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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.


Peptides generated by acid hemoglobinolysis. Peptides were identified by LC-MS/MS after hemoglobinolysis with native BmAP (A), BmCL1 (B) or both enzymes (C). Digestion was performed in 100 mM citrate-phosphate buffer pH 4.5 at 37°C for up to 4 h, as described in Methods.
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Figure 7: Peptides generated by acid hemoglobinolysis. Peptides were identified by LC-MS/MS after hemoglobinolysis with native BmAP (A), BmCL1 (B) or both enzymes (C). Digestion was performed in 100 mM citrate-phosphate buffer pH 4.5 at 37°C for up to 4 h, as described in Methods.

Mentions: Figure 7 summarizes the cleavage specificities of native BmAP, recombinant BmCL1 and both enzymes against either alpha or beta subunit of bovine hemoglobin in vitro. Mass spectrometric analysis of the peptides generated by BmAP identified 20 peptide sequences, with molecular masses ranging from 1085 Da to 3257 Da; sixteen were derived from the α subunit and four from the β subunit of bovine hemoglobin (Figure 7, panel A). BmAP preferentially hydrolysed hemoglobin at sites containing non-polar amino acids at P1 (81% of the cleavage sites), of which the most prevalent residues were Leu and Phe (62%) and predominantly at sites containing hydrophobic residues at P1' (65%). Cleavage sites were found in alpha helical and random coil regions of hemoglobin and resulted in the generation of several peptides previously shown to be antimicrobial [25], such as α1-29, α1-32, α84-98 and β1-13 (Figure 7, panel A) as well as potential antimicrobials, such as α33-43 and α129-141, due to their sequence similarity with the antimicrobials α34-46 and α133-141, respectively [25].


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)

Peptides generated by acid hemoglobinolysis. Peptides were identified by LC-MS/MS after hemoglobinolysis with native BmAP (A), BmCL1 (B) or both enzymes (C). Digestion was performed in 100 mM citrate-phosphate buffer pH 4.5 at 37°C for up to 4 h, as described in Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Peptides generated by acid hemoglobinolysis. Peptides were identified by LC-MS/MS after hemoglobinolysis with native BmAP (A), BmCL1 (B) or both enzymes (C). Digestion was performed in 100 mM citrate-phosphate buffer pH 4.5 at 37°C for up to 4 h, as described in Methods.
Mentions: Figure 7 summarizes the cleavage specificities of native BmAP, recombinant BmCL1 and both enzymes against either alpha or beta subunit of bovine hemoglobin in vitro. Mass spectrometric analysis of the peptides generated by BmAP identified 20 peptide sequences, with molecular masses ranging from 1085 Da to 3257 Da; sixteen were derived from the α subunit and four from the β subunit of bovine hemoglobin (Figure 7, panel A). BmAP preferentially hydrolysed hemoglobin at sites containing non-polar amino acids at P1 (81% of the cleavage sites), of which the most prevalent residues were Leu and Phe (62%) and predominantly at sites containing hydrophobic residues at P1' (65%). Cleavage sites were found in alpha helical and random coil regions of hemoglobin and resulted in the generation of several peptides previously shown to be antimicrobial [25], such as α1-29, α1-32, α84-98 and β1-13 (Figure 7, panel A) as well as potential antimicrobials, such as α33-43 and α129-141, due to their sequence similarity with the antimicrobials α34-46 and α133-141, respectively [25].

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.