Limits...
Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides.

Cruz CE, Fogaça AC, Nakayasu ES, Angeli CB, Belmonte R, Almeida IC, Miranda A, Miranda MT, Tanaka AS, Braz GR, Craik CS, Schneider E, Caffrey CR, Daffre S - Parasit Vectors (2010)

Bottom Line: 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'.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil. sidaffre@icb.usp.br.

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.

No MeSH data available.


Nucleotide and deduced amino acid sequence of R. (B.) microplus aspartic proteinase cDNA. The predicted 20-aa signal peptide is underlined. The conserved residues involved in catalysis are in rectangles. The regions identified by LC-MS/MS are shaded. The cysteine residues involved in dissulfide bond formation are in black. The GenBank accession number for this sequence is FJ655904.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2921360&req=5

Figure 3: Nucleotide and deduced amino acid sequence of R. (B.) microplus aspartic proteinase cDNA. The predicted 20-aa signal peptide is underlined. The conserved residues involved in catalysis are in rectangles. The regions identified by LC-MS/MS are shaded. The cysteine residues involved in dissulfide bond formation are in black. The GenBank accession number for this sequence is FJ655904.

Mentions: Using specific oligonucleotides designed from the identified contig #3, a full-length cDNA was obtained. Its nucleotide sequence encodes a protein containing 392 amino-acid residues, with a predicted molecular mass of 42.2 kDa (Figure 3). Using Signal P, the most likely signal peptide cleavage site was identified between residues 20 (Ala) and 21 (Leu) of the preprotein, resulting in a 372 amino-acid mature polypeptide with a molecular mass of 40.2 kDa and a theoretical isoelectric point of 8.2.


Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides.

Cruz CE, Fogaça AC, Nakayasu ES, Angeli CB, Belmonte R, Almeida IC, Miranda A, Miranda MT, Tanaka AS, Braz GR, Craik CS, Schneider E, Caffrey CR, Daffre S - Parasit Vectors (2010)

Nucleotide and deduced amino acid sequence of R. (B.) microplus aspartic proteinase cDNA. The predicted 20-aa signal peptide is underlined. The conserved residues involved in catalysis are in rectangles. The regions identified by LC-MS/MS are shaded. The cysteine residues involved in dissulfide bond formation are in black. The GenBank accession number for this sequence is FJ655904.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2921360&req=5

Figure 3: Nucleotide and deduced amino acid sequence of R. (B.) microplus aspartic proteinase cDNA. The predicted 20-aa signal peptide is underlined. The conserved residues involved in catalysis are in rectangles. The regions identified by LC-MS/MS are shaded. The cysteine residues involved in dissulfide bond formation are in black. The GenBank accession number for this sequence is FJ655904.
Mentions: Using specific oligonucleotides designed from the identified contig #3, a full-length cDNA was obtained. Its nucleotide sequence encodes a protein containing 392 amino-acid residues, with a predicted molecular mass of 42.2 kDa (Figure 3). Using Signal P, the most likely signal peptide cleavage site was identified between residues 20 (Ala) and 21 (Leu) of the preprotein, resulting in a 372 amino-acid mature polypeptide with a molecular mass of 40.2 kDa and a theoretical isoelectric point of 8.2.

Bottom Line: 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'.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil. sidaffre@icb.usp.br.

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.

No MeSH data available.