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.


Purification of aspartic proteinases from tick midgut homogenates. Purification was accomplished in three chromatographic steps: anion exchange chromatography (A), hydrophobic interaction chromatography (B) and pepstatin affinity chromatography (C) in FPLC system. Enzyme activity was assayed with SF 29-35 (-o-). Absorbance was monitored at 280 nm (- -). NaCl gradients (---) were developed as described in Methods. (D) SDS-PAGE of active fraction eluted from the pepstatin affinity chromatography (see also Additional File 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Purification of aspartic proteinases from tick midgut homogenates. Purification was accomplished in three chromatographic steps: anion exchange chromatography (A), hydrophobic interaction chromatography (B) and pepstatin affinity chromatography (C) in FPLC system. Enzyme activity was assayed with SF 29-35 (-o-). Absorbance was monitored at 280 nm (- -). NaCl gradients (---) were developed as described in Methods. (D) SDS-PAGE of active fraction eluted from the pepstatin affinity chromatography (see also Additional File 1).

Mentions: BmAP was purified to apparent homogeneity in three chromatographic steps. Midgut homogenate supernatant was initially resolved using a Mono Q column. The eluted fractions were screened with SF 29-35 and a single activity peak, eluted at 80 mM NaCl, was detected and inhibited by pepstatin (Figure 1A). This active fraction was collected and further resolved by hydrophobic interaction chromatography. A single activity peak was eluted at 1.2 M (NH4)2SO4 (Figure 1B). As a last purification step, active fractions were pooled and loaded onto a pepstatin affinity column and a single active fraction was obtained by a stepwise elution with 0.1 M Tris-HCl, 1 M NaCl, pH 8.6 (Figure 1C). Enzyme recoveries after each purification step were 67%, 30% and 21%, respectively. The purified enzyme was designated BmAP.


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)

Purification of aspartic proteinases from tick midgut homogenates. Purification was accomplished in three chromatographic steps: anion exchange chromatography (A), hydrophobic interaction chromatography (B) and pepstatin affinity chromatography (C) in FPLC system. Enzyme activity was assayed with SF 29-35 (-o-). Absorbance was monitored at 280 nm (- -). NaCl gradients (---) were developed as described in Methods. (D) SDS-PAGE of active fraction eluted from the pepstatin affinity chromatography (see also Additional File 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Purification of aspartic proteinases from tick midgut homogenates. Purification was accomplished in three chromatographic steps: anion exchange chromatography (A), hydrophobic interaction chromatography (B) and pepstatin affinity chromatography (C) in FPLC system. Enzyme activity was assayed with SF 29-35 (-o-). Absorbance was monitored at 280 nm (- -). NaCl gradients (---) were developed as described in Methods. (D) SDS-PAGE of active fraction eluted from the pepstatin affinity chromatography (see also Additional File 1).
Mentions: BmAP was purified to apparent homogeneity in three chromatographic steps. Midgut homogenate supernatant was initially resolved using a Mono Q column. The eluted fractions were screened with SF 29-35 and a single activity peak, eluted at 80 mM NaCl, was detected and inhibited by pepstatin (Figure 1A). This active fraction was collected and further resolved by hydrophobic interaction chromatography. A single activity peak was eluted at 1.2 M (NH4)2SO4 (Figure 1B). As a last purification step, active fractions were pooled and loaded onto a pepstatin affinity column and a single active fraction was obtained by a stepwise elution with 0.1 M Tris-HCl, 1 M NaCl, pH 8.6 (Figure 1C). Enzyme recoveries after each purification step were 67%, 30% and 21%, respectively. The purified enzyme was designated BmAP.

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.