Evaluation of proteases and protease inhibitors in Heterodera glycines cysts obtained from laboratory and field populations

Abstract

Proteases and protease inhibitors were evaluated in preparations ofHeterodera glycinescysts (nHglCE) obtained from glasshouse cultures (GH) and a field (LR) population. Using a FRET-peptide library comprising 512 peptide substrate pools that detect four endoprotease types (aspartic, cysteine, metallo- and serine), we found that the relative distributions of six endoproteases within the four catalytic types were similar among GH and LR preparations. However, levels of mean protease activity ( s−1(μg nHglCE)−1) across all 512 pools varied nearly eight-fold among the preparations. This variation was not related to cyst source. These qualitative (type distribution) and quantitative relationships persisted when analysis was restricted to the top 40% (activity) pools. Analysis of the top 4% of activity pools revealed some substrate cleavage site preferences between the GH and LR proteases. GH and LR preparations also differed significantly in digestion rates of trypsin and matrix metalloprotease (MMP) substrates, with LR rates two-fold greater than GH rates for each protease. By contrast, inhibition of trypsin activity inMeloidogyne incognitaextracts by heat-denatured preparations (hHglCE) was 1.6-fold greater with GH hHglCE than with LR hHglCE. Inhibition of MMP activity was the same (>60%) for each hHglCE. Fractionation of GH and LR hHglCE preparations by RP-HPLC (CH3CN/0.1% TFA system) yielded protease inhibition profiles that were similar for GH and LR. MMP inhibitors eluted at 35-40% CH3CN, and trypsin inhibitors eluted at both 5% and 35-40% CH3CN, suggesting the possibility of peptide as well as other small molecular weight inhibitors. These discoveries illustrate the importance of examiningH. glycinescysts as a source of materials for novel nematode controls.