title: Molecular Characterization of Two Monoclonal Antibodies against the Same Epitope on B-Cell Receptor Associated Protein 31.
abstract: Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assays also showed that 144-A8 had higher antigen binding capacity than 297-D4. Affinity measurement revealed that 144-A8 had 1.54-fold higher binding affinity than 297-D4. Analysis of the heavy- and light-chain variable region sequences of two MAbs revealed that both MAbs belonged to the same heavy chain (Igh-V3660 VH3) and light chain subgroup (IGKV21) with just two amino acid differences in each framework region, indicating that both MAbs arise from the same germline origin. Seven amino acid differences were found between the complementarity determining regions (CDRs) of the two MAbs. Molecular modeling of the epitope-paratope complexes revealed that the epitope appeared to reside in closer proximity to the CDRs of 144-A8 than to those of 297-D4 with the stronger hydrogen bond interactions with the former than the latter. More interestingly, an additional hydrophobic interaction appeared to be established between the leucine residue of epitope and the paratope of 144-A8, due to the substitution of H-Tyr101 for H-Phe101 in 144-A8. Thus, the different binding specificity and affinity of 144-A8 appeared to be due to the different hydrogen bonds and hydrophobic interaction induced by the alterations of amino acids in CDRs of 144-A8. The results provide molecular insights into how the binding specificities and affinities of antibodies evolve with the same epitope in different microenvironments.
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Won-Tae
authors.initials: WT
authors.lastname: Kim
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Saemina
authors.initials: S
authors.lastname: Shin
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Hyo Jeong
authors.initials: HJ
authors.lastname: Hwang
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Min Kyu
authors.initials: MK
authors.lastname: Kim
authors.affiliation: Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Korea.|Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR.
authors.forename: Han-Sung
authors.initials: HS
authors.lastname: Jung
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Hwangseo
authors.initials: H
authors.lastname: Park
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Chun Jeih
authors.initials: CJ
authors.lastname: Ryu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D000915:Antibody Affinity; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000737:chemistry* / Q000276:immunology; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000276:immunology; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D051379:Mice; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D011947:Receptors, Antigen, B-Cell / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment
chemical_list: D000911:Antibodies, Monoclonal; C091247:BCAP31 protein, human; D022801:Complementarity Determining Regions; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D011947:Receptors, Antigen, B-Cell