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A recombinant Trypanosoma cruzi trans-sialidase lacking the amino acid repeats retains the enzymatic activity
MOLECULAR
ELSEVIER
Molecular and Biochemical Parasitology 64 (1994) 337 340
BIOCHEMICAL PARASITOLOGY
Short Communication
A recombinant Trypanosoma cruzi trans-sialidase lacking the amino acid repeats retains the enzymatic activity Oscar E. Campetella, Antonio D. Uttaro, Armando J. Parodi, Alberto C.C. Frasch* lnstituto de Investigaciones Bioquimicas, Fundaci6n Carnpomar, Av. Patricias Argentinas 435, 1405 Buenos Aires, Argentina Received I October 1993; accepted 17 January 1994
Key words'." Trypanosoma cruzi; Trans-sialidase; Neuraminidase; SAPA antigen; Sialic acid
Trans-sialidase (TS) is an enzymatic activity found in Trypanosoma cruzi, the agent of American trypanosomiasis or Chagas disease, and, recently, in the African parasite Trypanosoma brucei [1-3]. This enzyme differs from mammalian sialyltransferases as the latter employs only CMPsialic acid as sugar donor. TS employs glycolipids and glycoproteins as donors [4-5], with sialic acid linked c~(2 3) to a terminal galactose in either Nor O-linked oligosaccharides, in gangliosides or in lactose [1-2]. Molecules accepting sialic acid must have a terminal galactose present in N- or Olinked oligosaccharides, gangliosides or N-acetyllactosamine [1-2]. The T. cruzi enzyme has been related to cell invasion (reviewed in [6]), where
r~orresponding author. Fax: (54-1) 8652246 or (54-1) 881916. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession No. L26499. Abbreviations." NA, neuraminidase; TS, trans-sialidase; SAPA, shed acute phase antigen; PCR, polymerase chain reaction. 0166-6851/94/$7.00 ~'~ 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 6 8 5 1 ( 9 4 ) 0 0 0 3 6 - M
sialic acid seems to play a key role. The TS from T. cruzi trypomastigotes contains a C-terminal and an N-terminal domains [7]. The C-terminal domain contains variable number of 12-amino acid-long repeats in tandem, that constitutes the SAPA antigen [8 10]. These repeats were suggested not to have any influence on the enzymatic domain of the molecule [7]. Antibodies bound to the amino acid repeats did not affect enzymatic activity [2,7]. On the other hand, TS is inhibited by antibodies directed to the N-terminus (Leguizam6n et al, submitted). Chimeric protein constructs suggested a putative region of the gene necessary for enzymatic activity within the Nterminal portion [11]. However, a clear-cut localization of the enzymatic activity in the N-terminus and of the absence of effect of the SAPA domain on the TS activity is lacking. There are several TSrelated molecules which are encoded by different genes (reviewed in [12]), only some of them having TS activity [11]. Very recently, a TS present in the epimastigote stage derived from axenic cultures has been described [13]. It does not contain the SAPA repeats, but information on the C-ter-
O.E. Campetella et al./Molecular and Biochemical Parasitology 64 (1994) 337-340
338
