Structure of yeast external invertase Man8-14GlcNAc processing intermediates by 500-megahertz 1H NMR spectroscopy

Abstract

A series of high mannose oligosaccharides with the size range Man8-14GlcNAc was purified from Saccharomyces cerevisiae invertase, and the composition of each was determined by chemical analysis. Purity and composition were verified by 1H NMR spectroscopy at 500 MHz, and structures were assigned on the basis of chemical shifts in C1-H and C2-H protons of similarly substituted compounds of known structure. Such analyses showed that these invertase oligosaccharides were a homologous series of homogeneous compounds, each related to the next member by addition of 1 mol of mannose in a specific alpha-linked configuration. Man8GlcNAc purified from the total glycoprotein fraction of disrupted yeast was the smallest species found and had the same homogeneous structure as that previously reported for the Man8GlcNAc from invertase (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). Digestion of Man8-13GlcNAc species from invertase with Aspergillus satoi alpha 1,2-mannosidase provided products that were consistent with the structures assigned by 1H NMR as did fast atom bombardment-mass spectroscopy fragmentation analysis of the Man9,10GlcNAc oligosaccharides. These results lead to the proposal that Man8GlcNAc is the only trimming intermediate in Saccharomyces sp., and the remaining Man9-14GlcNAc oligosaccharides are biosynthetic intermediates which define the principal pathway of single-step mannose addition in the formation of the inner core of yeast mannan.