SAREC), the Consejo Nacionai de lnvestigaciones Cientificas y T6cnicas and the University of Buenos Aires, Argentina and the Programa Regional de Biotecnologia PNUD/UNESCO/ONUDI. O.C., D.O.S. and A.C.C.F. are researchers from the Consejo Nacional de lnvestigaciones Cientificas y T~cnicas, Argentina.
References 1 Frasch, A. C. C., Cazzulo, J. J., ,Aslund, L. and Pettersson, U. (1991) Comparison of genes encoding Trypanosoma cruzi antigens. Parasitol. Today 7, 148 151. 2 Fouts, D. L., Ruef, B. J., Ridley, P. T., Wrightsman, R. A., Peterson, D. S. and Manning, J. E. (1991) Nucleotide sequence and transcription of a trypomastigote surface antigen gene of Tr)Tanosoma cruzi. Mol. Biochem. Parasitol. 46, 189-200. 3 Takle, G. B. and Cross, G. A. M. (1991) An 85 kDa surface antigen gene family of Trypanosoma cruzi encodes polypeptides homologous to bacterial neuraminidases. Mol. Biochem Parasitol. 48 185 198. 4 Kahn, S., Colbert, T. G., Wallace, J. C., Hoagland, N. A. and Eisen, H. (1991)The major 85-kDa surface antigen of the mammalian-stage forms of Tr)'panosoma cruzi is a family of sialidases. Proc. Natl. Acad. Sci. USA. 88, 4481- 4485. 5 Pereira, M. E. A., Santiago Mejia, J., Ortega-Barria, E., Matzilevich, D. Prioli, R. P. (1991) The Trypanosoma cruzi neuraminidase contains sequences similar to bacterial neuraminidases, to YWTD repeats of the
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6
7
8
9
10
I1
LDL receptor and to type II1 modules of fibronectin. J. Exp. Med. 174, 179-192. Pollevick,G D., Affranchino, J. L., Frasch, A. C. C. and Sanchez, D. O. (1991) The complete sequence of SAPA, a shed-acute phase-antigen of Trypanosoma cruzi. Mol. Biochem. Parasitol. 47, 247 250. Roggentin, P., Rothe, B., Kaper, J. B., Galen, J., Lawrisuk, L., Vimr, E. R. and Schauer, R. (1989) Conserved sequences in bacterial and viral neuraminidases. Glycoconjugate J. 6, 349-356. Parodi, A., Pollevick, G., Mautner, M., Buschiaz~.o, A., Sanchez, D.O. and Frasch, A.C.C. (In Press) Identification of the gene(s) encoding the trans-sialidase of Trypanosorna cruzi. EMBO J. ibafiez, C., Affranchino, J.L. and Frasch, A.C.C. (1987) Antigenic determinants of Trypanosoma cruzi defined by cloning of parasite DNA. Mol. Biochem. Parasitol. 25 175 184. lbfifiez, C. F., Affranchino, J. L., Macina, R.°A., Reyes, M. B., Leguizam6n, S., Camargo, M. E., Aslund, L., Petterson U. and Frasch, A. C. C. (1988) Multiple Trypanosoma cruzi antigens containing tandemly repeated amino acid sequence motifs. Mol. Biochem. Parasitol. 30, 27 34. Frasch, A. C. C. and Reyes, M. B. (1990) Diagnosis of Chagas disease using recombinant DNA technology. Parasitol. Today 6, 137 140.