minus, together with its sequence is not available yet. Here we show that the amino acid repeats are not necessary for the enzymatic activity of TS. The 5' region of a SAPA gene contained in a genomic D N A fragment from T. cruzi RA strain was subcloned by PCR and expressed in p K K 233-3 (Pharmacia-LKB, Uppsala, Sweden). The 5'-end primer was designed from the mature protein [15] and an A T G codon was included: 5' ATGCTGGCACCCGGATCGAGCCGAGTTGA G C T G 3'. The 3'-end primer includes a third of the first repeat followed by a stop codon and an EcoRI restriction site: 5' G T G G A A T T C A G G C A C T C G T G T C G C T G C T G C T G T C 3'. The construct (Tc-TS1N1) was made from a gene that also encodes the SAPA repeats. Thus, it might be expressed only in the trypo.mastigote stage because the TS recently described in the epimastigote stage has no such repeats [13]. Employing sialyllactose as donor and ]4C-labeled lactose as acceptor molecules in a Tc-TS1Nl-catalyzed reaction, a labeled product with the same electrophoretic mo-
bility as the sialyllactose marker and sensitive to Clostridium perfringens neuraminidase (NA) treatment was detected, as expected from a trans-sialidase reaction (not shown). This clone also displayed NA activity when assayed on 2'-(4-methylumbelliferyl)-~-D-N-acetylneuraminic acid fluorogenic substrate (0.190 for Tc-TSIN1 vs. 0.037 for an inactive control; values are in fluorescence arbitrary units) [2]. These results clearly demonstrate experimentally that the amino acid repeats are not necessary for the NA and/or TS activity. To test if amino acid repeats might modify the activity, some kinetic and specificity parameters were assayed. The apparent Km for sialyllactose of Tc-TS1N1 and the natural enzyme purified from supernatants of Vero cell cultures [15] 0.23 mM and 0.19 mM, respectively. The recombinant enzyme, as well as the native one, transferred ~(2,3)- but not ~(2,6)-linked sialyl units from fetuin-derived glycopeptides to asialofetuin (Table 1). Both enzymes also transferred sialyl units from glycoli-
Table 1 Donor specificities of the natural and recombinant (Tc-TSIN1) trans-sialidases~' Assay
Enzyme activity (Units)
Donor
Enzyme
Sialyl-~-(2,3)glycopeptides Sialyl-~-(2,6)glycopeptides Sialyl-~-(2,3)gl'ycopeptides Sialyl-~-(2,6)glycopeptides
Natural Natural Tc-TSIN1 Tc-TS1NI
64 0 9.2 0
GM3 GM2 GM3 GM2
Natural Natural Tc-TS1N 1 Tc-TS I N 1
250 3.8 48 2
Acceptor specificity of recombinant (Tc-TSIN1) trans-sialidase b Acceptor
Enzyme
Enzyme activity (Units)
[galactose-~4C]~-methylgalactoside [galactose-14C][3-methylgalactoside
Tc-TS 1N 1 Tc-TS 1N I
0.35 79.3
gangliosi.de ganglioside ganglioside ganglioside
Assays were performed in duplicate, the enzyme activity units are expressed as fmol of sialic acid transferred min-~. The low radioactivity values in the absence of enzyme were subtracted. About I /~g of natural and 0.2 2 /tg of recombinant enzymes were employed, as estimated from silver-stained polyacrylamide gels (see text). ~tThe donors in the assay 1 were [sialic acid-14C]sialyl-glycopeptides and unlabeled GM3 or GM2 gangliosides in assay 2. The acceptors were asialofetuin in assay 1 and N-[N-acetylglucosamine-14C]acetyllactosamine in assay 2. For further details see [5]. Reactions were performed under the conditions previously described [2]. hThe donor was sialyllactose, both acceptor galactosides were 300 Ci m o l - ~. Conditions of the assay were as in [5}. Reaction products were submitted to paper electrophoresis in I M pyridine acetate buffer pH 6.5 and radioactivity in charged products quantitated.
