Structural Characterization of Different Isomers of Lipid-linked Hexa-

A previous study (vijay, I. K., Perdew, G. H., and Lewis, D. E. (1980) J. BioL Chem. 255, 11210-11220) indicated that incubation of a membrane preparation from the lactating bovine mammary tissue with UDPISH]GlcNAc and GDP-[’“C]Man synthesized a series of lipid-linked saccharides that appeared to be related to one another as precursor products in the biosynthesis of asparagine-linked glycoproteins of this tissue. The structure of monoisomeric species of tri-, tetra-, penta-, and undecasaccharide were characterized. Utilizing the substrate specificity of endo-b-N-acetylglucosaminidase D and H for oligomannosylchitobiose type of saccharides, two isomers in the hexaand at least three isomers in the heptasaccharide fraction could be sorted out. Based on digestion with a-mannosidase, endo-/3-N-acetylghcosaminidase L, and &mannosidase, and on Smith periodate degradation, acetolysis, and methylation analysis, the proposed structures of two isomers of the lipid-linked hexasaccharide are Manal-, 3Manal-+ 6(Manal-+ 3)Manfil4 (3)GlcNAcfl1-+4(3)GIcNAc (Hexa-I) and Mana1-+2Manal+ 3(ManaI-+6)Man/3l-*4(3)GlcNAc/31-+4(3)GlcNAc (Hexa-II). Both of the hexasaccharides appear to be biosynthetic intermediates rather than products of a lipid-linked saccharide processing. The two isomers of lipid-linked heptasaccharide that were characterized are Mana1+2Manal~3Mana1+6(Manal-t3)Man~l+ 4(3)GlcNAc/31-+4(3)GlcNAc(Hepta-I) and Manal-2Mana1-,2Manal-+3(Manal-t6)Man~1-+4(3)GlcNAc/31*4(3)GlcNAc (Hepta-II). Hepta-11, presumably derived from Hexa-11, appears to be the intermediate in the major dolichol phosphate-mediated pathway for the lipid-linked assembly of the saccharides; Hepta-I could be a biosynthetic product of Hexa-I in a minor pathway, or might result from lipid-linked processing of higher saccharides. The precise structure and origin of a third, heterogeneous fraction of heptasaccharide isomer(s) is presently unclear.