Peptide ligand interaction with maltose-binding protein tagged to the calcitonin gene-related peptide receptor: The inhibitory role of receptorN-glycosylation

The powerful vasodilator and neuropeptide calcitonin gene-related peptide CGRP is abundant in trigeminal ganglion neurons, and is released from the peripheral nerve and central nerve terminals as well as being secreted within the trigeminal ganglion. CGRP has a proven role in migraine and selective antagonists and antibodies are now reaching the clinic for treatment of migraine. Indeed, a new class of drugs to treat patients with frequent, episodic, and/or chronic migraine headaches acts by antagonism of the calcitonin gene-related peptide (CGRP) pathway. This is the first category of pharmaceuticals developed as targeted therapy for migraine prevention.

RAMPs are an example of membrane-spanning auxiliary proteins that can change the function of G protein-coupled receptors (GPCRs). RAMPs are small families of three proteins that have the potential to introduce functional variety by interacting with GPCRs. RAMPs have a single transmembrane spanning domain with an extracellular N-terminal region of 90-100 amino acids and a short intracellular C-terminal domain of 9 amino acids. Maltose-binding protein (MBP) was employed as a tagged protein in several crystal structures of the CGRP and AM receptor extracellular domain (ECD). It was predicted that N-glycosylation of the CGRP receptor ECD would inhibit MBP from docking to the altered peptide ligands.

In a new study published in the journal Peptides by Dr. Sangmin Lee from Fred Wilson School of Pharmacy at High Point University investigated the role of CGRP receptor N-glycosylation in the interaction with the MBP tag. He postulated that CLR N-glycosylation would push the MBP tag away from the CGRP receptor’s peptide binding site based on known crystal structures. According to the author N-glycosylation of CLR ECD N123 decreased MBP interaction with peptide ligands, suggesting that N-glycosylation plays a substantial role in peptide ligand binding for the MBP-tagged CGRP receptor ECD.

Dr. Lee employed FITC-labeled AM(37-52) with S45W/Q50W mutations for FP peptide binding and its affinity for the MBP tagged, or MBP-free CGRP receptor ECD (tested as RAMP1-CLR ECD fusion protein) has previously been published. Although these affinity values were obtained from two separate tests, the inclusion of the N-terminal MBP tag resulted in a 5-fold greater affinity of the peptide ligand. This matches the MBP interaction with the peptide ligands. MBP did not have any N-glycan in the crystal structures, as expected from its sequence. CLR ECD of the MBP-tagged RAMP1-CLR ECD fusion protein has three N-glycosylation sequons, which are N66, N118, and N123 of CLR ECD. The author findings show that N-glycosylation of the RAMP2-CLR ECD fusion protein increases peptide ligand binding considerably. He also demonstrated that N-glycosylation of the AM receptor ECD improved peptide ligand binding much and CLR N123 was the primary cause of this impact. He also showed that the conformational dynamics of RAMP1- or RAMP2-CLR complexes might be a significant driver of different receptor phenotypes.

According to Dr. Lee   MBP N-terminally linked to the CGRP receptor ECD offered an extra interaction site for mutant peptide ligands, increasing their affinity for the CGRP receptor ECD. However, these MBP tags can be easily removed. Moreover, CGRP can be expressed using other tags such as GST, would the affinity binding affected, something that was not studied and lacking in the study. Furthermore, the argument that MBP will affect affinity binding is not strong and it depends on expression system where glycosylation can change using different expression system. Common screening methods are are largely based on measuring CGRP-mediated changes in cAMP which will not be affected by MBP tag. Moreover, a successful advancement in the migraine treatment was the development of Erenumab which is the first FDA approved antibody therapeutic against a G-protein-coupled receptor, the canonical receptor of calcitonin gene related peptide (CGRP-R). The researchers at Amgen created a  novel, epitope-focused antigen  to reconstruct the extracellular domains of the CGRP-R in a stable conformation. The study is weak with contradictory results and we believe the published paper on the effect of MBP on binding affinity of CGRP is NOT of importance in migraine drug development.