Mucin synthesis: Conversion of R₁‐β1‐3Gal‐R₂ to R₁‐β‐3(GlcNAcβ1‐6)Gal‐R₂ and of R₁‐β1‐3GalNAc‐R₂ to R₁‐β1‐3(GlcNAcβ1‐6)GalNAc‐R₂ by a β6‐N‐acetylglucosaminyltransferase in pig gastric mucosa

A UDP‐GlcNAc:R₁‐β1‐3Gal(NAc)‐R₂ [GlcNAc to Gal(NAc)] β6‐N‐acetylglucosaminyltransferase activity from pig gastric mucosa microsomes catalyzes the formation of GlcNAcβ1‐3(GlcNAcβ1‐6)Gal‐R from GlcNAcβ1‐3Gal‐R where ‐R is ‐β1‐3GalNAc‐α‐benzyl or ‐β1‐3(GlcNAcβ1‐6)GalNAc‐α‐benzyl. This enzyme is therefore involved in the synthesis of the I antigenic determinant in mucin‐type oligosaccharides. The enzyme also converts Galβ1‐3Galβ1‐4Glc to Galβ1‐3(GlcNAcβ1‐6)Galβ1‐4Glc. The enzyme was stimulated by Triton X‐100 at concentrations between 0 and 0.2% and was inhibited by Triton X‐100 at 0.5%. There is no requirement for Mn²⁺ and the enzyme activity is reduced to 65% in the presence of 10 mM EDTA. Enzyme products were purified and identified by proton NMR, methylation analysis and β‐galactosidase digestion. Competition studies suggest that this pig gastric mucosal β6‐GlcNAc‐transferase activity is due to the same enzyme that converts Galβ1‐3GalNAc‐R to mucin core 2, Galβ1‐3(GlcNAcβ1‐6)GalNAc‐R, and GlcNAcβ1‐3GalNAc‐R to mucin core 4, GlcNAcβ1‐3(GlcNAcβ1‐6)GalNAc‐R. Substrate specificity studies indicate that the enzyme attaches GlcNAc to either Gal or GalNAc in β(1‐6) linkage, provided these residues are substituted in β(1‐3) linkage by either GlcNAc or Gal. The insertion of a GlcNAcβ1‐3 residue into Galβ1‐3GalNAc‐R to form GlcNAcβ1‐3Galβ1‐3GalNAc‐R prevents insertion of GlcNAc into GalNAc. These studies establish several novel pathways in mucin‐type oligosaccharide biosynthesis.