Premium
Alteration of amino acid residues at the L‐chain N‐terminus and in complementarity‐determining region 3 increases affinity of a recombinant F(ab) for the human N blood group antigen
Author(s) -
Song ShuhChyuny,
Czerwinski Marcin,
Wojczyk Boguslaw S.,
Spitalnik Steven L.
Publication year - 2004
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1111/j.1537-2995.2004.00625.x
Subject(s) - complementarity determining region , recombinant dna , antibody , amino acid , chemistry , biochemistry , glycopeptide , mutagenesis , microbiology and biotechnology , peptide sequence , mutant , stereochemistry , biology , genetics , gene , antibiotics
BACKGROUND: To examine the fine specificity of glycopeptide‐specific antibodies, this study focused on the human MN blood group system. F(ab) phage display methods were previously used to construct an F(ab) family in which the H‐chain Fd fragment was held constant whereas the L chains were “shuffled.” This yielded two related F(ab), N92 and NNA7, with low and high affinity for N, respectively. Although their L‐chain sequences are very similar, sharing 92 percent amino acid identity, there are intriguing differences at the N‐terminus and in complementarity‐determining region 3 (CDR3) at positions 89, 91, 92, and 96. STUDY DESIGN AND METHODS: Site‐directed mutagenesis, ELISA, and hemagglutination were used to examine the contributions of these variations to antibody affinity. RESULTS: Studies with the N92‐S91G and NNA7‐G91S mutants demonstrated that the Gly at position 91 was critically important for ensuring high affinity. Indeed, the affinity of N92‐S91G was almost as high as N92TM, in which all four CDR3 residues were changed to match NNA7. N‐terminal L‐chain differences were surprisingly important in determining affinity. For example, when the N‐terminus of N92 was changed to match that of NNA7, affinity increased approximately 30‐fold. CONCLUSION: Specific residues at the L‐chain N‐terminus and in CDR3 significantly affected F(ab) affinity for N. Future structural studies of these F(ab), alone and complexed with this glycopeptide antigen, will provide further insights into these phenomena.