Site-specific characterization of the N-linked glycans of murine prion protein by high-performance liquid chromatography/electrospray mass spectrometry and exoglycosidase digestions.
The murine prion protein PrP gene encodes a protein of 254 amino acids with two consensus sites for Asn-linked glycosylation at codons 180 and 196. A partial site-specific study of the N-linked glycans from hamster PrP has previously been carried out by mass spectrometry [Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame, A. L., and Prusiner, S. B. (1993) Biochemistry 32, 1991-2002] and revealed that the glycosylation at Asn-181 (equivalent to mouse 180) is heterogeneous, comprising over 30 glycoforms. The identification of the glycosylated peptide spanning Asn-197 was not reported. Recent technical advances in electrospray mass spectrometry now provide the sensitivity to detect low femtomole quantities of glycopeptides with >5000 mass resolution and 30 ppm mass measurement [Medzihradszky, K. F., Besman, M. J., and Burlingame, A. L. (1998) Rapid Commun. Mass Spectrom. 12, 472-478]. This performance coupled with stepwise exoglycosidase digestion has been employed to establish the differential nature of the structural complexity (glycoforms) of the glycans at Asn-180 and Asn-196 from a single strain infected with the ME7 strain. Some sixty structures have been found characterized by neutral and sialylated bi-, tri-, and tetraantennary complex-type bearing outer-arm alpha(1-3)-fucosylation (the Lewisx and sialyl-Lewisx epitopes), core alpha(1,6) fucosylation, and the presence of terminal HexNAc residues. The Lewisx trisaccharide is the major nonreducing structure at Asn-180, and significant amounts of both Lewisx and sialyl Lewisx epitopes are observed at Asn-196. The abundance of the Lewisx and sialyl Lewisx epitopes on murine PrPSc may indicate a role for these structures in the normal function of PrPC or the pathophysiology of PrPSc.
Stimson
E
E
Ludwig Institute for Cancer Research, 91 Riding House Street, London, U.K.
Hope
J
J
Chong
A
A
Burlingame
A L
AL
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011328:Prions / Q000737:chemistry / Q000378:metabolism*
D011134:Polysaccharides; D011328:Prions; D001216:Asparagine; D006026:Glycoside Hydrolases