O.E. Campetella et al./Molecular and Biochemical Parasitology 64 (1994) 337-340
Tc-TSINI
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L P A L V N V D Q V N V A I
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repeats
Fig. 1. C o m p a r i s o n o f p u t a t i v e l y e n c o d e d p r o t e i n s f r o m T c - T S I N 1 , T C N A u p to t h e first r e p e a t [16] a n d a v a i l a b l e s e q u e n c e s o f c l o n e s 154 ( T S a c t i v e ) a n d 151/121 ( T S i n a c t i v e ) [I 1]. A m i n o a c i d i d e n t i t i e s a r e i n d i c a t e d b y -. G a p s m a d e for a best a l i g n m e n t a r e i n d i c a t e d w i t h stars. R o m a n n u m e r a l s i n d i c a t e t h e p o s i t i o n o f t h e f o u r c o m p l e t e l y o r p a r t i a l l y c o n s e r v e d S X D X G X T W bacterial neuraminid a s e s m o t i f s [17]. T h e p r o p o s e d s u b t e r m i n a l c o n s e n s u s m o t i f f o r t h e s u p e r f a m i l y o f a n t i g e n s [18] is u n d e r l i n e d . T h e a r r o w i n d i c a t e s the start of the SAPA repeats.
pids to N-acetyllactosamine only when the former residues were linked to terminal galactose units (Table 1). Similarly to natural TS[5], Tc-TS1N1 recognized fl- but not a-linked galactose units as acceptors (Table 1). Besides, the specific activfty at 37°C of the natural TS, determined with sialyllactose as donor and lac-labeled lactose as sialic acid acceptor, was 0.01 /~mol of sialic acid transferred m i n - l (mg protein)-I. When the itaduced recombinant Escherichia coli cell-free extracts were run in SDS-PAGE and silver stained, comparison of the intensity of the 70 kDa containing the recombinant trans-sialidase band with standards suggests that the specific activity of the recombinant TS was in the range 0.005~).05 #mol
m i n - I (mg protein)-I and thus comparable to that of the natural enzyme. Therefore, no differences were observed in the donor acceptor specificity and the specific activities seem to be similar between the natural and Tc-TS1NI enzymes in spite of the absence of repeats and the high mannose-type glycosylation. Two sequences from active genes have been published, a partial one encoding an active TS (clone 154) [11] and a related sequence (TCNA) encoding a molecule with NA activity [16]. The latter was never reported to have TS activity. Therefore, no complete sequence of an active TS molecule has been previously described. TcTSIN1 was sequenced and the deduced amino
340
O.E. Campetella et al./Molecular and Biochemical Parasitology 64 (1994) 33~340
acid composition was compared with those sequences (Fig. 1). The total or partially conserved four S X D X G X T W motifs from bacterial neuraminidases [17] and the proposed subterminal V T V X N V f L Y N R family motif [18] are indicated. T C N A is included only up to the first repeat that defines the SAPA antigen. The variation in amino acids 390~,35 of Tc-TS1N1 with respect to T C N A is due to a reading frame change in this region. Concerning the region previously reported as essential for the activity in clone 154 [11], three amino acids are changed in T c - T S I N I " K to E at position 219, A to D at 226 and L to V at 265, suggesting that none of them is related to activity. The Y at position 343, suggested to be necessary for activity, is also present in Tc-TS1N1.
Acknowledgements We are indebted to Dr. J.J. Cazzulo for critical reading of the manuscript. This work was supported by grants from the UNDP/World Bank/ WHO Special Programme for Research and Training in Tropical Diseases (TDR), the Swedish Agency for Research Cooperation with Developing Countries (SAREC), the Consejo Nacional de Investigaciones Cientificas y Tbcnicas and the University of Buenos Aires, Argentina.
References [1] Schenkman, S., Jiang, M.S., Hart G. W. and Nussenzwieg V. (t991) A novel cell surface trans-sialidase ot" Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells. Ceil 65, 1117 1126. [2] Parodi, A. J., Pollevick, G. D., Mautner, M., Buschiazzo, A., Sanchez, D.O. and Frasch, A.C.C. (1992) Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi. EMBO J. I1, 1705 1710. [3] Engstler M and Schauer R. (1993) Sialidases from african trypanosomes. Parasitol. Today 9, 222-225. [4] Schenkman, S., Pontes de Carvalho, L, and Nussenzweig, V. (1992) Trypanosoma cruzi trans-sialidase and neuraminidase activities can be mediated by the same enzymes. J. Exp. Med. 175, 567 575. [5] Garcia Ferrero, M. A., Trombetta, S. E., Sanchez, D. O., Reglero, A., Frasch A.C.C. and Parodi A. J. (1993) The action of Trypanosoma cruzi trans-sialidase on glycolipids and glycoproteins. Eur. J. Biochem. 213, 765 771.
