Abstract
There are 14 potential Asn-X-Thr/Ser glycosylation sites, or sequons, in the yeast external invertase sequence. Of these, 13 are wholly or partially glycosylated to give an average of 9-10 oligosaccharides/subunit (Reddy, V. A., Johnson, R. S., Biemann, K., Williams, R. S., Ziegler, F. D., Trimble, R. B., and Maley, F. (1988) J. Biol. Chem. 263, 6978-6985). On digestion of native holoenzyme by endo-beta-N-acetylglucosaminidase H (Endo H) an average of about seven oligosaccharides per subunit are released without affecting enzyme activity (Trimble, R. B., and Maley, F. (1977) J. Biol. Chem. 252, 4409-4412). To determine whether the remaining Endo H-resistant chains were restricted to a limited number of unique sequons or were randomly distributed on all 13, Endo H-treated native invertase was digested with either thermolysin or trypsin and the resultant glycopeptides isolated by reversed-phase high pressure liquid chromatography and gel filtration. It was found that the oligosaccharides attached to Asn92, Asn247, and Asn350 were partially resistant to Endo H, while those at Asn45 and Asn337) were completely resistant. Bio-Gel P-4 analysis revealed the Endo H-resistant oligosaccharides at Asn45, Asn92, Asn247, and Asn337 to be Man8-14GlcNAc, while the minor residual carbohydrate at Asn350 was Man greater than 50GlcNAc. The Endo H-susceptible oligosaccharides at Asn4, Asn146, and Asn256 were Man greater than 50GlcNAc while all other glycosylation sites contained Man8-14GlcNAc. Based on a hydropathic analysis of invertase, the two most Endo H-resistant oligosaccharides at Asn45 and Asn337 were located in the more hydrophobic regions of the molecule. These may form part of the folded protein structure or interacting subunit surfaces, thus restricting their accessibility to Endo H.
Highlights
Chem. 252,4409-4412).To determine whether the remaining Endo H-resistant chains were restricted to a limited number of unique sequons or were randomly distributed on all 13,Endo H-treated native invertase wasdigested with eitherthermolysin or trypsin and the resultantglycopeptides isolated by reversed-phase high pressure liquid chromatography and gel filtration
As shown by the employedto confirm the thermolysin data and tiodentify any Sepharose 6B profile in Fig. 1(Miniprint), about 20% of the additional Endo H-resistant glycopeptide attachment sites, mannose remained with this preparationof invertase, which, should they exist
The residual carbohydrate(Table I), wasdissolved in 88% with a window of seven amino acid stretches wasused to formic acid and diluted 10-foldwith H,O before HPLC
Summary
As shown in the accompanyingpaper (l), 13 of the 14 AsnX-Thr/Ser sequons found in theprotein sequence of secreted yeast invertase are glycosylateddespite chemical analyses that consistently reveal the presence of only 9-10 asparaginelinked oligosaccharides per peptide chain. 13 of the 14 AsnX-Thr/Ser sequons found in theprotein sequence of secreted yeast invertase are glycosylateddespite chemical analyses that consistently reveal the presence of only 9-10 asparaginelinked oligosaccharides per peptide chain. $ T o whom correspondence should he addressed
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