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Proteolytic cleavage of IgG and other protein substrates by Dirofilaria immitis microfilarial enzymes
Abstract:
Proteases were detected in aqueous extracts of Dirofilaria immitis microfilariae. Enzymes within the extract were capable of hydrolyzing Azocoll, a general protease substrate, at pH's 7, 8, and 9. Sensitivities to a variety of protease inhibitors indicated that multiple azocollytic enzymes were present in the extract, most prominent of which appear to belong to the serine class of proteases. By incorporating various substrates into the matrices of polyacrylamide gels, 2 SDS-resistant, mercaptoethanol-sensitive proteases in the MF extract were identified at 22 and 76 kDa. These proteases showed differential abilities to digest casein, fibrinogen, hemoglobin, and IgG. The MF extract hydrolyzed radiolabeled IgG into 8-10-kDa fragments following a 20-hr incubation. A similar degree of digestion was observed in 2 hr when viable microfilariae were used. The potential significance of these proteases in the evasion of host effector mechanisms is discussed.
Insights
Proteases from Dirofilaria immitis microfilariae were identified, including serine proteases. These enzymes degrade host proteins like IgG, potentially aiding parasite evasion of immune responses.
Area of Science:
- Parasitology
- Biochemistry
- Molecular Biology
Background:
- Dirofilaria immitis microfilariae (MF) are the larval stage of the heartworm parasite.
- Parasitic worms often employ secreted enzymes to interact with and manipulate their hosts.
Purpose of the Study:
- To identify and characterize proteases present in Dirofilaria immitis microfilariae extracts.
- To investigate the enzymatic activity and potential role of these proteases in host-parasite interactions.
Main Methods:
- Aqueous extracts of D. immitis microfilariae were prepared and assayed for protease activity using Azocoll substrate.
- Protease inhibitors were used to determine enzyme classes.
- Gel electrophoresis with incorporated substrates identified specific protease molecular weights.
- Digestion of various protein substrates, including radiolabeled IgG, was analyzed.
Main Results:
- Multiple azocoll-hydrolyzing enzymes were detected in MF extracts, active at neutral to alkaline pH.
- Serine proteases were identified as a prominent class.
- Two major proteases (22 and 76 kDa) were characterized by their resistance to SDS and sensitivity to mercaptoethanol.
- These proteases exhibited differential substrate specificity, degrading casein, fibrinogen, hemoglobin, and IgG.
- MF extract and viable microfilariae efficiently degraded host IgG.
Conclusions:
- Dirofilaria immitis microfilariae possess a repertoire of proteases, notably serine proteases.
- These enzymes can degrade key host proteins like IgG.
- The identified proteases likely contribute to the parasite's ability to evade host immune defenses.