[6] Schenkman, S. and Eichinger, 1~. (1993) Trypanosoma cruzi trans-sialidase and cell invasion. Parasitol. Today 9, 218-222. [7] Cazzulo, J.J. and Frasch, A.C.C. (1992) SAPA/transsialidase and cruzipain: two antigens from Trypanosoma cruzi contain immunodominant but enzymatically inactive domains. FASEB J. 6, 3259 3264. [8] Affranchino, J. L., Ib~iez, C. F., Luquetti, A. O., Rassi, A., Reyes, M. B., Macina, A., Aslund, L., Pettersson, U. and Frasch, A. C. C. (1989) Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas disease. Mol. Biochem. Parasitol 34, 221 228. [9] Reyes, M. B., Lorca, M., Muoz, P. and Frasch, A. C. C. (1990) Fetal lgG specificities against Trypanosoma cruzi antigens in infected newborns. Proc. Natl. Acad. Sci. USA 87, 2846 2850. [10] Leguizamon, M. S., Campetella, O. E., Reyes, M. B., Ibb,ez, C. F., Basombr~o, M. A., Rinc6n, J., rn, A. and Frasch, A. C. C. (1991) Bloodstream Trypanosoma cruzi parasites from mice simultaneously express antigens that are markers of acute and chronic human Chagas disease. Parasitology 102, 379 385. [11] Uemura, H., Schenkman, S., Nussenzweig, V. and Eichinger, D. (1992) Only some members of a gene family in T~vpanosoma cruzi encode'proteins that express both trans-sialidase and neuraminidase activities. EMBO J. 11, 3837 3844. [12] Campetella, O., Stinchez, D. O., Cazzulo, J.J. and Frasch, A.C.C. (1992) A superfamily of Trvpanosoma cruzi surface antigens. Parasitol. Today 8, 378 381. [13] Chaves, L. B., Briones M. R. S. and Schenkman, S. (1993) Trans-sialidase from Trvpanosoma cru=i epimastigotes is expressed at the stationary phase and is different from the enzyme expressed in trypomastigotes. Mol. Biochem. Parasitol. 61, 97-106. [14] Pollevick, G. D., Affranchino, J. L., Frasch, A. C. C. and Stinchez, D. O. (199.1) The complete sequence of SAPA, a shed acute phase-antigen of Trvpanosoma cruzi. Mol. Bioch~/n. Parasitol. 47, 247-250. [15] Pollevick, G. D., Sanchez, D. O., Campetella, O., Trombetta, S., Sousa, M., Henriksson, J., Hellman, U., Pettersson, U, Cazzulo, J.J., Frasch, A. C. C. (1993) Members of the SAPA/trans-sialidase protein family have identical N-terminal sequences and a putative signal peptide. Mol. Biochem. Parasitol. 59, 171 174. [16] Pereira, M. E. A., Santiago Mejia, J., Ortega-Barria, E., Matzilevich, D. and Prioli, R. P. (1991) The Trypanosoma cruzi neuraminidase contains sequences similar to bacterial neuraminidases, to YWTD repeats of the LDL receptor and to type Ill modules of fibronectin. J. Exp. Med. 174, 179 192. [17]'Roggentin, P., Rothe, B., Kaper, J.B., Galen, J., Lawrisk, L., Vimr, E.R, and Schauer, R. (1989) Conserved sequences in bacterial and viral neuraminidases. Glycoconjugate J. 6, 349 356. [18] Cross, G.A.M. and Takle, G.B. (1993) The surface transsialidase family of Tr)panosoma cruzi. Annu. Rev. Microbiol. 47, 385 411.
