Identification of essential residues of CTLA-2alpha for inhibitory potency.
To identify functionally essential sequences and residues of CTLA-2alpha, in vitro mutagenesis was carried out. The coefficient of inhibition (K(i)) was determined towards rabbit cathepsin L using Z-Phe-Arg-MCA as the substrate. Recombinant CTLA-2alpha inhibited the enzyme potently (K(i) = 15 nM). A truncated mutant, lacking the N- and C-terminal Ala1-Asp9 and Leu80-Glu109 regions, was also a potent inhibitor (K(i) = 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 (alpha1, alpha2 and alpha3) and sequences that interact with the cognate enzyme. Alanine scanning showed that replacement of one of three conserved Trp residues increased the K(i) 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-2alpha 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-2alpha 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-2alpha.
Deshapriya
R M C
RM
Department of Veterinary Science, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan.
Yuhashi
Sho
S
Usui
Masaru
M
Kageyama
Takashi
T
Yamamoto
Yoshimi
Y
D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056668:Cathepsin L / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D000943:Antigens, Differentiation; D003374:Coumarins; D004151:Dipeptides; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C102076:cytotoxic T-lymphocyte antigen-2, mouse; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; D056668:Cathepsin L