Identification of the epitopes of calcitonin gene‐related peptide (CGRP) for two anti‐CGRP monoclonal antibodies by 2D NMR

Abstract

The interactions between calcitonin gene-related peptide and FAB fragments prepared from two different high-affinity anti-CGRP monoclonal antibodies (CB3 and CDI) have been studied at physiological pH using the ability of 'H NMR to detect selectively regions of dynamic flexibility. The 37-residue peptide retains considerable flexibility in regions of its sequence when bound to both antibodies; in each case, more than half of the residues can be seen to have linewidths little perturbed from those of the free peptide. However, the regions where substantial broadening of resonances occur, attributed to substantially reduced motional freedom of the peptide resulting from interactions within the antibody combining site, differ greatly in the two cases. In the complex with CB3 the results indicate that the restricted residues lie exclusively within the C-terminal half of the peptide, and include residues 25 to 32 and the terminal two residues (36 and 37). By contrast, in the complex with CDI, the conformationally restricted residues appear to lie predominantly within the N-terminal half of the CGRP molecule, particularly residues 4-16, although several residues in the middle section of the sequence (22-31) have reduced conformational freedom. These findings, consistent with the results from immunological assays, add considerably to our knowledge of the epitopes.Keywords: antibody peptide interactions; calcitonin gene-related peptide; epitope mapping; NMR Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide with potent vasodilatory and cardioexcitatory properties whose effects are receptor mediated (Abello et al., Raynaud et al., 1994). Little is known about the active conformation of the peptide. The N-terminal region, which is constrained by a disulphide bond, is required for activity, although N-terminal fragments by themselves have only low activity (Maggi et al., 1990) Abbreviarions: CGRP, calcitonin gene-related peptide; CD, circular dichroism spectroscopy; CPMG, Cam Purcell Meiboom Gill; COSY, correlated spectroscopy: DQF, double quantum filtered: ELISA, enzyme linked immunoassay; FAB, fast atom bombardment mass spectrometry; HOHAHA, homonuclear Hartmann-Hahn: NMR, nuclear magnetic resonance: NOE, nuclear Overhauser effect; NOESY, two-dimensional nuclear Overhauser spectroscopy; RELAY COSY, two-dimensional relayed coherence transfer spectroscopy: TOCSY. total correlation spectroscopy: lD, one-dimensional: 2D, two-dimensional. ture of the peptide is flexible and disordered in solution, although an amphiphilic helix is formed in the middle of the peptide between residues 8 and 18 in trifluoroethanol/H20 mixtures (Breeze et al., Hubbard et al., 1991). There are some indications of a preference for a turn-type conformation in the C-terminal half of the peptide in the TFE-induced conformation and also in C-terminal fragments of CGRP (Sagoo et al., 1991). The requirement for activity, if any, for these regions of secondary structure remains to be determined.Eighteen monoclonal antibodies have been...

Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling

“…The CGRP molecule retained considerable flexibility in regions that were not directly involved in the interaction with antibodies. The most striking result was that the two antibodies differ almost completely in the regions of the CGRP molecule that retain flexibility in the complex (Hubbard et al, 1997).…”

“…NMR and far-UV CD spectroscopy indicate that CGRP does not adopt a unique structure, but rather is highly flexible or disordered in aqueous solutions (Hubbard et al, 1991(Hubbard et al, , 1997). The ability of 1 H-NMR to detect specific regions of dynamic flexibility has been employed to analyze the interactions between CGRP and Fab fragments prepared from two different high-affinity anti-CGRP monoclonal antibodies (Hubbard et al, 1991).…”