Specific glycosidase activity isolated from a random phage display antibody library.
Carbohydrates serve as key receptor sites in various cellular events such as viral attachment, tumor formation, and tissue inflammation. A potential route to control these events is to manipulate targeted carbohydrate structures in vivo using specifically designed glycohydrolases. Here we show that a stereospecific catalytic activity designed toward a particular sugar and linkage can be readily isolated from a phage display antibody library derived from a nonimmunized host. The activity was isolated using a transition-state analogue mimicking an alpha-glucosidasic linkage as antigen and showed a 20-fold specificity for that sugar and linkage. The DNA sequence, however, contains a large deletion in the antibody gene, which also changes the downstream reading frame, resulting in a translated sequence containing only 57 amino acids that has a predominantly hydrophobic amino terminal and a strongly hydrophilic carboxy terminal. The isolated catalytic activity has a strong pH dependence, attributable to one or more of the numerous potentially charged groups in the carboxyl terminal. While the protein readily forms more stable multimers, the 7.3-kD monomer represents by far the smallest glycosidase enzyme reported to date and can provide substantial new information toward understanding and modifying glycosidase activity.
Goud
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GN
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA.
Artsaenko
O
O
Bols
M
M
Sierks
M
M
D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism*; D001435:Bacteriophages / Q000201:enzymology*; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002384:Catalysis; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D000906:Antibodies; D004274:DNA, Recombinant; D006026:Glycoside Hydrolases