title: Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus.
abstract: BACKGROUND
  Chagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas disease have unpleasant side effects and may be ineffective, therefore, alternative control techniques are required. Knowledge of the T. cruzi interactions with the triatomine host needs extending and new targets/strategies for control identified. Serine and cysteine peptidases play vital roles in protozoan life cycles including invasion and entry of T. cruzi into host cells. Peptidase inhibitors are, therefore, promising targets for disease control.
  
  
  METHODS
  SDS PAGE and chromatograpy detected and isolated a P. megistus serpin which was peptide sequenced by mass spectrometry. A full amino acid sequence was obtained from the cDNA and compared with other insect serpins. Reverse transcription PCR analysis measured serpin transcripts of P. megistus tissues with and without T. cruzi infection. Serpin homology modeling used the Swiss Model and Swiss-PDB viewer programmes.
  
  
  RESULTS
  The P. megistus serpin (PMSRP1) has a ca. 40 kDa molecular mass with 404 amino acid residues. A reactive site loop contains a highly conserved hinge region but, based on sequence alignment, the normal cleavage site for serine proteases at P1-P1' was translocated to the putative position P4'-P5'. A small peptide obtained corresponded to the C-terminal 40 amino acid region. The secondary structure of PMSRP1 indicated nine α-helices and three β-sheets, similar to other serpins. PMSRP1 transcripts occurred in all tested tissues but were highest in the fat body and hemocytes. Levels of mRNA encoding PMSRP1 were significantly modulated in the hemocytes and stomach by T. cruzi infection indicating a role for PMSRP1 in the parasite interactions with P. megistus.
  
  
  CONCLUSIONS
  For the first time, a constitutively expressed serpin has been characterized from the hemolymph of a triatomine. This opens up new research avenues into the roles of serine peptidases in the T. cruzi/P. megistus association. Initial experiments indicate a role for PMSRP1 in T. cruzi interactions with P. megistus and will lead to further functional studies of this molecule.
authors.forename: Carlos J C
authors.initials: CJ
authors.lastname: Moreira
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Waniek
authors.forename: Richard H
authors.initials: RH
authors.lastname: Valente
authors.forename: Paulo C
authors.initials: PC
authors.lastname: Carvalho
authors.forename: Jonas
authors.initials: J
authors.lastname: Perales
authors.forename: Denise
authors.initials: D
authors.lastname: Feder
authors.forename: Reinaldo B
authors.initials: RB
authors.lastname: Geraldo
authors.forename: Helena C
authors.initials: HC
authors.lastname: Castro
authors.forename: Patricia
authors.initials: P
authors.lastname: Azambuja
authors.affiliation: Laboratório de Biologia de Insetos, Universidade Federal Fluminense, Morro do, Valonguinho s/n, Cx Postal 100436, Niterói, RJ CEP 24001-970, Brazil. n.a.ratcliffe@swansea.ac.uk.
authors.forename: Norman A
authors.initials: NA
authors.lastname: Ratcliffe
authors.forename: Cícero B
authors.initials: CB
authors.lastname: Mello
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006458:Hemolymph / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D010203:Panstrongylus / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D020543:Proteome; D016415:Sequence Alignment; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D020543:Proteome; D015843:Serpins