The WD40-repeat proteins serve as a platform coordinating partner proteins and

The WD40-repeat proteins serve as a platform coordinating partner proteins and are involved in a range of regulatory cellular functions. of ADF/cofilin-bound actin filaments. However the Aip1 itself exerts very weak activity on actin filament dynamics. The WD40-repeat cDNA (CsWD1) was cloned and its expression was FRP-2 characterized from metacercariae. The CsWD1 protein comprising of 7 WD40-repeats was predicted to fold into anti-parallel β-sheets then into a β-propeller. The CsWD1 protein was expressed stage-specifically in the tegumental syncytium of the metacercariae but not in the adults (Cho et Cisplatin al. 2007 cDNAs of partner proteins interacting with the CsWD1 protein were cloned using yeast two-hybrid screening and extended with EST sequences retrieved from the EST pools. The annotated partner proteins were 4 signal proteins 3 transporters 1 protease and 1 muscle protein (Kim et al. 2007 In life cycle of adults (Rim 2005 In the metacercariae the CsWD1 protein is expected to function as a platform holding proteins close together and to render protein-protein interactions. The present study was performed to clone out partner proteins and to get more information on molecular function of the WD40-repeat protein in the metacercariae. To identify interacting partner proteins immunoprecipitation experiments were performed using anti-CsWD1 monoclonal antibody and metacercaria soluble extract. The soluble extract was prepared by grinding and sonicating the metacercariae with 2 volumes of 1× MB buffer (10 mM K-HEPES 20 mM KCl 1 mM EGTA 3 mM MgCl2 1 mM DTT) containing protease inhibitor cocktail and by spinning them at 4℃. To remove non-specific proteins binding to the Protein A 50 μl of soluble extract of metacercaiae was mixed with 50 μl of Protein A resin in 500 μl of PBS and stirred at 4℃ for 2 hr. After spinning Cisplatin the pellet of the Protein A resin was taken out from the extract mix. This metacercarial extract 100 μl was mixed with 100 μl of anti-CsWD1 monoclonal antibody concentrated using Protein A column. After adding Protein A resin the mix was stirred further for 2 hr and washed with PBS. The CsWD1-partner protein complex was eluted with 2-DE rehydration buffer. IPG strips rehydrated with the prepared samples were electrofocused using Ettan IPGphor Isoelectric Focusing System (Amersham Biosciences Inc. Uppsala Sweden) then loaded on 2-dimension SDS-polyacrylamide gel. Spots of the putative partner proteins and CsWD1 were excised from the silver-stained gel. The proteins were digested with trypsin and eluted from the gel slices. Tryptic peptides of the partner protein were analyzed by LC/ESI-MS using Q-TOF2 micromass spectrometer (Micromass Manchester UK) at Cisplatin Korea Basic Science Institute (Daejeon Korea). Control samples were run in parallel without the metacercarial soluble extract. Two dimensional gel electrophoresis of the CsWD1 complex revealed 6 putative partner proteins (Figs. 1A 1 Spots 1 and 2 produced tryptic peptide sequences (GYSFTTTAER EITALAPSTMK EITALAPSTMK + Oxidation (M) QEYDESGPGIVHR + Pyro-glu QEYDESGPGIVHR SYELPDGQVITIGNER VAPEEHPVLLTEAPLNPK DLYANTVLSGGTTMFPGIADR) matching actin-2 (“type”:”entrez-protein” attrs :”text”:”P53471″ term_id :”1703114″ term_text :”P53471″P53471) of and Aip1 and F212 and D217 of the CsWD1 appeared at corresponding sites of the Aip1. E126 of the Aip1 residing in blade 3 is spatially close to D168 and F192 which are also conserved in the CsWD1. The E126 residue is crucial for filament disassembly but not F-actin-binding. An E126A mutant showed binding activity to F-actin (Mohri et al. 2004 The Aip1 in which E126 was substituted with glycine at the equivalent position binds F-actin in pollen grains (Allwood et al. 2002 In Cisplatin the CsWD1 an alanine residue resides at the equivalent position. These results suggest that the CsWD1 has 3 conserved actin-interacting residues on its putative functional surface. Based on the above evidence the CsWD1 is proposed to Cisplatin be an actin-interacting protein of C. sinensis. Footnotes This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST;.