ELSEVIER
Molecular and Biochemical Parasitology 64 (1994) 337 340
BIOCHEMICAL PARASITOLOGY
Short Communication
A recombinant Trypanosoma cruzi trans-sialidase lacking the amino acid repeats retains the enzymatic activity Oscar E. Campetella, Antonio D. Uttaro, Armando J. Parodi, Alberto C.C. Frasch* lnstituto de Investigaciones Bioquimicas, Fundaci6n Carnpomar, Av. Patricias Argentinas 435, 1405 Buenos Aires, Argentina Received I October 1993; accepted 17 January 1994
Key words'." Trypanosoma cruzi; Trans-sialidase; Neuraminidase; SAPA antigen; Sialic acid
Trans-sialidase (TS) is an enzymatic activity found in Trypanosoma cruzi, the agent of American trypanosomiasis or Chagas disease, and, recently, in the African parasite Trypanosoma brucei [1-3]. This enzyme differs from mammalian sialyltransferases as the latter employs only CMPsialic acid as sugar donor. TS employs glycolipids and glycoproteins as donors [4-5], with sialic acid linked c~(2 3) to a terminal galactose in either Nor O-linked oligosaccharides, in gangliosides or in lactose [1-2]. Molecules accepting sialic acid must have a terminal galactose present in N- or Olinked oligosaccharides, gangliosides or N-acetyllactosamine [1-2]. The T. cruzi enzyme has been related to cell invasion (reviewed in [6]), where
r~orresponding author. Fax: (54-1) 8652246 or (54-1) 881916. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession No. L26499. Abbreviations." NA, neuraminidase; TS, trans-sialidase; SAPA, shed acute phase antigen; PCR, polymerase chain reaction. 0166-6851/94/$7.00 ~'~ 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 6 8 5 1 ( 9 4 ) 0 0 0 3 6 - M
sialic acid seems to play a key role. The TS from T. cruzi trypomastigotes contains a C-terminal and an N-terminal domains [7]. The C-terminal domain contains variable number of 12-amino acid-long repeats in tandem, that constitutes the SAPA antigen [8 10]. These repeats were suggested not to have any influence on the enzymatic domain of the molecule [7]. Antibodies bound to the amino acid repeats did not affect enzymatic activity [2,7]. On the other hand, TS is inhibited by antibodies directed to the N-terminus (Leguizam6n et al, submitted). Chimeric protein constructs suggested a putative region of the gene necessary for enzymatic activity within the Nterminal portion [11]. However, a clear-cut localization of the enzymatic activity in the N-terminus and of the absence of effect of the SAPA domain on the TS activity is lacking. There are several TSrelated molecules which are encoded by different genes (reviewed in [12]), only some of them having TS activity [11]. Very recently, a TS present in the epimastigote stage derived from axenic cultures has been described [13]. It does not contain the SAPA repeats, but information on the C-ter-
O.E. Campetella et al./Molecular and Biochemical Parasitology 64 (1994) 337-340
338
minus, together with its sequence is not available yet. Here we show that the amino acid repeats are not necessary for the enzymatic activity of TS. The 5' region of a SAPA gene contained in a genomic D N A fragment from T. cruzi RA strain was subcloned by PCR and expressed in p K K 233-3 (Pharmacia-LKB, Uppsala, Sweden). The 5'-end primer was designed from the mature protein [15] and an A T G codon was included: 5' ATGCTGGCACCCGGATCGAGCCGAGTTGA G C T G 3'. The 3'-end primer includes a third of the first repeat followed by a stop codon and an EcoRI restriction site: 5' G T G G A A T T C A G G C A C T C G T G T C G C T G C T G C T G T C 3'. The construct (Tc-TS1N1) was made from a gene that also encodes the SAPA repeats. Thus, it might be expressed only in the trypo.mastigote stage because the TS recently described in the epimastigote stage has no such repeats [13]. Employing sialyllactose as donor and ]4C-labeled lactose as acceptor molecules in a Tc-TS1Nl-catalyzed reaction, a labeled product with the same electrophoretic mo-
