ANALYSIS OF THE CONSERVED GLYCOSYLATION SITE IN THE NICOTINIC ACETYLCHOLINE-RECEPTOR - POTENTIAL ROLES IN COMPLEX ASSEMBLY

Background: Assembly of the functional nicotinic acetylcholine recepto r (nAChR) is dependent on a series of exquisitely coordinated events i ncluding polypeptide synthesis and processing, side-chain elaboration through post-translational modifications, and subunit oligomerization. A 17-residue sequence that includes a cystine disulfide and an N-link ed glycosylation site is conserved in the extracellular domain of each of the nAChR subunits, and is involved in intersubunit interactions t hat an critical for assembly of intact, pentameric complexes. A polype ptide representing the relevant sequence from the alpha-subunit oi the nAChR l-Thr-His-Phe-Pro-Phe-Asp-Gln-Gln-Asn-Cys-Thr-NH2) is small eno ugh to allow detailed structural analysis, which may provide insight i nto the role of glycosylation in the maturation process that leads to ion-channel assembly. We therefore investigated the effect of N-linked glycosylation on the structure of this heptadecapeptide. Results: The rmodynamic analysis shows that glycosylation alters disulfide formatio n in the loop peptide, shifting the equilibrium in favor of the disulf ide. Spectroscopic studies reveal that the cis/trans amide isomer rati o of the proline is also affected by the modification, with a resultan t shift in the equilibrium in favor of the trans isomer, even though t he proline is several residues removed from the glycosylation site. Tw o-dimensional NMR analysis of the glycopeptide does not indicate the p resence of any specific interactions between the carbohydrate and the peptide. Conclusions: These studies demonstrate that glycosylation can have a significant influence on disulfide formation and proline isome rization in a local peptide sequence. As both these processes are cons idered slow steps in protein folding, it is evident that N-linked glyc osylation has important indirect roles that influence the folding of t he receptor subunit and assembly of the pentameric complex.