Insect prophenoloxidases (PPOs) are important immunity proteins for defending against the invading pathogens and parasites. As a Type-Ⅲ copper-containing proteins, unlike Homo sapiens tyrosinases, the insect PPOs and most bacterial tyrosinases contain no signal peptides for unknown reason, however they can still be released. To this end, we fused different signal peptides to Drosophila melanogaster PPOs for in vitro and in vivo expression, respectively. We demonstrate that an artificial signal peptide can help PPO secretion in vitro. The secreted PPO appeared larger than wild-type PPO on molecular weight sizes due to glycosylation when expressed in S2 cells. Two asparagine residues for potential glycosylation in PPO1 were identified when a signal peptide was fused. After purification, the glycosylated PPO1 lost zymogen activity. When PPO1 containing a signal peptide was over-expressed in Drosophila larvae, the glycosylation and secretion of PPO1 was detected in vivo. Unlike insect PPO, human tyrosinase needs a signal peptide for protein expression and maintaining enzyme activity. An artificial signal peptide fused to bacterial tyrosinase had no influence on the protein expression and enzyme activity. These Type-Ⅲ copper-containing proteins from different organisms may evolve to perform their specific functions. Intriguingly, our study revealed that the addition of calcium inhibits PPO secretion from the transiently cultured larval hindguts in vitro, indicating that the calcium concentration may regulate PPO secretion. Taken together, insect PPOs can maintain enzyme activities without any signal peptide.

College of Life Sciences Shangrao Normal University Shangrao 334001 China

College of Life Sciences Shangrao Normal University Shangrao 334001 China; Key Laboratory of Insect Developmental and Evolutionary Biology CAS Center for Excellence in Molecular Plant Sciences Shanghai Institute of Plant Physiology and Ecology Chinese Academy of Sciences Shanghai 200032 China

Institute of Entomology Biology Centre CAS Branisovska 31 370 05 Ceske Budejovice Czech Republic

Key Laboratory of Insect Developmental and Evolutionary Biology CAS Center for Excellence in Molecular Plant Sciences Shanghai Institute of Plant Physiology and Ecology Chinese Academy of Sciences Shanghai 200032 China

Key Laboratory of Insect Developmental and Evolutionary Biology CAS Center for Excellence in Molecular Plant Sciences Shanghai Institute of Plant Physiology and Ecology Chinese Academy of Sciences Shanghai 200032 China; College of Advanced Agricultural Sciences Zhejiang Agriculture and Forestry University Hangzhou 311300 China

Key Laboratory of Insect Developmental and Evolutionary Biology CAS Center for Excellence in Molecular Plant Sciences Shanghai Institute of Plant Physiology and Ecology Chinese Academy of Sciences Shanghai 200032 China; Innovative Academy of Seed Design Chinese Academy of Sciences Beijing 100093 China

Key Laboratory of Insect Developmental and Evolutionary Biology CAS Center for Excellence in Molecular Plant Sciences Shanghai Institute of Plant Physiology and Ecology Chinese Academy of Sciences Shanghai 200032 China; Shanghai Majorbio Bio pharm Technology Co Ltd Shanghai 201318 China

Life Science Institute Jinzhou Medical University Jinzhou 121001 China

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