Biosynthesis of mammary glycoproteins. Partial characterization of the sequence for the assembly of lipid-linked saccharides

Incubation of a membrane preparation from the lactating bovine mammary gland with UDP-[³H]GlcNAc, GDP-[¹⁴C]Man, and UDP-[³H]Glc results in the biosynthesis of 15 lipid-linked saccharides that differ from one another by a monosaccharide unit. Pulse and chase kinetics indicate that these glycolipids are related to one another as precursor products for the biosynthesis of asparagine-linked glycoproteins of this tissue. [Man-¹⁴C]- and [Man-¹⁴C, GlcNAc-³H]saccharides were prepared from corresponding glycolipids by mild acid hydrolysis. . Following extensive purification by paper and gel filtration chromatography, structural characterization was conducted on tri-, tetra-, penta-, and undecasaccharides via size determination on calibrated columns of Bio-Gel P-2 and P-4, compositional analysis, exo- and endoglycosidase digestions, methylation, Smith degradation, and acetolysis. These structures were identified as: Manβ1→4(3)GlcNacβ1→4(3)Glc-Nac, Manα1→3Manβ1→4(3)GlcNAcβ1→4(3)GlcNAc, Manα1→3(Manα1→6)Manβ1→4(3)GlcNAcβ1→4(3)Glc-NAc, and Manα1→2Manα1→2Manα1→3(Manα1→2Manα1→6[Manα1→2Manα1→3]Manα1→6)Manβ1→4(3)GlcNAcβ1→4(3)GlcNAc.