Structure, oligosaccharide structures, and posttranslationally modified sites of the nicotinic acetylcholine receptor.
Using mass spectrometry, we have examined the transmembrane topography of the nicotinic acetylcholine receptor, a five-subunit glycosylated protein complex that forms a gated ion channel in the neuromuscular junction. The primary sequences of the four polypeptide chains making up the acetylcholine receptor from Torpedo californica contain many possible sites for glycosylation or phosphorylation. We have used liquid secondary ion mass spectrometry to identify posttranslationally modified residues and to determine the intact oligosaccharide structures of the carbohydrate present on the acetylcholine receptor. Asparagine-143 of the alpha subunit (in consensus numbering) is shown to be glycosylated with high-mannose oligosaccharide. Asparagine-453 of the gamma subunit is not glycosylated, a fact that bears on the question of the orientations of putative transmembranous helices M3, MA, and M4. The structures of the six major acetylcholine receptor oligosaccharides are determined: the major components (70%) are of the high-mannose type, with bi-, tri-, and tetraantennary complex oligosaccharides making up approximately equal to 22 mol% of the total carbohydrate. This application of a multichannel array detector mass spectrometer provided a breakthrough in sensitivity that allowed us to identify the site of attachment of, and the sequence of, oligosaccharides on a 300-kDa membrane protein from only 5 pmol of the isolated oligosaccharide.
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
L
L
Poulter
J P
JP
Earnest
R M
RM
Stroud
A L
AL
Burlingame
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000378:metabolism; D006031:Glycosylation; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009469:Neuromuscular Junction / Q000378:metabolism; D009844:Oligosaccharides / Q000302:isolation & purification*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000302:isolation & purification; D014101:Torpedo
D046911:Macromolecular Substances; D009844:Oligosaccharides; D011978:Receptors, Nicotinic