Functional analysis of the CXXC motif using phage antibodies that cross-react with protein disulphide-isomerase family proteins.
Polyclonal antibodies that had been raised against particular PDI (protein disulphide-isomerase) family proteins did not cross-react with other PDI family proteins. To evade immune tolerance to the important self-motif Cys-Xaa-Xaa-Cys, which is present in PDI family proteins, we used the phage display library [established by Griffiths, Williams, Hartley, Tomlinson, Waterhouse, Crosby, Kontermann, Jones, Low, Allison et al. (1994) EMBO J. 13, 3245-3260] to isolate successfully the phage antibodies that can cross-react with human and bovine PDIs, human P5, human PDI-related protein and yeast PDI. By measuring the binding of scFv (single-chain antibody fragment of variable region) to synthetic peptides and to mutants of PDI family proteins in a surface plasmon resonance apparatus, we identified clones that recognized sequences containing the CGHC motif or the CGHCK sequence. By using the isolated phage antibodies, we demonstrated for the first time that a lysine residue following the CXXC motif significantly increases the isomerase activities of PDI family proteins. Moreover, we demonstrated that the affinity of isolated scFvs for mutant PDI family proteins is proportional to the isomerase activities of their active sites.
Kimura
Taiji
T
Department of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan.
Nishida
Ai
A
Ohara
Nobutoshi
N
Yamagishi
Daisuke
D
Horibe
Tomohisa
T
Kikuchi
Masakazu
M
D020816:Amino Acid Motifs / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D000918:Antibody Specificity; D002417:Cattle; D003429:Cross Reactions; D006801:Humans; D007128:Immunoglobulin Fragments; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D019704:Protein Disulfide-Isomerases / Q000276:immunology* / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D019704:Protein Disulfide-Isomerases; D008239:Lysine