12 Souto-Padron T, Rcyes M B, Leguizamon S, Campetella O E, Frasch A C C and de Souza W. (1989)Trypanosoma cruzi proteins which are antigenic during human infections are located in defined regions of the parasite.. Eur. J. Cell Biol. 50, 272-278. 13 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. (USA) 74, 5463 5467. 14 Laird, PW. (1989) Transplicing in trypanosomes archaism or adaptation?. Trends Genet. 5, 204 208. 15 Huang, J. and Van der Ploeg, L.H.T. (1991) Requirement of a polypyrimidine tract for trans-splicing in trypanosomes: discriminating the PARP promoter from the immediately adjacent 3' splice acceptor site. EMBO J. 10, 3877 3885.. 16 Henriksson J, Aslund L, Macina R.A, Franke de Cazzulo, B M, Cazzulo J J, Frasch A C C and Pettersson U. (1990) Chromosomal localization of seven cloned antigens provides evidence of diploidy and further demonstration of karyotype variability in Trypanosoma cruzi. Mol. Biochem. Parasitol. 42, 213 224. 17 Hoft D E, Kim K S, Otsu K, Moser D R, Yost W J, Blumin J H, Donelson J E and Kirchhoff L V (1989) TrkTanosorna cruzi expresses diverse repetitive protein antigens. Infect. Immun. 57. 1959-1967.
125
MOLBIO 01797
Short Communication
Sequence of the gene for a Trypanosoma cruzi protein antigenic
during the chronic phase of human Chagas disease A l e j a n d r o Buschiazzo, Oscar E. C a m p e t e l l a , R o b e r t o A. M a c i n a , S u s a n a Salceda, A l b e r t o C.C. F r a s c h a n d Daniel O. Sanchez lnstituto de Investigaciones Bioquimicas Fundaci6n Carnpomar, Buenos Aires, Argentina (Received 14 May 1992; accepted 15 May 1992)
Key words: Trypanosoma cruzi; Antigen; D N A .sequence: Gene cloning
DNA sequencing studies showed two major characteristics of Trypanosoma cruzi trypomastigote antigens. Firstly, a large number of them contain repeated amino acid motifs (see ref. 1 for a review) and secondly, analyzing the non-repeated region of several molecules it became clear that they belong to a major family of surface antigens [2-6]. Members of this family have an amino acid sequence homology ranging from 20% to 80% and contain copies of the sequence Ser-X-Asp°XGly-X-Thr-Trp, which is conserved among bacterial neuraminidases [7]. Proteins belonging to this major antigen family are expressed in the trypomastigote stage of the parasite, have a size of around 85 kDa or 130-200 kDa and are located on the parasite surface or shed into the medium. A member of this antigen family is SAPA, a shed-acute-phase-antigen homologous to the T. cruzi neuraminidase [5] and able to mediate neuraminidase and transsialidase activity [8]. One of the genes that we have previously Correspondence address." Alejandro Buschiazzo, Instituto de Investigaciones Bioquimicas Fundacion Campomar, Antonio Machado 151, 1405 Buenos Aires, Argentina.
Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M92049.
