4252 Nucleic Acids Research, Vol. 18, No. 14
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A putative flagellar Ca2+-binding protein of the flagellum of trypanosomatid protozoan parasites Mary Gwo-Shu Lee, Jifan Chen, Allan W.M.Ho, Philip A.D'Alesandro and Lex H.T.Van der
Ploeg'
Division of Tropical Medicine, School of Public Health and 1Department of Genetics and Development, Columbia University, New York, NY 10032, USA Submitted June 13, 1990
Several cDNA clones of the protozoa Trypanosoma brucei and Trypanosoma lewisi were isolated as a reult of selection for evolutionarily conserved genes encoding cell-surface proteins (Lee and Van der Ploeg, unpublished). cDNA clone Th-17 had a predicted open reading frame, encoding a 23 kDa protein (Figure IA). This protein shared significant homology with a flagellar Ca2+ binding protein (FCaBP) previously identified in Trypanosonw cruzi (1, Figure IB). The predicted Th-17 protein encoded the 'E-F hand' Ca2+ binding site homologous to the Ca2+ binding site of Calmodulin in T. brucei (2, 3). Th-17 is expressed at a relatively high level in both the bloodstream form and the insect form of T. brucei (data not shown). The evolutionary conservation, the presumed flagellar location and its potential Ca2+ binding ability, suggest that this protein may play a role in cell motility or in Ca2+ transport.
ACKNOWLEDGEMENTS We thank Sylvia Le Blancq for critical reading of the report. This work was supported by Grants from the NIH, no. AI 21784 to LHTVDP and AI-13387 to PAD and by a Grant from the John D. and Catherine T.MacArthur Foundation to LHTVDP. LHTVDP is a Burroughs Weilcome Scholar in Molecular Parasitology.
EMBL accession
IIINT-ISYON
GTAAAATAAACAAGC AAG GAT GGT GCC GAC CGG AAG GTT AAG GAT GCT GAG GAC ACG AAC GGG TGC TAC AGT ATA ATT GTC CAG CGT AAG GGC GTG GGT ATG CTT TGC TAC ACC ATG GAT AAG GAA GCC TTG CCG ACC ACT GTG TTT TTC GAT GAA TTC GGC GAT CCT GAT AAT GCA GGG GAT
no.
X53464
LGTTAATCAAGCAAAGCCAA 7Likil-AGTTTCTGTALCTATJO GGA TOG AAG AAC GCA TCT AAC CCC
ATG GGT TGC TCA GCC AGT AAG CGT GCG TGG GAG CGT TCG AAA AGC CGT ACT GGA AAA CTC CTC AAG TTG GAT GCG TTT GAC AAA GAA GAG GAT CTT ATC TAT GAT ATC GAT GGA AGT TTG AAA TTG AAG GAG AAT GAG ATC GAC TCA TGC TGG GCT
GGA AAG GAC GGG AAA ACT ACT GCG ATT CGG TGT GCG ATT CCC CGT GAC CGT ATT GAA CTC TTC AAG CAG TTT GGC TTC AGG GAG GTG CTT GAT GGT GAG TTC ACG ACT CAC CTT CCT GAC GCG AAG GAC CTC GGC AAC AAG GTA GTG GAG TTC CTT GAA TTC CGC CTG TTT GAG TTA ACT GTA ATC TTC GAC TTG CTT GAG CTA CAC GAG TTC AAG TGG GGT GTT GAT ATC ACT GAC GCT ACT AAT GGA TCT GGT GTT GTG ACA GTC ACT AAA AAG TTG CAG GTA TCT GAT GAG GAG AAC GGA GCA AAT GAA GGT GAC GGG GCA GGA GTT CCT GCG GCT GAG GGT TCT GCC TAGTGTTGAGA - 16 ., ,.V%. ra..I.I.Wom ~~~~~~~~~CGTAGAGATCTG G2TCIS TTTTTPT PAAG. mATGCTTT TGTTCTTTTT1ACCA CCCCICGGTCCATATTATTTAATAATAT TGTTTCA mGTG I 1 l' AAA&CTCA1ATGTTTTTTTTTGTCTTCTT?'JAT?CTTTTTTTTAC TTGTCTC CGACTGC CTAI( AC _TT TTm GTT CTT VWG ATTTATG hATAX ITCCTl=T] I =C ~T ITC GCT CGGTGACATCTGGGACCAGTAGA GTGCTGC RITCG. AAGCACACATTTCGCG GAGTGCG CCAT( AG AAG GAGMA _ CGACACACTCGTGG WA
_GTACGTGATCTCTCATGTCA TTTGTAATCTTTTTCGTTCTAGCAAAATAGTTTTTTTATTT CCGGTTGTGTAATAGATATACGCTCCATGGGCCTGTTAGGGTAAAA AAAAAMAAA
Figure IA. DNA sequence analysis of cDNA clone Tb 17 from T. brucei. Boxed region is mini-exon; single spaced sequences represent untranslated extensions of the mRNA.