bility as the sialyllactose marker and sensitive to Clostridium perfringens neuraminidase (NA) treatment was detected, as expected from a trans-sialidase reaction (not shown). This clone also displayed NA activity when assayed on 2'-(4-methylumbelliferyl)-~-D-N-acetylneuraminic acid fluorogenic substrate (0.190 for Tc-TSIN1 vs. 0.037 for an inactive control; values are in fluorescence arbitrary units) [2]. These results clearly demonstrate experimentally that the amino acid repeats are not necessary for the NA and/or TS activity. To test if amino acid repeats might modify the activity, some kinetic and specificity parameters were assayed. The apparent Km for sialyllactose of Tc-TS1N1 and the natural enzyme purified from supernatants of Vero cell cultures [15] 0.23 mM and 0.19 mM, respectively. The recombinant enzyme, as well as the native one, transferred ~(2,3)- but not ~(2,6)-linked sialyl units from fetuin-derived glycopeptides to asialofetuin (Table 1). Both enzymes also transferred sialyl units from glycoli-
Table 1 Donor specificities of the natural and recombinant (Tc-TSIN1) trans-sialidases~' Assay
Enzyme activity (Units)
Donor
Enzyme
Sialyl-~-(2,3)glycopeptides Sialyl-~-(2,6)glycopeptides Sialyl-~-(2,3)gl'ycopeptides Sialyl-~-(2,6)glycopeptides
Natural Natural Tc-TSIN1 Tc-TS1NI
64 0 9.2 0
GM3 GM2 GM3 GM2
Natural Natural Tc-TS1N 1 Tc-TS I N 1
250 3.8 48 2
Acceptor specificity of recombinant (Tc-TSIN1) trans-sialidase b Acceptor
Enzyme
Enzyme activity (Units)
[galactose-~4C]~-methylgalactoside [galactose-14C][3-methylgalactoside
Tc-TS 1N 1 Tc-TS 1N I
0.35 79.3
gangliosi.de ganglioside ganglioside ganglioside
Assays were performed in duplicate, the enzyme activity units are expressed as fmol of sialic acid transferred min-~. The low radioactivity values in the absence of enzyme were subtracted. About I /~g of natural and 0.2 2 /tg of recombinant enzymes were employed, as estimated from silver-stained polyacrylamide gels (see text). ~tThe donors in the assay 1 were [sialic acid-14C]sialyl-glycopeptides and unlabeled GM3 or GM2 gangliosides in assay 2. The acceptors were asialofetuin in assay 1 and N-[N-acetylglucosamine-14C]acetyllactosamine in assay 2. For further details see [5]. Reactions were performed under the conditions previously described [2]. hThe donor was sialyllactose, both acceptor galactosides were 300 Ci m o l - ~. Conditions of the assay were as in [5}. Reaction products were submitted to paper electrophoresis in I M pyridine acetate buffer pH 6.5 and radioactivity in charged products quantitated.
O.E. Campetella et al./Molecular and Biochemical Parasitology 64 (1994) 337-340
Tc-TSINI
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8 FR
L P A L V N V D Q V N V A I
I
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repeats
Fig. 1. C o m p a r i s o n o f p u t a t i v e l y e n c o d e d p r o t e i n s f r o m T c - T S I N 1 , T C N A u p to t h e first r e p e a t [16] a n d a v a i l a b l e s e q u e n c e s o f c l o n e s 154 ( T S a c t i v e ) a n d 151/121 ( T S i n a c t i v e ) [I 1]. A m i n o a c i d i d e n t i t i e s a r e i n d i c a t e d b y -. G a p s m a d e for a best a l i g n m e n t a r e i n d i c a t e d w i t h stars. R o m a n n u m e r a l s i n d i c a t e t h e p o s i t i o n o f t h e f o u r c o m p l e t e l y o r p a r t i a l l y c o n s e r v e d S X D X G X T W bacterial neuraminid a s e s m o t i f s [17]. T h e p r o p o s e d s u b t e r m i n a l c o n s e n s u s m o t i f f o r t h e s u p e r f a m i l y o f a n t i g e n s [18] is u n d e r l i n e d . T h e a r r o w i n d i c a t e s the start of the SAPA repeats.
