Nucleic Acids Research, Vol. 19, No. 20 5789
Nucleotide sequence of the gene coding for the 85-B antigen of Mycobacterium leprae Leila de Mendon9a Lima, Jean Content1, Hugo van Heuverswyn2 and Wim Degrave* Department of Biochemistry and Molecular Biology and Laboratory of Hanseniasis, Department of Tropical Medicine, Oswaldo Cruz Institute, FIOCRUZ, Av. Brasil 4365, Rio de Janeiro, RJ, CEP 21045, Brasil, 1Institut Pasteur du Brabant, Department of Virology, B-1 180 Brussels and 2Innogenetics, Industriepark Zwijnaarde 7, box 4, B-9052 Gent, Belgium Submitted August 20, 1991
EMBL accession no. X60934
ACKNOWLEDGEMENTS
Antigens of the 85 complex, present in culture supernatants of a variety of mycobacteria, play an important role in humoral and cellular immune responses (1). As no data on the structure of the corresponding M. leprae proteins were available, we decided to clone the genes coding for the different antigens belonging to this complex. Using a Eag I/BstE II 900 bp DNA fragment coding for part of the M.tuberculosis 32 kDa protein (antigen 85-A; 2) we screened a M.leprae genomic library constructed in the vector lambda-dash (3). Several clones were isolated and were shown, through hybridization and sequence analysis, to code for antigens 85-A (32 kDa), 85-C (33 kDa) and 85-B (28 kDa, alpha antigen), as judged from sequence homology with the corresponding M.bovis BCG (4, 5), M.tuberculosis (1, 2) and M. kansasii (6) genes. Here we show that the presumably secreted M. leprae 85-B antigen shares 86.3% and 84.5% homology, respectively, to the M.kansasii and M.bovis BCG corresponding mature antigens (Figure iB). A presumable 38 amino acid signal peptide, as judged by sequence homology and hydrophobicity, precedes the 289 amino acid long mature protein. In the 5' upstream sequence, putative promoter and ribosome binding site sequences could be identified which are homologous to the E. coli consensus (Figure 1A).
This work received financial support from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases. L.M.L. was supported by a training grant from TDR and by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico - CNPq, Brasil. We thank Dr R.J.W.Rees (NIMR, London) for his kind gift of M.leprae infected Armadillo liver and Dr J.Colston (NIMR, London) for his help in M.leprae DNA preparation.
REFERENCES 1. Content,J., de la Cuvellerie,A., De Wit,L., Vincent-Levy-Frebault,V., Ooms,J. and De Bruyn,J. (1991) Infec. Immun., 59, in press. 2. Borremans,M., De Wit,L., Volckaert,G., Ooms,J., De Bruyn,J., Huygen,K., Van Vooren,J., Stelandre,M., Verhofstadt,R. and Content,J. (1989) Infec. Immun. 57, 3123-3130. 3. Santos,A., Miranda,A., Mendonca-Lima,L., Suffys,P. and Degrave,W., submitted. 4. De Wit,L., de la Cuvellerie,A., Ooms,J. and Content,J. (1990) Nucl. Acids Res. 19, 3995. 5. Matsuo,K., Yamaguchi,R., Yamazaki,A., Tasaka,H. and Yamada,T. (1988) J. Bacteriol. 170, 3847-3854. 6. Matsuo,K., Yamaguchi,R., Yamazaki,A., Tasaka,H., Terasaka,K. and Yamada,T. (1990) Infec. Immun. 58, 550-556. -35
A
M. 1 prae
M. kanea i M.bovi a BCG
AC00TCG0ATCCIOACQG0TAACCGATACAGAACTOAGATCQCTCWrCATTCOGACAO ..C.. T..TG.c.
........
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.
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N. I *pra. M. karJaca i M.bovi
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M. kanecii M. bovia CO
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-G.
AG. C. A... C. -C ......
MIDVSG2IRAWGRWLLVAAAT--LP8LISLAGSATASAF8RPGLPVIYLQSPSTKFRTVQSPAVLLDOLRAQDDNDINT8AZEKrrQSGL8WM .T .. R. 0. ..P . I. A .8. S.DN AAA. .O.VQ P ...D. 8 .. . P. I. .TR R. R.MI.T ..AW o.va a0. PVWOSFY8nGfYSPACTYGTLT8tLSANRVK ....................................
.................
.
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.................. ............ .......... P
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A. .
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...
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VR90GPPNDPAQRNDPIL4QAGKLvANNTLNVYCoTPSZLaoNVPAzFLzNFvHGsNLKFQDAY__swHntNArWQNAPDLv A. A. D.N .... G AS..R---.8. S.HIPZ ..... R.I RS. N .... A.. . KP . FPPN ..0 ... SS.G. R ..DD .. .... STQ..P . R .
Figure 1. A. Alignment of the 5' upstream sequences of the 85-B genes from M. leprae, M. kansasii (6) and M. bovis BCG (5). Dots represent homology to the M. leprae sequence. Possible regulatory regions are indicated. B. Alignment of 85-B amino acid sequences. The arrow indicates the probable beginning of the mature M. leprae protein.
