A distinct 8 kDa calcium-binding protein (CaBP) is preferentially expressed at the cercarial stage during the life-cycle of the schistosome. Available data indicate that this CaBP may be associated with tissue/organ remodelling (involving protein degradation and synthesis of new proteins) during transformation of the cercariae from free-living form in water to parasitic life in the vertebrate host. Many CaBP molecules (e.g. calmodulin) show Ca(++)-dependent interaction with target proteins and thus modulate their activity. Accordingly, the parasite 8 kDa CaBP was used as a probe to clone and identify putative target protein(s) directly by binding interaction. Screening of schistosome lambdagt11 expression library with radio-iodinated CaBP yielded several overlapping clones showing Ca(++)-dependent binding of the CaBP. Sequence analyses revealed that these clones encode the S5a/Rpn10 multiubiquitin-binding protein which is a component of the regulatory 19S subunit of the 26S proteasome. The schistosome molecule, designated SmS5a, is 420 amino acids long. The nearly full length molecule (Gln3-Ser420) as well as the amino terminal (N-S5a, Gln3-Gly200) and carboxyl-terminal (C-S5a, Asp225-Ser420) portions were synthesized in bacteria, purified, and antibodies to the parasite SmS5a were prepared. Interaction between SmS5a and the 8 kDa CaBP in a Ca(++)-dependent manner was found under various experimental conditions: CaBP-Sepharose bound soluble SmS5a, immobilized SmS5a bound soluble CaBP, and complex formation was found when both molecules were in solution. Furthermore, it was shown that the C-terminal portion of SmS5a, but not the N-terminal portion of the molecule, reacted with the CaBP. SmS5a synthesized in a cell-free system and Western blots revealed 2 species, conceivably corresponding to the naked molecule (approximately 50 kDa) and the molecule subjected to post-translational modification (approximately 70 kDa). The present studies suggest that proteasome activity may be modulated by calcium, and this modulation is mediated via CaBP molecule(s).

• PMID: 14636020
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Ram D, Ziv E, Lantner F, Schechter I

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