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.

Enhanced expression of cell-surface B-cell receptor-associated protein 31 contributes to poor survival of non-small cell lung carcinoma cells

“…The similar results were obtained with A549 sphere cells ( Fig 4D, 4E and 4F ). The same phenomenon was also obtained with hESCs where csBAP31 are expressed on the cell surface [ 6 – 8 ] ( S4 Fig ). Thus, the results suggest that enhanced cell surface expression of csBAP31 may contribute to apoptosis.…”

“…BAP31 is cleaved by caspase-8, and its cleaved product, p20, is an important inducer of apoptosis [ 4 , 5 ]. Although BAP31 is mainly localized to the ER membrane, recent studies have shown that BAP31 is also present on the cell surface of human embryonic stem cells (hESCs) [ 6 – 8 ]. It seems that cell surface BAP31 (csBAP31) promotes cell survival through the regulation of cell adhesion to extracellular matrix in hESCs [ 6 , 7 , 9 ].…”