Analysis of the conserved glycosylation site in the nicotinic acetylcholine receptor: potential roles in complex assembly.
BACKGROUND
Assembly of the functional nicotinic acetylcholine receptor (nAChR) is dependent on a series of exquisitely coordinated events including 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-linked glycosylation site is conserved in the extracellular domain of each of the nAChR subunits, and is involved in intersubunit interactions that are critical for assembly of intact, pentameric complexes. A polypeptide representing the relevant sequence from the alpha-subunit of the nAChR (Ac-Tyr-Cys-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Gln-Gln Asn-Cys-Thr-NH2) is small enough to allow detailed structural analysis, which may provide insight into 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
Thermodynamic analysis shows that glycosylation alters disulfide formation in the loop peptide, shifting the equilibrium in favor of the disulfide. Spectroscopic studies reveal that the cis/trans amide isomer ratio of the proline is also affected by the modification, with a resultant shift in the equilibrium in favor of the trans isomer, even though the proline is several residues removed from the glycosylation site. Two-dimensional NMR analysis of the glycopeptide does not indicate the presence 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 isomerization in a local peptide sequence. As both these processes are considered slow steps in protein folding, it is evident that N-linked glycosylation has important indirect roles that influence the folding of the receptor subunit and assembly of the pentameric complex.
Rickert
K W
KW
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
Imperiali
B
B
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D017124:Conserved Sequence; D004220:Disulfides; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D007700:Kinetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000378:metabolism*; D013552:Swine; D013816:Thermodynamics
D004220:Disulfides; D006020:Glycopeptides; D011978:Receptors, Nicotinic