Th-17 MGCAGSKNASNPKDGAASKGGKDGXTTADGKVAWEGIRCAIPRDKDAESKSRRIELFXQ TcFCaBP
Tl- 17
MGACGSKGSISDKGLASD****KAK*R*E***R**Q****E*T**A*Q*******K RRSKNSSSSK****AAKTARLRGRLLRQT***ERTD*ARQ***D***K
FDTNGTGKLGFREVLDGCYSILKLDFT'THLPDIVQRAFDKAKDLGNKVKGVGEEDLVEFLEFR **K*E****CYD**NS**LEV*******PRVR**TK******RA**S*LENX*S**F*******
REFERENCES 1. Engman,D.M. et al. (1989) J. Biol. Chem. 264, 18627-18631. 2. Tschudi,C. et al. (1985) Proc. Natl. Acad. Sci. USA 82, 3998-4002. 3. Persechini,A. et al. (1989) TINS 12, 462-467.
**K*NS***SYD**YR**IDE********RVR**TK***N****N****EKT*S**F******* * b*s* I* ****D TIT* **V*O***T LIMLCYIYDIFELTVNFDThDKDSLIDITDATTVFNEIDTNGSGVV ********F********EI*AS*NN*VDZZ*L*V***ZA**AV*PAAL*K*L*K**T*S* ********Y********KI*TS*Nm**DDJ**IK*V***EK**LK* X**XQL*XK***** "Z-F hand" Ca -binding site-i 'E-F hand" Ca"-
TFDEFSCWAVTKKLQVSGDPDDZZNGANRGDGANAGDGVPAAEGSA *****AA**SAV**DAD****NVPFS* *****AA**SA***DAE****K
Binding sit--2
Figure 1B. Amino-acid sequence comparison of the predicted Ca2+ binding protein from T. brucei (Tb-17), the partial sequence from T. lewisi (TI-17), T. cruzi (TcFCaBP; 1) and the Ca2+ binding sites of Calmodulin from T. brucei (boxed sequence; 2). The underlined sequences indicate the 'E-F hand' Ca2+ binding sites (3). Asterisks mark identical amino acid residues.
_
A putative flagellar Ca2+-binding protein of the flagellum of trypanosomatid protozoan parasites Mary Gwo-Shu Lee, Jifan Chen, Allan W.M.Ho, Philip A.D'Alesandro and Lex H.T.Van der
Ploeg'
Division of Tropical Medicine, School of Public Health and 1Department of Genetics and Development, Columbia University, New York, NY 10032, USA Submitted June 13, 1990
Several cDNA clones of the protozoa Trypanosoma brucei and Trypanosoma lewisi were isolated as a reult of selection for evolutionarily conserved genes encoding cell-surface proteins (Lee and Van der Ploeg, unpublished). cDNA clone Th-17 had a predicted open reading frame, encoding a 23 kDa protein (Figure IA). This protein shared significant homology with a flagellar Ca2+ binding protein (FCaBP) previously identified in Trypanosonw cruzi (1, Figure IB). The predicted Th-17 protein encoded the 'E-F hand' Ca2+ binding site homologous to the Ca2+ binding site of Calmodulin in T. brucei (2, 3). Th-17 is expressed at a relatively high level in both the bloodstream form and the insect form of T. brucei (data not shown). The evolutionary conservation, the presumed flagellar location and its potential Ca2+ binding ability, suggest that this protein may play a role in cell motility or in Ca2+ transport.
ACKNOWLEDGEMENTS We thank Sylvia Le Blancq for critical reading of the report. This work was supported by Grants from the NIH, no. AI 21784 to LHTVDP and AI-13387 to PAD and by a Grant from the John D. and Catherine T.MacArthur Foundation to LHTVDP. LHTVDP is a Burroughs Weilcome Scholar in Molecular Parasitology.
EMBL accession
IIINT-ISYON
GTAAAATAAACAAGC AAG GAT GGT GCC GAC CGG AAG GTT AAG GAT GCT GAG GAC ACG AAC GGG TGC TAC AGT ATA ATT GTC CAG CGT AAG GGC GTG GGT ATG CTT TGC TAC ACC ATG GAT AAG GAA GCC TTG CCG ACC ACT GTG TTT TTC GAT GAA TTC GGC GAT CCT GAT AAT GCA GGG GAT
no.
