Abstract
The formation of the isoaspartate (isoAsp) is one of spontaneous degradation processes of proteins, affecting their stability and activity. Here, we report for the first time the crystal structures of an antibody Fab that contains isoAsp in the complementarity-determining region (CDR), along with biochemical studies to detect isoAsp. By comparing the elution profiles of cation-exchange chromatography, it was clarified that the antibody 64M-5 Fab is converted from the normal form to isoAsp form spontaneously and time-dependently under physiological conditions. The isoAsp residue was identified with tryptic peptide mapping, N-terminal sequencing, and the protein isoaspartyl methyltransferase assay. Based on the fluorescence quenching method, the isoAsp form of 64M-5 Fab shows a one order of magnitude lower binding constant for its dinucleotide ligand dT(6–4)T than the normal form. According to the structure of the isoAsp form, the conformation of CDR L1 is changed from the normal form to isoAsp form; the loss of hydrogen bonds involving the Asn28L side-chain, and structural conversion of the β-turn from type I to type II’. The formation of isoAsp leads to a large displacement of the side chain of His27dL, and decreased electrostatic interactions with the phosphate group of dT(6–4)T. Such structural changes should be responsible for the lower affinity of the isoAsp form for dT(6–4)T than the normal form. These findings may provide insight into neurodegenerative diseases (NDDs) and related diseases caused by misfolded proteins.
Highlights
The formation of the isoaspartate is one of spontaneous degradation processes of proteins, affecting their stability and activity
Many therapeutic antibodies are in clinical use, there have been many reports about deamidation of Asn residues of complementarity-determining regions (CDRs)[26,27,28,29,30,31]
The purified Fr. 2 isoform was incubated under physiological conditions (0.1 M HEPES-NaOH pH 7.5, 37 °C) for up to a month, and elution profiles on a Mono S column were compared (Fig. 2B)
Summary
The formation of the isoaspartate (isoAsp) is one of spontaneous degradation processes of proteins, affecting their stability and activity. We report for the first time the crystal structures of an antibody Fab that contains isoAsp in the complementarity-determining region (CDR), along with biochemical studies to detect isoAsp. By comparing the elution profiles of cation-exchange chromatography, it was clarified that the antibody 64M-5 Fab is converted from the normal form to isoAsp form spontaneously and time-dependently under physiological conditions. The formation of isoAsp leads to a large displacement of the side chain of His27dL, and decreased electrostatic interactions with the phosphate group of dT(6–4)T Such structural changes should be responsible for the lower affinity of the isoAsp form for dT(6–4)T than the normal form. Many therapeutic antibodies are in clinical use, there have been many reports about deamidation of Asn residues of complementarity-determining regions (CDRs)[26,27,28,29,30,31] Such alterations decreases relative binding affinity for their ligands[26,28]. This is the first report to determine the structure of isoAsp in CDRs of monoclonal antibodies and discuss the structural basis of the decreased affinity of the antibodies due to the formation of isoAsp
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