*
To whom correspondence should be addressed
Nucleotide sequence of the gene coding for the 85-B antigen of Mycobacterium leprae Leila de Mendon9a Lima, Jean Content1, Hugo van Heuverswyn2 and Wim Degrave* Department of Biochemistry and Molecular Biology and Laboratory of Hanseniasis, Department of Tropical Medicine, Oswaldo Cruz Institute, FIOCRUZ, Av. Brasil 4365, Rio de Janeiro, RJ, CEP 21045, Brasil, 1Institut Pasteur du Brabant, Department of Virology, B-1 180 Brussels and 2Innogenetics, Industriepark Zwijnaarde 7, box 4, B-9052 Gent, Belgium Submitted August 20, 1991
EMBL accession no. X60934
ACKNOWLEDGEMENTS
Antigens of the 85 complex, present in culture supernatants of a variety of mycobacteria, play an important role in humoral and cellular immune responses (1). As no data on the structure of the corresponding M. leprae proteins were available, we decided to clone the genes coding for the different antigens belonging to this complex. Using a Eag I/BstE II 900 bp DNA fragment coding for part of the M.tuberculosis 32 kDa protein (antigen 85-A; 2) we screened a M.leprae genomic library constructed in the vector lambda-dash (3). Several clones were isolated and were shown, through hybridization and sequence analysis, to code for antigens 85-A (32 kDa), 85-C (33 kDa) and 85-B (28 kDa, alpha antigen), as judged from sequence homology with the corresponding M.bovis BCG (4, 5), M.tuberculosis (1, 2) and M. kansasii (6) genes. Here we show that the presumably secreted M. leprae 85-B antigen shares 86.3% and 84.5% homology, respectively, to the M.kansasii and M.bovis BCG corresponding mature antigens (Figure iB). A presumable 38 amino acid signal peptide, as judged by sequence homology and hydrophobicity, precedes the 289 amino acid long mature protein. In the 5' upstream sequence, putative promoter and ribosome binding site sequences could be identified which are homologous to the E. coli consensus (Figure 1A).
This work received financial support from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases. L.M.L. was supported by a training grant from TDR and by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico - CNPq, Brasil. We thank Dr R.J.W.Rees (NIMR, London) for his kind gift of M.leprae infected Armadillo liver and Dr J.Colston (NIMR, London) for his help in M.leprae DNA preparation.
REFERENCES 1. Content,J., de la Cuvellerie,A., De Wit,L., Vincent-Levy-Frebault,V., Ooms,J. and De Bruyn,J. (1991) Infec. Immun., 59, in press. 2. Borremans,M., De Wit,L., Volckaert,G., Ooms,J., De Bruyn,J., Huygen,K., Van Vooren,J., Stelandre,M., Verhofstadt,R. and Content,J. (1989) Infec. Immun. 57, 3123-3130. 3. Santos,A., Miranda,A., Mendonca-Lima,L., Suffys,P. and Degrave,W., submitted. 4. De Wit,L., de la Cuvellerie,A., Ooms,J. and Content,J. (1990) Nucl. Acids Res. 19, 3995. 5. Matsuo,K., Yamaguchi,R., Yamazaki,A., Tasaka,H. and Yamada,T. (1988) J. Bacteriol. 170, 3847-3854. 6. Matsuo,K., Yamaguchi,R., Yamazaki,A., Tasaka,H., Terasaka,K. and Yamada,T. (1990) Infec. Immun. 58, 550-556. -35
A
M. 1 prae
M. kanea i M.bovi a BCG
AC00TCG0ATCCIOACQG0TAACCGATACAGAACTOAGATCQCTCWrCATTCOGACAO ..C.. T..TG.c.
........
TC
A
.
-10 CATCAAAT CC C. A C. a
GA. .... C. T CCA... -T. .C.CGA.T .. C. CCA.A. C...
A
-COACAC
a0.. OCa Tao.CC.A... .0.
SD M. M.
1.prao
keanaaali
N.boviJ DCG
B
N. 1epra. . kansasii
M.bovia C
N. I *pra. M. karJaca i M.bovi
N. I *pra.
BC
M. kanecii M. bovia CO
ATAC--GATAA-aOG0TATTAO0TrP7AY .. .. .. ..
-0. C ..-...
C.
-G.
AG. C. A... C. -C ......
MIDVSG2IRAWGRWLLVAAAT--LP8LISLAGSATASAF8RPGLPVIYLQSPSTKFRTVQSPAVLLDOLRAQDDNDINT8AZEKrrQSGL8WM .T .. R. 0. ..P . I. A .8. S.DN AAA. .O.VQ P ...D. 8 .. . P. I. .TR R. R.MI.T ..AW o.va a0. PVWOSFY8nGfYSPACTYGTLT8tLSANRVK ....................................
.................
.
L.
AL8MA8SALI AAYHPDQFIYAOSLSALtD89QG1 PQLI A1Q I .Q.8.. a 8
.................. ............ .......... P
Q.
A.
.... Al .
A. .
Q.
...
L.P ...S.
VR90GPPNDPAQRNDPIL4QAGKLvANNTLNVYCoTPSZLaoNVPAzFLzNFvHGsNLKFQDAY__swHntNArWQNAPDLv A. A. D.N .... G AS..R---.8. S.HIPZ ..... R.I RS. N .... A.. . KP . FPPN ..0 ... SS.G. R ..DD .. .... STQ..P . R .
Figure 1. A. Alignment of the 5' upstream sequences of the 85-B genes from M. leprae, M. kansasii (6) and M. bovis BCG (5). Dots represent homology to the M. leprae sequence. Possible regulatory regions are indicated. B. Alignment of 85-B amino acid sequences. The arrow indicates the probable beginning of the mature M. leprae protein.
*
To whom correspondence should be addressed