pids to N-acetyllactosamine only when the former residues were linked to terminal galactose units (Table 1). Similarly to natural TS[5], Tc-TS1N1 recognized fl- but not a-linked galactose units as acceptors (Table 1). Besides, the specific activfty at 37°C of the natural TS, determined with sialyllactose as donor and lac-labeled lactose as sialic acid acceptor, was 0.01 /~mol of sialic acid transferred m i n - l (mg protein)-I. When the itaduced recombinant Escherichia coli cell-free extracts were run in SDS-PAGE and silver stained, comparison of the intensity of the 70 kDa containing the recombinant trans-sialidase band with standards suggests that the specific activity of the recombinant TS was in the range 0.005~).05 #mol
m i n - I (mg protein)-I and thus comparable to that of the natural enzyme. Therefore, no differences were observed in the donor acceptor specificity and the specific activities seem to be similar between the natural and Tc-TS1NI enzymes in spite of the absence of repeats and the high mannose-type glycosylation. Two sequences from active genes have been published, a partial one encoding an active TS (clone 154) [11] and a related sequence (TCNA) encoding a molecule with NA activity [16]. The latter was never reported to have TS activity. Therefore, no complete sequence of an active TS molecule has been previously described. TcTSIN1 was sequenced and the deduced amino
340
O.E. Campetella et al./Molecular and Biochemical Parasitology 64 (1994) 33~340
acid composition was compared with those sequences (Fig. 1). The total or partially conserved four S X D X G X T W motifs from bacterial neuraminidases [17] and the proposed subterminal V T V X N V f L Y N R family motif [18] are indicated. T C N A is included only up to the first repeat that defines the SAPA antigen. The variation in amino acids 390~,35 of Tc-TS1N1 with respect to T C N A is due to a reading frame change in this region. Concerning the region previously reported as essential for the activity in clone 154 [11], three amino acids are changed in T c - T S I N I " K to E at position 219, A to D at 226 and L to V at 265, suggesting that none of them is related to activity. The Y at position 343, suggested to be necessary for activity, is also present in Tc-TS1N1.
Acknowledgements We are indebted to Dr. J.J. Cazzulo for critical reading of the manuscript. This work was supported by grants from the UNDP/World Bank/ WHO Special Programme for Research and Training in Tropical Diseases (TDR), the Swedish Agency for Research Cooperation with Developing Countries (SAREC), the Consejo Nacional de Investigaciones Cientificas y Tbcnicas and the University of Buenos Aires, Argentina.
References [1] Schenkman, S., Jiang, M.S., Hart G. W. and Nussenzwieg V. (t991) A novel cell surface trans-sialidase ot" Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells. Ceil 65, 1117 1126. [2] Parodi, A. J., Pollevick, G. D., Mautner, M., Buschiazzo, A., Sanchez, D.O. and Frasch, A.C.C. (1992) Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi. EMBO J. I1, 1705 1710. [3] Engstler M and Schauer R. (1993) Sialidases from african trypanosomes. Parasitol. Today 9, 222-225. [4] Schenkman, S., Pontes de Carvalho, L, and Nussenzweig, V. (1992) Trypanosoma cruzi trans-sialidase and neuraminidase activities can be mediated by the same enzymes. J. Exp. Med. 175, 567 575. [5] Garcia Ferrero, M. A., Trombetta, S. E., Sanchez, D. O., Reglero, A., Frasch A.C.C. and Parodi A. J. (1993) The action of Trypanosoma cruzi trans-sialidase on glycolipids and glycoproteins. Eur. J. Biochem. 213, 765 771.
