title: Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides.
abstract: BACKGROUND
  Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins.
  
  
  RESULTS
  An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'.
  
  
  CONCLUSIONS
  BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.
authors.affiliation: Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil. sidaffre@icb.usp.br.
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Cruz
authors.forename: Andréa C
authors.initials: AC
authors.lastname: Fogaça
authors.forename: Ernesto S
authors.initials: ES
authors.lastname: Nakayasu
authors.forename: Cláudia B
authors.initials: CB
authors.lastname: Angeli
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Belmonte
authors.forename: Igor C
authors.initials: IC
authors.lastname: Almeida
authors.forename: Antônio
authors.initials: A
authors.lastname: Miranda
authors.forename: Maria Terêsa M
authors.initials: MT
authors.lastname: Miranda
authors.forename: Aparecida S
authors.initials: AS
authors.lastname: Tanaka
authors.forename: Glória R
authors.initials: GR
authors.lastname: Braz
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: Eric
authors.initials: E
authors.lastname: Schneider
authors.forename: Conor R
authors.initials: CR
authors.lastname: Caffrey
authors.forename: Sirlei
authors.initials: S
authors.lastname: Daffre