Characterisation of the effects on proteases of Heterodera glycines and Meloidogyne incognita second-stage juveniles by inhibitors obtained from cysts of H. glycines

Abstract

Summary The protease inhibitor component of Heterodera glycines cyst contents was explored using a battery of peptide substrates and H. glycines and Meloidogyne incognita second-stage juveniles as enzyme sources. Protease inhibitors were prepared by heat-denaturing H. glycines cyst-egg extract (hHglCE), which was used in all inhibition exploration. Eight substrates targeting four endoprotease groups (aspartic, cysteine, metallo- and serine proteases) revealed that protease inhibition by hHglCE varied significantly between H. glycines and M. incognita with seven of the eight substrates. Only cysteine protease activity was inhibited equally between H. glycines and M. incognita. Aspartic protease activity was inhibited more strongly in H. glycines and serine protease activity was inhibited more strongly in M. incognita. Digestion of five matrix metalloprotease (MMP) substrates was inhibited more strongly in H. glycines (two substrates) and M. incognita (three substrates). These variations were particularly intriguing given the potential association of MMP proteases with developing embryos. Inhibition of digestion of nematode FMRFamide-like peptides (FLPs) showed less variation between nematode species than the targeted substrates, but inhibition did vary significantly across substrates within each species. Digestion of FLP-6 was the least affected by hHglCE but was inhibited significantly more in M. incognita than in H. glycines. Residue differences between two FLP-14 sequences significantly affected inhibition of FLP-14 digestion in both H. glycines and M. incognita. RP-HPLC fractionation of hHglCE clearly demonstrated the presence of high (Fr No.5) and low (Fr No.14) polarity inhibitor components. Potency of inhibition of M. incognita serine protease activity, based upon IC50 values (1.68 and 2.78 hHglCEeq reaction−1 for Fr No.5 and Fr No.14, respectively), was reduced significantly from unfractionated hHglCE (IC50 = 0.61), suggesting inhibitor dilution, loss of component synergy, or both, due to fractionation.