In previous chapters expression data, substrate specifies, pH-stability and inhibitory susceptibility of root PLCPs was analyzed. The publicly available expression data of roots was collected and normalized to leaf expression levels (2.20A). PLCPs were clustered based on their relative expression pattern in roots versus leaves. As described earlier, most of the PLCPs are higher expressed in roots than in leaves with CP1C showing the highest relative root expression with a 6-fold increase. Interestingly, CP1A and two of the SA-associated PLCPs, B4FYA3 and Q10716 show higher expression levels in leaves than in roots.
Differential affinities towards the tested commercial substrates were observed for CP1-like and other PLCPs, which may be explained by unequal substrate accessibility to their active sites (2.20B). Summarizing PLCPs in three clusters for the substrate specificities confirmed that all tested PLCPs show highest cleavage capacity for the LR-substrate as seen for RAF but differ in their cleavage ca- pacities towards substrates carrying different residues at the P2-position. The RR-substrate was not cleaved by any PLCPs except for CP1A and CP1B. The FVR substrate was also non-favorable for most PLCPs where again CP1A and CP1B show best results of cleavage. As Val is structurally and biochemically similar to Leu, it was surprising to see that the FVR-substrate was cleaved drastically less efficient than the LR-substrate. FVR was the only substrate consisting of three amino acids prior the N-terminal AMC which already made it structurally more difficult for interaction with the enzymes due to its size. The relatively low cleavage of this substrate despite the small Val in the P2-position could be explained by the specific additional amino acid in the P3-position (Richau et al., 2012). The bulky Phe might sterically hinder the interaction and cleavage of the substrate as seen for the FR-substrate. The FR-substrate was second best for CP1A and CP1B, but still unfavorable for most of the PLCPs. Apart from CP1A and CP1B also CP1D and the SA-associated PLCPs B4FYA3 and Q10716 are capable to cleave the FR-substrate to some extend (2.20B). Three-dimensional modelling of all PLCPs revealed an outward orientation of the catalytic His for B4FYA3 and Q10716 as seen for CP1D. Reorientation of the His rest is widening the active site and may facilitate interac- tions with bulky substrates carrying the Phe in the P2-position. An inward orientation of the catalytic His as seen for CP1A was observed for the remaining PLCPs. The tested PLCPs were clustered after their cleavage specificities in three clusters. Cluster I comprises of CP1A and CP1B, Cluster II contains B4FYA3, Q10716 and CP1D and Cluster III is built of CP1C, CathB, CP2, XCP2 and B4FS65. Cluster I cleaves the substrates FR, FVR, LR and RR. Cluster II cleaves the substrates FR and LR and Cluster III only cleaves the LR substrate. Interestingly, CP1C does not clusters together with other CP1-like PLCPs but with the phylogenetically distant CathB and XCP2 in cluster III which belong to different subfamilies of PLCPs (subfamily 9 and 3, respectively). The observed sim- ilarities in substrate specificity may be correlated to similar target preferences for each cluster in vivo.
PLCPs (Richau et al., 2012). An even higher pH optimum of pH 7 for B4FS65 points more to a vacuolar localization (Pfanz et al., 1987; Grignon et al., 1991). CP1C and CP1D are most selective for their pH dependent activity which ranges mainly from pH 4 – 6. CP1A, CP1B and B4FYA3 are active starting at a pH of 3 – 7 and XCP2, CathB, Q10716, CP2 and B4FS65 are active from pH 4 up to pH 9 or even 10 (2.20C & 6.14).
