Abstract
Tyrosinase (TYR) was thought to play a critical role during trematode egg production. In this study, we analyzed two genes (SjTYR1 and SjTYR2), derived from Schistosoma japonicum genome databases, which encode proteins with significant homologies to mammalian TYR. They exhibited the typical TYR topology, including two copper-binding domains and a highly conserved cysteine-rich domain. Semi-quantitative reverse transcription polymerase chain reaction showed that two SjTYR genes were mainly expressed in the female adult worm. A complementary DNA coding the putative common copper center domain of each SjTYR was cloned and inserted into a pET-28a-c(+) prokaryotic expression vector. After purification, the recombinant proteins expressed in Escherichia coli were used to produce their specific antibodies. The native active SjTYRs enzyme appeared to function as a homodimer, the subunits of which were linked to each other via covalent disulfide bonds. Both female and male worms possessed monophenol oxidase and diphenol oxidase activities of TYR. The relative enzymatic activities were 0.165 min−1 mg−1 and 0.0805 min−1 mg−1, which were inhibited by a copper-chelating agent (allyl thiourea) and correlated with disruption of female egg production. Our results revealed that SjTYRs might play a significant role during eggshell formation.
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This study was supported by the National Natural Science Foundation Committee of China (nos. 30471509 and 30671966).
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Fig. S1
a Domain structures of trematode tyrosinase (TYR), mammalian TYR, and arthropod prophenol oxidase (PPO). The conserved copper binding sites (Cu(A) and Cu(B) are indicated, as well as other conserved domains. Both trematode and mammalian TYRs have a signal peptide, EGF-like domain, binuclear copper binding sites (Cu(A) and Cu(B)), and cysteine rich central region, except that the mammalian TYRs contain an additional transmembrane domain in their C-terminal tails. Arthropod PPO has two N-terminal cleavage sites (RF and REE sites), a putative thiolester site, and a conserved C-terminal site. Only the two binuclear copper binding sites are common to both TYR and PPO. b Alignment of sequences at the Cu(A) and Cu(B) binding sites of trematode TYRs, mammalian TYRs, and arthropod PPOs. Gaps (−) have been introduced to optimize the alignment. The putative copper ligands of histidine residues (H) conserved in all proteins are labeled with asterisks, those conserved in trematode and mammalian TYRs with filled arrowhead, and those conserved in arthropod PPO proteins with hollowed arrowhead. The identity of each analyzed sequence is distinguished by a GenBank accession number and abbreviated species name as following: Sj, Schistosoma japonicum; Sm, Schistosoma mansoni; Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Bm, Bombyx mori; Ag, Anopheles gambiae; Aa, Aedes aegypti and Dm, Drosophila melanogaster. The identical residues in the alignment were highlighted in black, while similar residues were shown in gray (PPT 79.5 kb).
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Cai, G., Bae, Y., Zhang, Y. et al. Expression and characterization of two tyrosinases from the trematode Schistosoma japonicum . Parasitol Res 104, 601–609 (2009). https://doi.org/10.1007/s00436-008-1236-5
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DOI: https://doi.org/10.1007/s00436-008-1236-5