Identification of essential residues of CTLA-2 for inhibitory potency
Source: The Journal of Biochemistry, Volume 147, Number 3, 6 March 2010 , pp. 393-404(12)
Publisher: Oxford University Press
Abstract: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 Ala1Asp9 and Leu80Glu109 regions, was also a potent inhibitor (Ki 10 nM). Subsequent short deletions in the central region (Asn10Ser79) showed three functionally essential distinct regions: Asn10Phe19, His30Ala44 and Ser55Ser79. 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 1520-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.
Document Type: Regular paper
Affiliations: 1: Department of Veterinary Science, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753‐8515; and , 2: Center for Human Evolution Modelling Research, Primate Research Institute, Kyoto University, Inuyama 484‐8506, Japan,
Publication date: 2010-03-06