Control of glycoprotein synthesis. IX. A terminal Manα1-3Manβ1- sequence in the substrate is the minimum requirement for UDP-N-acetyl-D-glucosamine:α-D- mannoside (GlcNAc to Manα1-3) β2-N-acetylglucosaminyltransferase I

Abstract

Twenty low molecular weight compounds were tested as substrates for UDP-GlcNAc:α-D-mannoside (GlcNAc to Manα1-3) β2-N-acetylglucosammyltransferase 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 Manα1-3(R1α1-6)Manβ1-R2 where R1 was Manα1-3(Manα1-6)Man- (Km = 0.20 mM) and R2 was -4GlcNAcβ1-4GlcNAc-Asn. Somewhat less effective were substrates in which R1 was Man- (Km = 0.4–0.6 mM) and R2 was either -4GlcNAc or -4GlcNAcβ1-4(Fucα1-6)GlcNAc-Asn. Removal of the Manα1-6 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 Galβ1-4GlcNAcβ1-2Man-. Any compounds lacking a Manα1-3- terminus or in which the β-linked Man had been replaced with an α-linked Man were totally inactive. It was concluded that a terminal Manα1-3Manβ1-sequence is a minimal structural requirement for a GlcNAc-transferase I substrate. The only effective substrate for partially purified UDP-GlcNAc:α-D-mannoside (GlcNAc to Manβ1-6) β2-N-acetylglucosaminyltransferase II (GlcNAc-transferase II) from bovine colostrum was R1GlcNAcβ1-2Manα1-3(Manα1-6)Manβ1-R2 where R1 = H-. The absence of a terminal GlcNAcβ1-2- residue or masking this residue by making R1 = Galβ1-4-, both prevented enzyme activity, indicating that GlcNAc-transferase I action must precede GlcNAc-transferase II action during biosynthesis of complex Asn-linked oligosaccharides.