SmMAK16, the Schistosoma mansoni homologue of MAK16 from yeast, targets protein transport to the nucleolus.

The SmMAK16 gene from Schistosoma mansoni was cloned by chance when an adult worm cDNA library was probed with antiserum to affinity-purified S. mansoni GSH S-transferases. SmMAK16 encodes a hydrophilic protein of 259 amino acids with a molecular mass of 31 kDa. The protein shares 43% sequence identity and 66% similarity to the nuclear protein MAK16 of Saccharomyces cerevisiae that has been implicated both in cell cycle progression and biogenesis of 60S ribosomal subunits. Both proteins display a similar degree of sequence similar to the hypothetical protein CeMAK16 from Caenorhabditis elegans. These proteins share a number of apparent protein motifs, including two nuclear localization signals (NLS), multiple sites for phosphorylation by protein kinase CK2 and four conserved cysteine residues that resemble a zinc binding domain. SmMAK16 mRNA is more highly expressed in adult female worm than males. Recombinant SmMAK16 was phosphorylated by human protein kinase CK2. When chimeric constructs containing SmMAK16 fused the green fluorescent protein (GFP) were transiently transfected into COS-7s cells, the reporter was localized not in nuclei, but exclusively in nucleoli. The yeast and nematode homologues were likewise able to direct nucleolar accumulation of the fluorescent reporter. The high degree of sequence conservation together with the ability to direct nucleolar protein transport supports the hypothesis that MAK16 proteins play a key role in the biogenesis of 60S subunits.

Schistosoma japonicum GSH S-transferase Sj26 is not the molecular target of praziquantel action.

It has been suggested that Sj26, a Schistosoma japonicum GSH S-transferase, is the molecular target of the antischistosomal drug praziquantel (McTigue et al., 1995, J. Mol. Biol. 246, 21-27). We tested this hypothesis by asking two questions: (1) does praziquantel inhibit Sj26 activity with a variety of model substrates; and (2) does praziquantel prevent the binding to Sj26 of physiologically relevant nonsubstrate ligands? High concentrations of praziquantel (up to 500 microM) did not inhibit Sj26 activity using the model substrates 1-chloro-2,4-dinitrobenzene, 3,4-dichloronitrobenzene, or ethacrynic acid. Sj26 had no measurable activity with two higher molecular weight GSH S-transferase substrates: 5-androsten-3,17-dione and sulfobromophthalein. We also assessed the ability of praziquantel to prevent the inhibition of Sj26 by a series of S-alkyl-GSH conjugates. The half-maximal inhibitory concentrations of S-hexyl-GSH, S-octyl-GSH, and S-decyl-GSH (10, 10, and 5 microM, respectively) for Sj26 were not affected by up to 500 microM praziquantel. This suggests that praziquantel does not compete with GSH for Sj26 binding. In order to determine if praziquantel disrupts binding of nonsubstrate ligands to Sj26, we tested praziquantel for its ability to prevent the inhibition of Sj26 by both bilirubin and hematin. Praziquantel (100 or 500 microM) did not alter inhibition of Sj26 by 3 microM bilirubin, but partially protected Sj26 against inhibition by hematin (0.1 to 2.0 microM). Interestingly, in a similar reaction, 100 microM S-methyl-GSH protected Sj26 from inhibition equally as well as praziquantel. Bovine serum albumin (5 microM) completely protected against inhibition by 1 microM hematin. These results indicate that although praziquantel partially protects Sj26 from hematin inhibition, this protection is neither specific to praziquantel nor physiologically relevant. Our results do not support the hypothesis that the mechanism of praziquantel action involves competitive inhibition of Sj26 catalytic activity or blocking binding of nonsubstrate ligands. We can, therefore, find no evidence that Sj26 is the molecular target of the antischistosomal activity of praziquantel.