Expression and characterization of two tyrosinases from the trematode Schistosoma japonicum.
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.
Cai
Guobin
G
Department of Medical Parasitology, School of Medicine, Wuhan University, Wuhan, 430071, China.
Bae
Youngan
Y
Zhang
Ying
Y
He
Yuan
Y
Jiang
Mingsen
M
He
Li
L
D000595:Amino Acid Sequence; D000818:Animals; D004156:Catechol Oxidase / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D015801:Helminth Proteins / Q000096:biosynthesis* / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012549:Schistosoma japonicum / Q000201:enzymology*; D016415:Sequence Alignment; D017385:Sequence Homology; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology
D004791:Enzyme Inhibitors; D015801:Helminth Proteins; C009842:allylthiourea; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase; D013890:Thiourea