Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface.
Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.
Okochi
Norihiko
N
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan.
Kato-Murai
Michiko
M
Kadonosono
Tetsuya
T
Ueda
Mitsuyoshi
M
D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000378:metabolism*; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*
D017151:Antibodies, Catalytic; D007147:Immunoglobulin Light Chains; D014660:Vasoactive Intestinal Peptide; D012697:Serine Endopeptidases