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

in Nematology
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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.

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

in Nematology



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    Characteristics of substrates and conditions of use.

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    Inhibition of protease activities in preparations of infective juveniles of Heterodera glycines and Meloidogyne incognita by heated H. glycines cyst content. A: Substrates used targeted 4 endoprotease types, metalloproteases (MMPs), aspartic protease (Asp), cysteine proteases (Cys), serine protease (Ser). Specific proteases detected by the eight substrates, including the MMP substrates identified by number, are given in Table 1; B: Substrates used to detect general protease activities were KHEYLRFa (A1), KSAYMRFa (A2), KHEFVRFa (USEM) and FMRFa. Data are presented as percent activity inhibition mean ± SEM for 5-11 independent reactions; H. glycines (black bars) and M. incognita (white bars). Means were compared using Student’s t-test. Between species for each substrate, means connected by bars are significantly different (*P<0.05, **P<0.01, ***P<0.005, ****P<0.001). Within species and across substrates, means associated with different letters are significantly different. In all assays, inhibitor concentration was 1 hHglCEeq reaction−1 with 96 ng hHglCE protein μl−1.

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    Relative potencies of Heterodera glycines heated cyst content (hHglCE) in inhibiting aspartic and cysteine proteases detected in preparations of infective juveniles of H. glycines and Meloidogyne incognita. Nematode extracts and inhibition reactions were prepared as described. hHglCE was diluted with assay buffer to provide indicated concentrations in each reaction. Aspartic protease substrate was Mca-GKPILFFRK-K(Dnp)R-NH2 (10 μM) and cysteine substrate was Z-VVR-AMC (20 μM). Each data point is expressed as the mean percent inhibition over 3-11 independent reactions. Lines were compared with non-linear regression.

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    Chromatographic profile of hHglCE from Heterodera glycines cysts showing distribution of serine protease inhibitors detected using Meloidogyne incognita second-stage juvenile (J2) extract. Heated cyst content (hHglCE) was dissolved in 0.1% TFA, injected onto the RP-HPLC system, and fractions were collected as described. Dried fractions were dissolved in 30 μl of M. incognita J2 extract prepared as described, and 24.5 μl of each fraction mixture was transferred to reaction plate wells. After 15 min incubation at 27°C, reactions were started by addition of 0.5 μl of serine protease substrate (Boc-NAR-AMC). Concentrations in reactions were 0.1 μg M. incognita extract protein μl−1 and 100 μM Boc-NAR-AMC. Input injected was 5.5 hHglCEeq. Solid bars indicate percent inhibition (reduction of Boc-NAR-AMC digestion) at 4.5 hHglCEeq reaction−1. Solid rectangles within selected bars (fractions 5 and 14-16) indicate percent inhibition at reduced (approx. 2hHglCEeq) doses. The UV trace (solid black line) represents absorbance at 210 nm. The dotted line indicates CH3CN gradient and the vertical dashed line indicates %CH3CN. Numbers in parentheses indicate the CH3CN elution range of selected fractions.

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    Relative potencies of Heterodera glycines heated cyst content (hHglCE) and RP-HPLC column fractions of hHglCE in inhibiting serine proteases detected in preparations of infective juveniles of Meloidogyne incognita. Nematode extracts and inhibition reactions were prepared as described. Input (unfractionated hHglCE, UF) and hHglCE column fractions were diluted with assay buffer to provide the indicated concentrations in each reaction. Each data point is expressed as the mean ± SEM of % inhibition over 4-9 independent reactions except for fraction 14 hHglCEeq doses of 0.38 and 0.75, where n = 2 for each. Lines were compared by linear regression. Dotted lines, unfractionated hHglCE; dashed lines, hHglCE fractions.

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