J Vet Sci.  2006 Sep;7(3):249-255. 10.4142/jvs.2006.7.3.249.

Characterization of HC58cDNA, a putative cysteine protease from the parasite Haemonchus contortus

Affiliations
  • 1College of Veterinary Medicine, Nanjing Agricultural University, Jiangsu 210095, P R China. cimuleke@yahoo.com

Abstract

Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite.

Keyword

cysteine protease activity; Haemonchus contortus; recombinant HC58 protein; synthetic peptide substrates

MeSH Terms

Animals
Caseins/metabolism
Cathepsin B/antagonists&inhibitors/*genetics/isolation & purification/*metabolism
Cysteine Proteinase Inhibitors/pharmacology
DNA, Complementary/genetics
Goat Diseases/*parasitology
Goats
Haemonchiasis/parasitology/*veterinary
Haemonchus/*enzymology/genetics/isolation & purification
Hemagglutination Tests/veterinary
Hemoglobins/metabolism
Hydrogen-Ion Concentration
Immunoglobulin G/metabolism
Leucine/analogs & derivatives/pharmacology
RNA, Helminth/chemistry/genetics
Recombinant Proteins/genetics/metabolism
Reverse Transcriptase Polymerase Chain Reaction/veterinary

Figure

  • Fig. 1 Prokaryotic expression of the recombinant HC58 protein. Gels were stained with Coomassie blue and 15-µl samples were loaded per lane. lane M: standard protein molecular weight marker; lane 1: pET-28a empty expression vector, without HC58 cDNA insert (negative control); lane 2: E. coli (BL21) extracts transformed into pET-28a, before induction with IPTG; lane 3 and 4: extracts of E. coli (BL21) transformed with pET-28a HC58cDNA, after 3 and 4 hours of induction with IPTG, respectively; lane 5: supernatants of HC58 inclusion bodies extracted with 8 M urea; lane 6: the purified HC58 protein inclusion body of approximately 27 kDa.

  • Fig. 2 Cathepsin B/L substrate specificity of the recombinant HC58 protein. Cathepsin B activity assays were performed using the Z-RR-AMC substrate and cathepsin L activity was assayed using Z-FR-AMC suspended in 0.1 M PBS pH 6.0, supplemented with 5 mM DTT. The activity was determined from triplicate assays done on 3 separate occasions with freshly prepared batches of recombinant HC58 protein. The cathepsin L substrate activity was significantly higher than the cathepsin B substrate activity at similar protein concentrations (p < 0.05).

  • Fig. 3 Digestion of hemoglobin with recombinant HC58 protein. Lane P: protein molecular marker; lane 1: HC58 (80 µg/ml) incubated with Hb (80 µg/ml); lane 2: HC58 protein (1 mg/ml) incubated with Hb (80 µg/ml); lane 3: HC58 protein (2 mg/ml) incubated with Hb (80 µg/ml); lane 4: Hb(80 µg/ml) incubated in the absence of the recombinant HC58 protein; lane 5: HC58 protein (1 mg/ml) incubated in the absence of Hb substrate and inhibitor E64; lane 6: electrophoretic profile of HC58 (1 mg/ml) and Hb (80 µg) incubated in the presence of inhibitor E64 (43 µM), presence of inhibitor E64 completely abolished Hb digestion, as indicated by prominent Hb bands in lane 6; lane 7: Hb(80 µg/ml), incubated with extracts of the host bacteria carrying the empty pET-28a vector (without HC58 cDNA insert). Note complete and partial digestion of Hb lanes 1, 2, and 3.

  • Fig. 4 Degradation of goat IgG by recombinant HC58 protein at pH 4. The outcome of overnight digestion (16 h) at pH 4 with varying protein concentrations analyzed on 12% SDS-PAGE under reducing conditions. lane 1: standard protein molecular marker; lane 2: bacterial extracts plus IgG (1.0 µl) without HC58; lane 3: 2 mg/ml HC58 protein plus IgG (1.0 µl) with E64 inhibitor; lane 4: IgG (1.0 µl) and 80 µg/ml HC58; lane 5: IgG (1.0 µl) and l mg/ml HC58; lane 6: IgG (1.0 µl) and 2 mg/ml of HC58; lane 7: IgG (1.0 µl) and buffer only control. Note the degradation of the IgG heavy chain in lanes 4, 5, and 6.

  • Fig. 5 The effect of pH on the degradation of protein substrates Hb, azocasein, fibrinogen and goat IgG by recombinant HC58 protein of adult H. contortus. The buffers 0.1 M acetate, 0.1 M phosphate, 0.1 M Tris and 0.1 M glycine with overlapping pH, in the ranges pH 3 to 11, supplemented with antibiotics were used.


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