Characterization of the glycosylation sites in yeast external invertase. I. N-linked oligosaccharide content of the individual sequons.
External invertase is the product of the SUC2 gene of Saccharomyces cerevisiae. The deduced sequence of this enzyme (Taussig, R., and Carlson, M. (1983) Nucleic Acid Res. 11, 1943-1954) reveals it to contain 14 potential N-linked glycosylation sites, or sequons, although only 9-10 appear to be glycosylated (Trimble, R. B., and Maley, F. (1977) J. Biol. Chem. 252, 4409-4412). To determine the location of the glycosylated sequons, external invertase was deglycosylated with endo-beta-acetylglucosaminidase H and its component peptides analyzed by both fast atom bombardment mass spectrometry (FABMS) and classical peptide isolation procedures. By use of the former technique most of the glucosamine-containing sequons could be located and by the latter sufficient amounts of small glucosamine-containing peptides were isolated to enable their quantitation. From the combined FABMS and glucosamine analyses, it was established that eight of the sequons in a subunit of invertase are either completely or almost completely glycosylated, while five others are glycosylated to the extent of about 50% or less. In the case of two overlapping sequons (4 and 5), which include Asn92-Asn93-Thr-Ser, only the first Asn was glycosylated. Thus, all but one of the sequons of external invertase are glycosylated to some extent, giving an appearance of only 9-10 N-linked oligosaccharides/subunit. The sequence identity of both external and internal invertase was verified by FABMS and by peptide sequence analysis. In only one site was an amino acid found to differ from that deduced from the DNA sequence of the SUC2 gene. This occurred at position 390 where a proline was found in place of alanine, which could result from a single base change in the triplet specifying the latter amino acid.
Reddy
V A
VA
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
Johnson
R S
RS
Biemann
K
K
Williams
R S
RS
Ziegler
F D
FD
Trimble
R B
RB
Maley
F
F
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D006026:Glycoside Hydrolases; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D043324:beta-Fructofuranosidase
D009844:Oligosaccharides; D010446:Peptide Fragments; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D003488:Cyanogen Bromide