Molecular and biochemical characterization ofParagonimus westermanityrosinase

Abstract

Trematode tyrosinases (TYRs) play a major role in the tanning process during eggshell formation. We investigated the molecular and biochemical features of Paragonimus westermani TYR (PwTYR). The PwTYR cDNA was composed of 1568-bp encompassing a 1422-bp-long open reading frame (474-amino acid polypeptide). A strong phylogenetic relationship with Platyhelminthes and Deuterostomian orthologues was evident. The recombinant PwTYR expressed in prokaryotic cells promptly oxidized diphenol substrates, with a preferential affinity toward ortho-positioned hydroxyl groups. It demonstrated fairly weak activity for monophenol compounds. Diphenol oxidase activity was augmented with an increase of pH from 5.0 to 8.0, while monophenol oxidase activity was highest at an acidic pH and gradually decreased as pH increased. Transcription profile of PwTYR was temporally upregulated along with worm development. PwTYR was specifically localized in vitellocytes and eggs. The results suggested that conversion of tyrosine to L-dihydroxyphenylalanine by PwTYR monophenol oxidase activity might be rate-limiting step during the sclerotization process of P. westermani eggs. The pH-dependent pattern of monophenol and diphenol oxidase activity further proposes that the initial hydroxylation might slowly but steadily progress in acidic secreted vesicles of vitellocytes and the second oxidation process might be rapidly accelerated by neural or weak alkaline pH environments within the ootype.

Comparative genomics and transcriptomics of 4 Paragonimus species provide insights into lung fluke parasitism and pathogenesis

“…10 ). These included (i) P. westermani and Paragonimus pseudoheterotremus cysteine proteases identified in 2 previous studies [ 65 , 66 ] (matching to the same protein targets from both studies in P. heterotremus and P. kellicotti ), 1 of which had high adult-stage expression levels in all 4 species [ 65 ]; (ii) 3 different tyrosine kinases (1 of which was identified in 2 different studies, in Clonorchis sinensis and in P. westermani [ 67 , 68 ]), all of which had relatively low gene expression levels in adult stages; (iii) a previously unannotated P. heterotremus ELISA antigen [ 69 ] with low expression across life cycle stages, which we now annotate as a saposin protein (which we found to rapidly evolve among flukes [Fig. 7C ] and which is strongly immunogenic in fascioliasis [ 50 ]); and (iv) eggshell proteins of P. westermani [ 70 ], for which we now provide full-length sequences.…”

Lineage-specific expansion and loss of tyrosinase genes across platyhelminths and their induction profiles in the carcinogenic oriental liver fluke, Clonorchis sinensis

“…The cross-linking reactions are accompanied by changes in the colour and autofluorescence of the eggshell components (Kelly and von Lichtenberg, 1970). Trematode tyrosinases display high degrees of similarity in their structural properties and spatial expression patterns, as has been demonstrated in Schistosoma mansoni (Fitzpatrick et al 2007), Schistosoma japonicum (Cai et al 2009), Clonorchis sinensis (Bae et al 2013), and Paragonimus westermani (Bae et al 2015).…”

“…The primary structures of platyhelminth tyrosinases were compared with those of other γ-subclass tyrosinases and tyrosinase-related proteins. The full amino acid sequences of these proteins, rather than narrow-region restrictions, were thoroughly aligned (data not shown; for examples, see Bae et al 2013, 2015). These proteins displayed similarities in their domain architecture, which comprised the Cys-rich epidermal growth factor-like domain (ELD) and a binuclear copper centre (Aguilera et al 2013; Bae et al 2013).…”

Codon Usage Patterns of Tyrosinase Genes in Clonorchis sinensis

“…The expression of tyrosinase genes was specific to the vitellocytes and thus, coincided with the sexual maturation in trematodes, including C. sinensis [10,32–34]. Structural and biochemical analyses of the Clonorchis tyrosinases did not show any evidence revealing functional diversification, while the relative expression levels were found to be quite different among them (GAA27975>GAA32069>GAA48883>GAA48882; [10]).…”