Title : Site-specific characterization of
the N-linked glycans of murine prion
protein by high-performance liquid chromatography/electrospray mass spectrometry and exoglycosidase digestions
Abstract :
- 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