Control of glycoprotein synthesis. IX. A terminal Man alpha l-3Man beta 1- sequence in the substrate is the minimum requirement for UDP-N-acetyl-D-glucosamine: alpha-D-mannoside (GlcNAc to Man alpha 1-3) beta 2-N-acetylglucosaminyltransferase I

Abstract

Twenty low molecular weight compounds were tested as substrates for UDP-GlcNAc:alpha-D-mannoside (GlcNAc to Man alpha 1-3) beta 2-N-acetylglucosaminyltransferase I (GlcNAc-transferase I) purified from bovine colostrum. This enzyme is at a key control point in the biosynthetic path leading to complex Asn-linked oligosaccharides. The highest activity was obtained with the substrate Man alpha 1-3(R1 alpha 1-6)Man beta 1-R2 where R1 was Man alpha 1-3(Man alpha 1-6)Man- (Km = 0.20 mM) and R2 was -4GlcNAc beta 1-4GlcNAc-Asn. Somewhat less effective were substrates in which R1 was Man- (Km = 0.4-0.6 mM) and R2 was either-4GlcNAc or -4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn. Removal of the Man alpha 1-6 arm (R1 = H-) or replacing R2 with an isopropyl group had no effect on Vmax but increased the Km about 10-fold, thereby leading to an 85% reduction in enzyme activity as measured under standard conditions. An 85% reduction in activity was also observed if R2 was replaced with N-acetylglucosaminitol. Enzyme activity was reduced 33% if R1 was Gal beta 1-4GlcNAc beta 1-2Man-. Any compounds lacking a Man alpha 1-3- terminus or in which the beta-linked Man had been replaced with an alpha-linked Man were totally inactive. It was concluded that a terminal Man alpha 1-3Man beta 1-sequence is a minimal structural requirement for a GlcNAc-transferase I substrate. The only effective substrate for partially purified UDP-GlcNAc:alpha-D-mannoside (GlcNAc to Man alpha 1-6) beta 2-N-acetylglucosaminyltransferase II (GlcNAc-transferase II) from bovine colostrum was R1-GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6)Man beta 1-R2 where R1 = H-. The absence of a terminal GlcNAc beta 1-2- residue or masking this residue by making R1 = Gal beta 1-4-, both prevented enzyme activity, indicating that GlcNAc-transferase I action must precede GlcNAc-transferase II action during biosynthesis of complex Asn-linked oligosaccharides.