identified is that coding for antigen number 2 (TcCA-2, ref. 9, 10). This protein is a major antigen during the chronic phase of the human Chagas disease [11]. It is trypomastigotespecific, and immunofluorescence experiments performed with living parasites support that it is located at the parasite surface [10]. Subsequent failure to detect TcCA-I by immunoelectron microscopy [12] is probably due to its unstability to several fixation procedures, like glutaraldehyde [12], formaldehyde, ethanol or methanol/acetic acid (unpublished observations). In this paper the sequence of TcCA-2 is presented. A DNA clone encoding TcCA-2 gene was identified by screening a genomic library constructed with DNA of the CA-1/65 clone of T. cruzi with a DNA probe containing the 3' end of the gene [10]. It was sequenced using the dideoxinucleotide chain termination procedure [13]. After 4 upstream stop codons there is an open reading frame of approximately 4000 bp (see Fig. 1). An in-frame methionine is present 72 bp after the last stop codon at the 5' end. There are two putative trans-splicing sites, 68 and 73 bp upstream of the ATG, conforming to the consensus sequence for trypanosomes [14,15]: UUUCU---44 bp (84% Py)---AG. The gene contains, starting from the 5' end, a non repeated region of 645 bp, then approximately
126
t TGCGCCRTRG G A C R A T ~ G GCCGT~AT TGTAATGTTT GCTTGRGGCA'CAATTICTAC CTTTTTITTT 121 TTTTRTTTTT GTTTTTCTGT TGTTGBTTGT TGTGOGGTCCCGTGRTTTAR TRARTRTGGC CTTGCGTCCC f l R L R P 241 TGGRCTTGCA 8GIITRCRGG RGCTCTGCCR GCRGGTGRGA CTGCTGCGCG CCRRTGTRCR RCGCCTTOCR G L R S L Q E L C O Q U R L L R A H U O R L R 361 TCRCCTGCRC CTOCCGTTGC RTRCTRATTT TCRTGTTGOC ATGCRGGflGR RGRAACCGGC RCCRGGAAGT H L H L P L H T H F H U G ~ O E K K P R P G S 481 AGACT GCACR CGTGGOTCGC ACAGGCTGCC CGROflRGGRA CGCCCCRCAC ACflTGATGTB CGCGGTGGGG 0 C T R G S H R L P E K E R P T R M M Y R U G 601 RTRCRRTGRA RGRGRRRAGR TRGRRGRRRR RRRRGRTRC~ RCRTCGTRCR CRCCCAGCRR CCCCTCCCRR Y It E R E K I E E K K O T T S Y T P S H P S O 721 CCRRCRRRTT RRRCCTCRTR TCRCRRRTRR TRRRCRARRR RCRRCRCTTG RRRRRRCTRR RRCRGRRCAR K P H ' T N H K 0 K T T L E K T K T E O o o 841 TGORCRGGCI GCTGCR RR ~ ~ G T G R C R G 0 KRA G 0 R 2q68 ACAGGCCGCC GCA~GTGRCfi RflCCRTCGCT ATTTGGRCRO G~CGCTGCA~ GTGRCRRRCC RCCRCCfTTT R|O 0 K P $ L F IG (~ R R Rl G 0 K e P P F 3088 A~G6TGRCR RR CCRTCRCCRT TTGGRCAOGC C G C T G C ~ G I GRCRRRCCflT CRCCRTTTGG RCRGGCCGCT P s e r ° ~ R r r~G 0 K P S P r G q R R I0 o K 3200 RTCRCCRTTT OC GRC CI~ RG GG G R CTGC RRA ~ GTGRO G CRR RTCRK GCP C S CC TGORCp BIT~ 0 oAGGCCGC B TGCAR ? R GTA G0 BCB G K S P F GCR~OTGRCR RflCCATCGCT RTTTGGRCBG GCCOCTGCA~O GTGflCRRACC RCCflCCRITT R ~G 0 K P S L F Gt . ql R R R| G 0 K P P P F 3~48 oro.c... cc~qTcRCCRT TTGOACROGC COCTGCOIGGT GRCflARCCRC CRCCRTTTG6 RCRGGCTGCT ~GG 0 K P S P F G 9 A fl R1 G 0 K P P F F G ~ ~ R 3568 TC~BAGCRCT G'GTTTGCGfl RTGCGCCTGG TGTTGCCCAG GTGTCGTTTG GAflRGCCRTC GACGRCGTTT R S T U F R H A P G U A Q J S F