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

Abstract

SummaryThe protease inhibitor component ofHeterodera glycinescyst contents was explored using a battery of peptide substrates andH. glycinesandMeloidogyne incognitasecond-stage juveniles as enzyme sources. Protease inhibitors were prepared by heat-denaturingH. glycinescyst-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 betweenH. glycinesandM. incognitawith seven of the eight substrates. Only cysteine protease activity was inhibited equally betweenH. glycinesandM. incognita. Aspartic protease activity was inhibited more strongly inH. glycinesand serine protease activity was inhibited more strongly inM. incognita. Digestion of five matrix metalloprotease (MMP) substrates was inhibited more strongly inH. glycines(two substrates) andM. 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 inM. incognitathan inH. glycines. Residue differences between two FLP-14 sequences significantly affected inhibition of FLP-14 digestion in bothH. glycinesandM. 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 ofM. incognitaserine protease activity, based upon IC50values (1.68 and 2.78 hHglCEeq reaction−1for 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.