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Lack of signal peptide in insect prophenoloxidase to avoid glycosylation to damage the zymogen activity

K. Wu, B. Yang, R. Chen, R. Majeed, B. Li, L. Gong, X. Wei, J. Yang, Y. Tang, A. Wang, S. Toufeeq, HA. Shaik, W. Huang, X. Guo, E. Ling

. 2024 ; 160 (-) : 105230. [pub] 20240717

Language English Country United States

Document type Journal Article

Persistent link   https://www.medvik.cz/link/bmc24018689

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.

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$a Lack of signal peptide in insect prophenoloxidase to avoid glycosylation to damage the zymogen activity / $c K. Wu, B. Yang, R. Chen, R. Majeed, B. Li, L. Gong, X. Wei, J. Yang, Y. Tang, A. Wang, S. Toufeeq, HA. Shaik, W. Huang, X. Guo, E. Ling
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$a 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.
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$a Yang, Bing $u Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China; Shanghai Majorbio Bio-pharm Technology Co., Ltd, Shanghai, 201318, China
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$a Chen, Rongbing $u Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China
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$a Majeed, Rafia $u Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China
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$a Li, Baoling $u Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China; College of Advanced Agricultural Sciences, Zhejiang Agriculture & Forestry University, Hangzhou, 311300, China
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$a Gong, Liyuan $u Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China
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$a Wei, Xuefei $u Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China
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$a Yang, Jingfeng $u Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China
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$a Tang, Yingyu $u Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China
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$a Wang, Aibin $u College of Life Sciences, Shangrao Normal University, Shangrao, 334001, China
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$a Toufeeq, Shahzad $u Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China
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$a Shaik, Haq Abdul $u Institute of Entomology, Biology Centre CAS, Branisovska 31, 370 05, Ceske Budejovice, Czech Republic
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$a Huang, Wuren $u Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China. Electronic address: huangwuren10@163.com
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$a Guo, Xuan $u Life Science Institute, Jinzhou Medical University, Jinzhou, 121001, China. Electronic address: guoxuan@jzmu.edu.cn
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$a Ling, Erjun $u 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. Electronic address: ejling@sibs.ac.cn
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