These PLCPs were also tested for their susceptibility to various PLCP inhibitors like E-64, cMIP, CC9, CC and CC1. Pit2, the precursor protein of UmcMIP (Mueller et al., 2013; Misas Villamil et al., 2019) were also included (2.20D). Graphs for inhibition of CathB, CP2 and XCP2 and inhibition of Pit2 versus CP1-like and SA-activated PLCPs, which have not been represented in previous figures are shown in 6.15 & 6.16, respectively. In general, it was seen that cMIP and Pit2 were weaker inhibitors towards the tested PLCPs than the other tested inhibitors. CP1A shows to be most resistant to E-64 from all tested CP1-like PLCPs but is highly exceeded in resistance by B4FYA3. Interestingly, CP1C seems to be least resistant towards E-64, an inhibitor produced and first isolated from the soil fungus Aspergillus japonicas (Hanada et al., 1978) which might indicate CP1C as a putative favorable target for fungal inhibition through inhibitors similar to E-64. B4FYA3 on the contrary might be induced as a E-64 resistant PLCP to ensure a proper defense reaction. CP1C is most resistant of all tested PLCPs to cMIP. CP1A is the least resistant CP1-like and CathB the overall least resistant PLCP to cMIP tested in this study. In contrast, CP1A is the most resistant CP1-like PLCP to Pit2. CP1B is the least resistant CP1-like PLCP. Q10716 and CP2 are the most and least resistant tested PLCPs to Pit2, respectively. Interestingly we observed Pit2 to be able to inhibit CathB which stands in contrast to previously published results by Mueller et al., (2013) that showed Pit2 to be unable to inhibit CathB. CP1D is the least resistant tested PLCP towards CC9 inhibition. CP1C is the most resistant CP1- like and XCP2 the overall most resistant PLCP. CP2 is most resistant to inhibition of the endogenous root cystatin CC1 and CP1A displays the least resistance. CP1B is the displays the most resistance to CC1 of the tested CP1-like PLCPs. CP1C is the least resistant tested PLCP to the commercial CC. CP1D shows highest resistance among CP1-like PLCPs to CC but the two SA-activated PLCPs B4FYA3 and Q10716 display the highest resistance to CC inhibition of all tested PLCPs. CP1D displayed intermediate susceptibilities to the tested inhibitors compared the other CP1-like PLCPs suggesting that the overall structural changes towards CP1A and CP1B and the specific orientation of His in the catalytic triad do not strongly influence inhibitory interactions. Compared to the clear
correlation between the structural feature of catalytic His orientation and substrate cleavage that we described above there was no clear pattern observable for inhibition susceptibility of PLCPs for the tested inhibitors. We could neither observe a pattern for inhibitor susceptibility based on catalytic His orientation nor based on sequence homology and phylogeny of the PLCPs.
Figure 2.20:Transcriptional and biochemical analysis of root apoplastic maize PLCPs. (A) Expression pattern of root apoplastic maize PLCPs. Relative expression of root apoplastic maize PLCPs in untreated B73 based on publicly available data (Winter et al., 2007; Sekhon et al., 2011; Andorf et al., 2016; Stelpflug et al., 2016). Mean expression of leaves and roots at different developmental stages was calculated and normalized to leaf expression for individual PLCPs. The heat map represents a one – to – one comparison for each PLCP. PLCPs were clustered based on their relative expression pattern to leaves. (B) Heat-map of relative substrate affinity. Recombinant PLCPs were tested in substrate cleavage assays using 10 µM of the following substrates: Z-FR-AMC (FR), BZ-FVR-AMC (FVR), Z-LR-AMC (LR) and Z-RR- AMC (RR) (Figure 13, 18, 21). PLCP activity was calculated for each PLCP and each substrate relative to the strongest activity. Strongest activity was set to 1 (red) and no activity was set to 0 (white). PLCPs were clustered based on their relative substrate affinity pattern.
(C) pH dependent PLCP activity. Substrate cleavage of heterologous in N. benthamiana produced PLCPs was tested at various pH (3 – 10) using the substrate Z-LR-AMC (LR) (6.14). PLCP activity at each pH was calculated relative to the strongest activity. Strongest activity was set to 1 (red) and no activity set to 0 (white). PLCPs were clustered based on their activity pattern at various pH.
(D) Inhibitory profiles of root apoplastic PLCPs. Apoplastic fluids of recombinant in N. benthamiana produced PLCPs were evaluated for their activity using 10 µM of the substrate Z-LR-AMC (LR) in the presence of an inhibitor. The inhibitory profile for E-64, cMIP, Pit2, CC9, CC1 and the commercial chicken cystatin (CC) was tested. IC50-values [nM] were calculated for each inhibitor-PLCP combination and displayed in an overview table.