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Interaction of the proteasome S5a/Rpn10 multiubiquitin-binding protein and the 8 kDa calcium-binding protein of Schistosoma mansoni

Published online by Cambridge University Press:  17 October 2003

D. RAM ,
E. ZIV ,
F. LANTNER  and
I. SCHECHTER
D. RAM
Affiliation:
Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel
E. ZIV
Affiliation:
Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel
F. LANTNER
Affiliation:
Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel
I. SCHECHTER
Affiliation:
Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel
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Abstract

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 λgt11 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 (~50 kDa) and the molecule subjected to post-translational modification (~70 kDa). The present studies suggest that proteasome activity may be modulated by calcium, and this modulation is mediated via CaBP molecule(s).

Keywords

Schistosoma mansoniproteasomeS5a/Rpn10multiubiquitin-binding proteincalcium-binding proteinstage-specific genes
Type
Research Article
Information
Parasitology , Volume 127 , Issue 4 , October 2003 , pp. 337 - 347
Copyright
2003 Cambridge University Press

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