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    • GS967 australia The characterization of Da CTSL shows

    2021-11-22

    The characterization of Da-CTSL1 shows that it preferentially cleaves Z-FR-AFC (Fig. 8), which has a GS967 australia phenylalanine at P2 and Da-proCTSL1 is autocatalyzed at pH 8.0 releasing an activated Da-CTSL1 that exhibits optimal activity at pH 8 indicating that the enzyme when secreted by the foregut and anterior midgut is only activated when it reaches the posterior midgut which has a basic pH (Figs. 9 and 1B, respectively), unlike other cathepsin Ls that have optimal activity in the acidic pH range (pH 5–6.5). Reports of basic cysteine proteases are rare (Otto and Schirmeister, 1997) and unusual for the cathepsins listed in the MEROPS database (Rawlings et al., 2012). Furthermore, no insect basic cathepsin L have been previously identified. Though Z-FR-AFC is an established cathepsin L substrate (Tchoupe et al., 1991), cathepsins B and H have affinities also for it. The general cathepsin inhibitor E-64 inhibits the activities of almost all cysteine proteases, including cathepsins B, H, and L (Barrett et al., 1982, Turk et al., 2012) but not cathepsin C (Rozman-Pungercar et al., 2003). Meanwhile, the inhibitor CA074 and its analogues specifically inhibit cathepsin B (Bogyo et al., 2000), whereas Z-Phe-Tyr(tBu)-diazomethylketone and analogues specifically inhibits cathepsin L (Shaw et al., 1993). Some of these inhibitors, among others, were used to characterize Da-CTSL1. To rule out cathepsin B, the specific cathepsin inhibitor, CA074, was assayed against Da-CTSL1. In the presence of CA074 (50μM), Da-CTSL1 still retained 75% of its activity (Fig. 10). The general cathepsin inhibitor E-64 and the specific cathepsin L inhibitor Z-FY(tBu)DMK only showed limited inhibition of Da-CTSL1 (Fig. 10). The general cathepsin inhibitor chymostatin was effective at inhibiting Da-CTSL1 (Fig. 10), perhaps because it also inhibits serine proteases that are active at a basic pH. Additionally, aprotinin, a sole serine protease inhibitor, did not inhibit Da-CTSL1 at the same concentration range at which it inhibits serine proteases. Da-CTSL1 did not hydrolyze Z-GPR-AMC, Z-VVR-AMC, Suc-AAPF-pNA and H-Arg-pNA.2HCl probably because these substrates do not fit in the active pocket of Da-CTSL1 (Fig. 3B) and thus cannot be hydrolyzed either at acidic or basic pH as was shown for Z-GPR-AMC and Z-VVR-AMC (Fig. 8). The 3D modeling of Da-proCTSL1 predicts that the propeptide becomes inserted into the groove preventing the enzyme’s substrate entry into the active groove and reaching C120 and H255 at the catalytic site (Fig. 3). The propeptide binding at the active groove is fortified by a network of fourteen hydrogen bonds (Fig. 3B and C). Our 3D model of Da-proCTSL1 is supported by a report that in B. mori incubation of a recombinant proregion of cathepsin L binds activated cathepsin L protecting it from denaturation in urea (Yamamoto et al., 1999). Mass spectrometry analysis (not shown) after SDS–PAGE analysis of purified Da-GST-proCTSL1 and Da-CTSL1 identified the cleavage site of the Da-proCTSL1from Da-CTSL1 at L109 (Fig. 2) as was predicted from homology studies with other cathepsins (Fonseca et al., 2012). Our report shows that D. abbreviatus synthesizes in its gut a unique and basic Da-CTSL1 and the enzyme is not affected by chymotrypsin-specific inhibitor TPCK (tosyl phenylalanyl chloromethyl ketone) (data not shown). Our results also suggest that 64% of all the gut total cathepsins is Da-CTSL1 that is probably activated and functions in the basic region of the insect’s gut. Unlike acidic cathepsins which denature at basic pH Da-CTSL1 is stable and maximally active at pH 8 which allows it to synergize with D. abbreviatus trypsin that is active at a basic pH and is also synthesized by the gut of D. abbreviatus (Yan et al., 1999). The interesting implication in finding an unusual cathepsin L1-like protease that comprises 64% of all the cathepsin Ls found in D. abbreviatus allows this insect to become a highly polyphagous and invasive insect and precludes inhibitors that are commonly synthesized by plants to guard against damage from feeding insects from inhibiting Da-CTSL1. This provides a mechanism for certain Coleoptera to support polyphagy and invasiveness.