Cathepsin B-like Cysteine Proteases Confer Intestinal Cysteine Protease Activity in Haemonchus contortus

Abstract

Cathepsin B-like cysteine protease genes (cbls) constitute large multigene families in parasitic and nonparasitic nematodes. Although expressed in the intestine of some nematodes, the biological and biochemical functions of the CBL proteins remain unresolved. Di-and tetra-oligopeptides were used as fluorogenic substrates and irreversible/competitive inhibitors to establish CBL functions in the intestine of the parasitic nematode Haemonchus contortus. Cysteine protease activity was detected against diverse substrates including the cathepsin B/L substrate FR, the caspase 1 substrate YVAD, the cathepsin B substrate RR, but not the CED-3 (caspase 3) substrate DEVD. The pH at which maximum activity was detected varied according to substrate and ranged from pH 5.0 to 7.0. Individual CBLs were affinity isolated using FA and YVAD substrates. pH influenced CBL affinity isolation in a substrate-specific manner that paralleled pH effects on individual substrates. N-terminal sequencing identified two isolated CBLs as H. contortus GCP-7 (33 kDa) and AC-4 (37 kDa). N termini of each began at a position consistent with proregion cleavage and protease activation. Isolation of the GCP-7 band by each peptide was preferentially inhibited when competed with a diazomethane-conjugated inhibitor, Z-FA-CHN 2 , demonstrating one functional difference among CBLs and among inhibitors. Substrate-based histological analysis placed CBLs on the intestinal microvilli. Data indicate that CBLs are responsible for cysteine protease activity described from H. contortus intestine. Results also support a role of CBLs in nutrient digestion.

Comprehensive Analysis of the Secreted Proteins of the Parasite Haemonchus contortus Reveals Extensive Sequence Variation and Differential Immune Recognition

“…Two spots represent homologues of OV39, a protein from the filarial nematode Onchocerca volvulus (39) that is possibly involved in the autoimmunity to ocular components because of its cross-reactivity with a retinal auto antigen (hr44). Ov39 is supposed to be involved in the autoimmune pattern of this parasitic disease that causes river blindness in humans (40,41 (42)(43)(44). Here we identified multiple spots matching four different types of proteases belonging to three different protease classes (metallo-, serine and aspartic proteases).…”

Immunoprotection in goats against Haemonchus contortus after immunization with cysteine protease enriched protein fractions

“…The PBS-TSBP antigens would be expected to contain cysteine proteases released from the intestine of the parasite as E/S products [27] and hence could interact with the host immune system. In the present study we found high levels of anti-PBS-TSBP antibodies from the third immunization onwards, but no significant increase was detected after the challenge with H. contortus infective larvae.…”

Cathepsin L-like cysteine proteases from Brugia malayi: cDNA cloning and comparison with Caenorhabditis elegans

“…The majority of the parasitic nematode cysteine proteases characterized to date are cathepsin B-like enzymes (16,17,27,29,32,35). They are pointed out to function in the protein digestion for nutrition (8,30), molting (24), etc.…”