G K P S I I F 3688 tGGTTCTATT TTRCAGARTG TTCATGCT~C ATTGCCTGCC GGARSCGCGT TGTCTCA;AT TGGARGTGCC L 0 ~ U H R T L P R 0 5 A L S H I G S A G S 38138 AAGTGGTCCG GCAATIGGTG GTOGTqfiACT ACCTGCRTTG 5RGOGflFGTG GGTTTGGTCA'GGCTTTTAGT S G P R I G G G K L P A L E G S G F G Q R F S 3928 AGGTGRGGGC RCCTITGGTR CT3TOGOAGC CCRTGGGRCG GGTTCCGCCA CCCCGITITC RARGRCCAGT T F G T U G f l H G T G S R T P F S K T S G E G 4048 TCTCCGCRRT TTR~GRGTGT TTTTGGTCCT CRTTGRTRTG CGGTTTTTtT TTRRTCTGR~ TTTITGCTTG L B H L R U ~ k Y L I 0 M R F F F ~ l ~ F C L 4168 TTGCTTOCTG RRfiGCCRTTT GTTTTTTGGT TGTTGTTGTT GTTGTTTTTG GCTCAAGGAP TABCATTTGG 4288 GRTGT RCCOR GTCTTTTqGR TCTTCTCAGG C R C C T G A C T G flTGCqCCGGC TOTGRCTGCA TCTGCGCTOC 4408 Ri ;CRCRGRC CRACAAGGCG TGTCGTRGGG R C I G C R T C T C TTTTTGTTGR ACCCAARTTT TCGCGCGGTG 4520 ~ 3 G C ~ R R G CTTGGGCRGG CCC~T'GRTG qCCTtGCRBT ATTGCCGT
0
E
Q
GTCRTCTIIG U i F TATRCCCCCC Y T P CCCflRCGCCC P i4 A
I
P
S
L
0
L
0
1
T
R
L
O
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OTGCTGRGTC GCTCCTTTCR CRCTGCRRRR GGCTCflCTGC G fl E S L L S 14 C K R L T R ATTflTCCCAA CfiGRRRRATG CCTTTGTRCR RITCflACCflA H ¥ P N R ~ ff P L Y H S T i~ flRRRflRGCTT TflRTCCTTGT QCGIGflCRRRT TGRflTRTRFIA Q K S F H P C T 0 K L H I 14
A~R~CfiGCGC"C'R-T'r'T'G))-C'R'G'G];'C'Gi~"T'O'C'R'[GOTGnCRmnC CRtCGCTRTT K
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CCflTCRCCOT TIGGflCRGGCCGCCGCfl~OTGRCRRGCCRT c R c c e r r r e e P s P F 0 ~ R ~ R 10 O K P S P F G GGRCRGGCCG C R G C ~ G T G R CRRRCCRTCG CTATTTGGRC RGGCCGCIGC i
GCR~°T G f l0C R0R IzRRCCRTCGCTp S L RTTTGGRCflGF ° q GCCGCTGCRIGR R RO / .G, GTGflGRRRCCIz P CCflTCRCCRT P S P GGRCRGGCCG G ~ A GCR~GTGRCR
R| O 0 GCAACCflCRT R T T S TTTGGTTCrC F G S GCTTTIGGCA A F O H ACCACATTTC T T F Q TGfiATGRT~A * GCATGTCGGR RGGCRRTTGC GAAAATCIGT
TTfiORCflGGC COCTOCRIGGI OflCRflRCCRC CRCCRTTI00 F 0 ~ R ~ A ~G 0 K P P P F fi CTGC ~ G T G R CRRRCCATCA CCRTTTOGRC RGGCCGCRGC A R| G 0 K P S P F G ~ A 8 G~CCRTCRCC RTTTGGRCRG GGRRC,~G~GT TTORTGCOTC
R P 0
P
CCTTTGCTGC F R R G CTGGRATGCR P G R X ATCfiTGCflTC H R S T ARTCTTCTTC S S S ~ TCAATTflATT
F G O ~_..T...U
TGGTGGTGGG G G F G TTCTGGCGGT $ G G RRCTGTTTTA V L G H TTTTTCCRGT S $ 1 A TGRRGTIGTT
F 0
fi
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TTTGGRGCAC CRRGTGCTTT R P S B F GCGTTTGGTG GRGCPGGTGT A F G G R G U 00RARTTTTG GTCflGRRRGR F G O K E RTTGCGGGAA RCIRCRRGGC G H V K A T;GGGGRTGT TTGTGCRRCT
TGRRTTGGGT GRACTGGAAT TPRGGGRRGT TGAGGCRCGT CGTCRRGCTG 3CATGGTAAC GGGRCRCfiTT GfiRGCATGTC GTTGTTGRTC
TGAflGRGGGG ARRGGTTTTT CCTCRTRTCG
Fig. 1. Nucleotide and deduced amino acid sequence of the TcCA-2 gene. Four upstrcam codons are underlined. Arrows indicate predicted trans-splicing cleavage sites. The repeat units of 12 amino acids are boxed. The shadowed box represent approximately 57 repeat units as estimated by Hhal and Pstl digestion analysis (sec text). The hatched lines denole degenerate repeat units.