[6] Schenkman, S. and Eichinger, 1~. (1993) Trypanosoma cruzi trans-sialidase and cell invasion. Parasitol. Today 9, 218-222. [7] Cazzulo, J.J. and Frasch, A.C.C. (1992) SAPA/transsialidase and cruzipain: two antigens from Trypanosoma cruzi contain immunodominant but enzymatically inactive domains. FASEB J. 6, 3259 3264. [8] Affranchino, J. L., Ib~iez, C. F., Luquetti, A. O., Rassi, A., Reyes, M. B., Macina, A., Aslund, L., Pettersson, U. and Frasch, A. C. C. (1989) Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas disease. Mol. Biochem. Parasitol 34, 221 228. [9] Reyes, M. B., Lorca, M., Muoz, P. and Frasch, A. C. C. (1990) Fetal lgG specificities against Trypanosoma cruzi antigens in infected newborns. Proc. Natl. Acad. Sci. USA 87, 2846 2850. [10] Leguizamon, M. S., Campetella, O. E., Reyes, M. B., Ibb,ez, C. F., Basombr~o, M. A., Rinc6n, J., rn, A. and Frasch, A. C. C. (1991) Bloodstream Trypanosoma cruzi parasites from mice simultaneously express antigens that are markers of acute and chronic human Chagas disease. Parasitology 102, 379 385. [11] Uemura, H., Schenkman, S., Nussenzweig, V. and Eichinger, D. (1992) Only some members of a gene family in T~vpanosoma cruzi encode'proteins that express both trans-sialidase and neuraminidase activities. EMBO J. 11, 3837 3844. [12] Campetella, O., Stinchez, D. O., Cazzulo, J.J. and Frasch, A.C.C. (1992) A superfamily of Trvpanosoma cruzi surface antigens. Parasitol. Today 8, 378 381. [13] Chaves, L. B., Briones M. R. S. and Schenkman, S. (1993) Trans-sialidase from Trvpanosoma cru=i epimastigotes is expressed at the stationary phase and is different from the enzyme expressed in trypomastigotes. Mol. Biochem. Parasitol. 61, 97-106. [14] Pollevick, G. D., Affranchino, J. L., Frasch, A. C. C. and Stinchez, D. O. (199.1) The complete sequence of SAPA, a shed acute phase-antigen of Trvpanosoma cruzi. Mol. Bioch~/n. Parasitol. 47, 247-250. [15] Pollevick, G. D., Sanchez, D. O., Campetella, O., Trombetta, S., Sousa, M., Henriksson, J., Hellman, U., Pettersson, U, Cazzulo, J.J., Frasch, A. C. C. (1993) Members of the SAPA/trans-sialidase protein family have identical N-terminal sequences and a putative signal peptide. Mol. Biochem. Parasitol. 59, 171 174. [16] Pereira, M. E. A., Santiago Mejia, J., Ortega-Barria, E., Matzilevich, D. and Prioli, R. P. (1991) The Trypanosoma cruzi neuraminidase contains sequences similar to bacterial neuraminidases, to YWTD repeats of the LDL receptor and to type Ill modules of fibronectin. J. Exp. Med. 174, 179 192. [17]'Roggentin, P., Rothe, B., Kaper, J.B., Galen, J., Lawrisk, L., Vimr, E.R, and Schauer, R. (1989) Conserved sequences in bacterial and viral neuraminidases. Glycoconjugate J. 6, 349 356. [18] Cross, G.A.M. and Takle, G.B. (1993) The surface transsialidase family of Tr)panosoma cruzi. Annu. Rev. Microbiol. 47, 385 411.