Figure 1.

Three mechanisms of insect PPO activation. The terminal serine protease cleaves PPO differently among insect species (Ashida and Brey, 1997; Lee et al., 1998; Kim et al., 2002; Kanost and Gorman, 2008). And the three mechanisms diverge based on these differences. Here, we consider only BmPPO1 (Accession: NP_001037335), MsPPO1 (Accession: O44249), and HdPPO1 (Accession: BAC15603) as a summary. The conserved bonds ⁵¹RF⁵² in BmPPO1 (A), ⁵¹RF⁵² in MsPPO1 (B), and ⁵⁰RF⁵¹ in HdPPO1 (C) were cleaved by serine proteases separately in the same way, as shown. F⁸⁵ in BmPPO1, F⁸⁵ in MsPPO1, and F⁸⁴ HdPPO1 (labeled in blue) function as the place holder in the respective active site pockets, and may block substrates until they are dislodged. In HdPPO1, ¹⁶²RA¹⁶³ was further cleaved to form a fragment at 60 kD with PO activity (C). The corresponding sequences in BmPPO1 (A) and MsPPO1 (B) are also shown in red. In B. mori, BmPPO was cleaved by PPAE to produce PO (Ashida and Brey, 1997) (A). In M. sexta, PAPs cleaved MsPPO in the same place as in B. mori to produce PO fragments with low enzyme activity. When SPHs were added, PO activity increased significantly (Kanost and Gorman, 2008) (B). In H. diomphalia, HdPPO was cleaved as in BmPPO and MsPPO by PPAF-I at the conserved bond ⁵⁰RF⁵¹. However, the large fragment had no PO activity unless PPAF-I, PPAF-II, and PPAF-III were combined, and was cleaved again at ¹⁶²RA¹⁶³ to produce a fragment at 60 kD; this fragment had PO activity (Lee et al., 1998; Kim et al., 2002) (C). In an in vitro assay, commercial α-chymotrypsin cleaved D. melanogaster recombinant PPO1 (Accession: AAF57775) (rPPO1) in at least three places to produce a fragment, also of ~60 kD, with direct enzyme activity (Lu et al., 2014a) (D).