75 tandemly arranged 36-base-long repeats and finally, a non-repeated region of 570 bp at the 3' end. The region encoding the repeated units was not completely sequenced. The number of repeat units is based on restriction endonuclease analysis, and is known to vary between allelic forms of the gene [16]. The genomic organization of TcCA-2 has been previously studied in several parasite strains, including another clone of CA-l, number 72 [16]. Only two copies of the gene per parasite located in different chromosomes, were detected. It is possible that both copies are expressed since we have detected two RNA species, at least in RA strain [9]. We cannot tell at present whether the sequence provided belongs to a gene which is actively expressed. The expected size of the protein as deduced from the D N A sequence is around 155kDa. We had previously found that the protein
TcCA-2 has an apparent molecular weight of 85kDa in Western blots, with some reacting material remaining in the slot. This discrepancy might be due to the fact that the antigen is unstable, being quickly degraded when preparing protein extracts from parasites or even when the protein is expressed in bacterial systems (unpublished observations). New protein extracts were prepared. Parasites from the RA strain were collected by centrifugation and immediately resuspended in cracking buffer and boiled. The CA-1/72 clone used was kept at - 7 0 ~ C for one week and thawed directly in crackin~ buffer. The volumes corresponding to 2 × 10 parasites per well, were layered in a 10% SDS-PAGE and electrophoresed. The Western blot was reacted with antibodies made in rabbits against the TcCA-2 antigen expressed in a recombinant D N A clone [10]. The results in Fig. 2 show that there are two major
127
KDa
1
M
2 slot.
180 116 -
other significant homology with any published sequence, including any major surface antigen gene from T. cruzi [2-6]. Furthermore, the amino acid sequence Ser-X-Asp-X-Gly-X-ThrTrp, characteristic of bacterial neuraminidases and members of T. cruzi antigens, is not present in TcCA-2.
Acknowledgements 4/1.5-
Fig. 2. Detection of TcCA-2 protein in trypomastigote extracts. Protein extracts from RA and CA-1/72 parasites were prepared as described in the text, electrophoresed in a 10% polyacrylamide gel and transferred to a nitrocellulose filter. The blot was reacted with a rabbit antiserum raised against the carboxy terminus of TcCA-2 protein [10].
bands of around 140-160kDa in RA strain and one band of around 180kDa in CA-1/72 clone of T. cruzi. These sizes are in good agreement with the expected molecular weight deduced from the DNA sequence. In addition, there is another reacting band of around 50 kDa. If parasites are kept at -20~C or, after thawing, they are not immediately resuspended in cracking buffer and boiled, bands of smaller molecular weight are observed (results not shown). Thus, we consider likely that the size we had previously reported for TcCA-2 was not the correct one, due to degradation of this unstable protein. Hoft et al. [17] reported a partial amino acid sequence deduced from DNA sequence of a T. cruzi gene (TCR39), that includes a few repeat units and the carboxy-terminus. Comparison of TcCA-2 with TCR39 shows that both proteins contain the same repeated motif but different carboxy-termini, with several stretches of amino acids being conserved. Sequence comparison, in the GenBank (release 65) and the EMBL (release 28) data bank, showed no
We thank Carlos Ibfifiez for his help with the first sequences of TcCA-2, Drs. J.J. Cazzulo, U. Pettersson and L. A,slund for useful discussions and L. Sferco and M. Guerin for technical assistance. This work was supported by grants from the UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical diseases, the Swedish Agency for Research Co-operation with Developing Countries (SAREC), the Consejo Nacionai de lnvestigaciones Cientificas y T6cnicas and the University of Buenos Aires, Argentina and the Programa Regional de Biotecnologia PNUD/UNESCO/ONUDI. O.C., D.O.S. and A.C.C.F. are researchers from the Consejo Nacional de lnvestigaciones Cientificas y T~cnicas, Argentina.
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