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Structure and transcription of genes encoding the surface glycoprotein antigens gp90 and gp82 of Trypanosoma cruzi metacyclic trypomastigotes

STRUCTURE AND TRANSCRIPTION OF GENES ENCODING THE SURFACE GLYCOPROTEIN ANTIGENS GP90 AND GP82 OF Trypanosoma cruzi METACYCLIC TRYPOMASTIGOTES* * This work was supported by grants from FAPESP, Pronex, UNDP/World Bank/WHO/TDR, Cyted (Ibero American Project of Biotechnology, SubProgram III, Spain).

MIRIAN S. CARMO1, JORGE E ARAYA2, MARIA I. CANO1, MARCEL I. RAMIREZ1, RENATA P. BAIDA1, RITA C. RUIZ1, MARCIA R. SANTOS1, MIGUEL A. CHIURILLO3, JOSÉ L. RAMIREZ3, NOBUKO YOSHIDA1, JOSÉ FRANCO DA SILVEIRA1

1Departamento de Micro, Imuno e Parasitologia, Escola Paulista de Medicina, UNIFESP, 04023-062 São Paulo, Brasil; 2Unidad de Parasitologia, Universidad de Antofagasta, Antofagasta, Chile;

3Instituto de Biologia Experimental, Universidad Central de Venezuela, Aptado 47525, Caracas 1041-A, Venezuela

Presented by LUIZ R. TRAVASSOS

The infective forms of T. cruzi are the trypomastigote stages found in the bloodstream of mammalian hosts or the metacyclic trypomastigotes present in the digestive tract of the insect. Metacyclic trypomastigotes express two stage-specific glycoproteins (gp90 and gp82) that have no counterpart in blood trypomastigotes [Teixeira & Yoshida 1986. Mol Biochem Parasitol 18: 271-282]. The gp90 and gp82 are involved in the penetration of the parasite into host cells (Yoshida et al. 1990), [Ramirez et al. 1993. Infect Immun 6: 3636-3641.], [Santori et al. 1996a. Mol Biochem Parasitol 78: 209-216.]. Gp82 can induce Ca2+ signal in target cells [Dorta et al. 1995. Mol Biochem Parasitol 73: 285-89.], [Ruiz et al. 1998. Biochem J 330: 505-511.], an event essential for T. cruzi internalization (Dorta et al. 1995). Gp90 and gp82 are also relevant immunologically. Immunization with gp90 or gp82 protects mice against acute infection by T. cruzi [Yoshida et al. 1993. Exp Parasitol 77: 405-413.], [Santori et al. 1996b. Infect Immun 64: 1093-1099.].

cDNA clones encoding gp82 and gp90 were isolated from expression libraries using specific monoclonal antibodies [Franco et al. 1993. Infect Immun 61: 4196-4201.]; [Araya et al. 1994. Mol Biochem Parasitol 65: 161-169.]. Comparison of sequences of gp90 and gp82 showed 40% identity at amino acid level, with homologous regions separated by sequences displaying significant amino acid differences (Franco et al. 1993; Araya et al. 1994). Sequence analysis of gp90 and gp82 also revealed 40% - 60% identity at amino acid level with members of T. cruzi gp85/sialidase family. Based on these structural features, gp90 and gp82,genes could be considered as members of gp85/sialidase family.

Large DNA fragments (40 - 400kb) containing gp90 and gp82 sequences were isolated from T. cruzi genomic libraries constructed in YAC and cosmid vectors [Ferrari et al. 1997. Mem Inst Oswaldo Cruz 92: 843-858.]. Gp90 and gp82 are present in multiple copies, distributed in several chromosomes, and this gene family can be divided into subsets on the basis of hybridization patterns obtained with probes derived from different regions of gp90 and gp82 genes (Araya et al. 1994); [Cano et al. 1995. Mol Biochem Parasitol 71: 273-278.]; [Santos et al. 1997. Mem Inst Oswaldo Cruz 92: 821-828.]. Many members of gp90 and gp82 gene family are closely linked to members of gp85/sialidase family at multiple sites in the genome of different T. cruzi strains. Hybridization patterns of gp90, gp82 and gp85 genes with T. cruzi chromosomal bands separated by pulsed field gel electrophoresis are very similar, suggesting that many of these genes could be linked in different chromosomal loci. This was confirmed by isolation of genomic DNA clones from YAC and cosmid libraries.

It is interesting to note that some several sub-telomeric regions are made of sequences associated to the gp90 and gp85 [Chiurillo et al. 1999. Mol Biochem Parasitol 100: 173-183.]. The presence of gp90 and gp85 at T. cruzi telomeres suggests that new variants of the gp85/sialidase family can continously be arising by duplication, mutation, and recombination of copies that have been transposed to the telomeres.

Northern blot and western blot analyses showed that gp90 and gp82 are preferentially transcribed and expressed in the metacyclic trypomastigote stage (Franco et al. 1993; Araya et al. 1994). Further studies on the transcription of these genes using ''run on'' and RNA-PCR assays showed the presence of gp82 and gp90 transcripts in epimastigotes and blood trypomastigotes. Taken together these results suggest that the expression of genes gp90 and gp82 is constitutive and may be regulated at post-transcriptional level, for instance, at translational level and/or mRNA stabilization.

The presence of multiple copies of genes in T. rangeli encoding products related to T. cruzi gp82, gp90 and gp85 was revealed when genomic T. rangeli DNA was hybridized at moderate and high stringencies with gp82, gp90 and gp85 genes. Even at high stringency conditions, both probes hybridized with several genomic fragments suggesting that gp82, gp90 and gp85 related sequences are interspersed in the genome rather than arranged in tandem repeats. Sequence analysis showed that many of hybridizing fragments contain sequences associated to the gp85/sialidase gene family. These results suggested that gp90, gp82 and gp85 genes have been originated from a common ancestral gene present in several member of Trypanosoma genus.

— ( September 14, 1999 ) .

  • *
    This work was supported by grants from FAPESP, Pronex, UNDP/World Bank/WHO/TDR, Cyted (Ibero American Project of Biotechnology, SubProgram III, Spain).
  • Publication Dates

    • Publication in this collection
      07 Aug 2000
    • Date of issue
      Mar 2000
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