X53464
LGTTAATCAAGCAAAGCCAA 7Likil-AGTTTCTGTALCTATJO GGA TOG AAG AAC GCA TCT AAC CCC
ATG GGT TGC TCA GCC AGT AAG CGT GCG TGG GAG CGT TCG AAA AGC CGT ACT GGA AAA CTC CTC AAG TTG GAT GCG TTT GAC AAA GAA GAG GAT CTT ATC TAT GAT ATC GAT GGA AGT TTG AAA TTG AAG GAG AAT GAG ATC GAC TCA TGC TGG GCT
GGA AAG GAC GGG AAA ACT ACT GCG ATT CGG TGT GCG ATT CCC CGT GAC CGT ATT GAA CTC TTC AAG CAG TTT GGC TTC AGG GAG GTG CTT GAT GGT GAG TTC ACG ACT CAC CTT CCT GAC GCG AAG GAC CTC GGC AAC AAG GTA GTG GAG TTC CTT GAA TTC CGC CTG TTT GAG TTA ACT GTA ATC TTC GAC TTG CTT GAG CTA CAC GAG TTC AAG TGG GGT GTT GAT ATC ACT GAC GCT ACT AAT GGA TCT GGT GTT GTG ACA GTC ACT AAA AAG TTG CAG GTA TCT GAT GAG GAG AAC GGA GCA AAT GAA GGT GAC GGG GCA GGA GTT CCT GCG GCT GAG GGT TCT GCC TAGTGTTGAGA - 16 ., ,.V%. ra..I.I.Wom ~~~~~~~~~CGTAGAGATCTG G2TCIS TTTTTPT PAAG. mATGCTTT TGTTCTTTTT1ACCA CCCCICGGTCCATATTATTTAATAATAT TGTTTCA mGTG I 1 l' AAA&CTCA1ATGTTTTTTTTTGTCTTCTT?'JAT?CTTTTTTTTAC TTGTCTC CGACTGC CTAI( AC _TT TTm GTT CTT VWG ATTTATG hATAX ITCCTl=T] I =C ~T ITC GCT CGGTGACATCTGGGACCAGTAGA GTGCTGC RITCG. AAGCACACATTTCGCG GAGTGCG CCAT( AG AAG GAGMA _ CGACACACTCGTGG WA
_GTACGTGATCTCTCATGTCA TTTGTAATCTTTTTCGTTCTAGCAAAATAGTTTTTTTATTT CCGGTTGTGTAATAGATATACGCTCCATGGGCCTGTTAGGGTAAAA AAAAAMAAA
Figure IA. DNA sequence analysis of cDNA clone Tb 17 from T. brucei. Boxed region is mini-exon; single spaced sequences represent untranslated extensions of the mRNA.
Th-17 MGCAGSKNASNPKDGAASKGGKDGXTTADGKVAWEGIRCAIPRDKDAESKSRRIELFXQ TcFCaBP
Tl- 17
MGACGSKGSISDKGLASD****KAK*R*E***R**Q****E*T**A*Q*******K RRSKNSSSSK****AAKTARLRGRLLRQT***ERTD*ARQ***D***K
FDTNGTGKLGFREVLDGCYSILKLDFT'THLPDIVQRAFDKAKDLGNKVKGVGEEDLVEFLEFR **K*E****CYD**NS**LEV*******PRVR**TK******RA**S*LENX*S**F*******
REFERENCES 1. Engman,D.M. et al. (1989) J. Biol. Chem. 264, 18627-18631. 2. Tschudi,C. et al. (1985) Proc. Natl. Acad. Sci. USA 82, 3998-4002. 3. Persechini,A. et al. (1989) TINS 12, 462-467.
**K*NS***SYD**YR**IDE********RVR**TK***N****N****EKT*S**F******* * b*s* I* ****D TIT* **V*O***T LIMLCYIYDIFELTVNFDThDKDSLIDITDATTVFNEIDTNGSGVV ********F********EI*AS*NN*VDZZ*L*V***ZA**AV*PAAL*K*L*K**T*S* ********Y********KI*TS*Nm**DDJ**IK*V***EK**LK* X**XQL*XK***** "Z-F hand" Ca -binding site-i 'E-F hand" Ca"-
TFDEFSCWAVTKKLQVSGDPDDZZNGANRGDGANAGDGVPAAEGSA *****AA**SAV**DAD****NVPFS* *****AA**SA***DAE****K
Binding sit--2
Figure 1B. Amino-acid sequence comparison of the predicted Ca2+ binding protein from T. brucei (Tb-17), the partial sequence from T. lewisi (TI-17), T. cruzi (TcFCaBP; 1) and the Ca2+ binding sites of Calmodulin from T. brucei (boxed sequence; 2). The underlined sequences indicate the 'E-F hand' Ca2+ binding sites (3). Asterisks mark identical amino acid residues.
