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Articles by S Yuhashi
Total Records ( 1 ) for S Yuhashi
  R.M.C Deshapriya , S Yuhashi , M Usui , T Kageyama and Y. Yamamoto
 

To identify functionally essential sequences and residues of CTLA-2, in vitro mutagenesis was carried out. The coefficient of inhibition (Ki) was determined towards rabbit cathepsin L using Z-Phe-Arg-MCA as the substrate. Recombinant CTLA-2 inhibited the enzyme potently (Ki = 15 nM). A truncated mutant, lacking the N- and C-terminal Ala1–Asp9 and Leu80–Glu109 regions, was also a potent inhibitor (Ki = 10 nM). Subsequent short deletions in the central region (Asn10–Ser79) showed three functionally essential distinct regions: Asn10–Phe19, His30–Ala44 and Ser55–Ser79. These regions cover sequences corresponding to three helices (1, 2 and 3) and sequences that interact with the cognate enzyme. Alanine scanning showed that replacement of one of three conserved Trp residues increased the Ki by 15–20-fold; whereas, replacement of two/three Trp residues at once caused complete loss of potency, as did replacing Cys75 with Ala or Ser. The proteins from wild-type (WT) CTLA-2 and mutant C75A were stable overnight when incubated with cathepsin L; whereas, proteins from mutants W12A, W15A and W35A were quickly digested. Incubation of cathepsin L/WT CTLA-2 formed a complex; whereas, C75S did not form a complex. Our overall results point to a critical role of W12, W15, W35 and Cys75 residues in CTLA-2.

 
 
 
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