Mucin synthesis. The action of pig gastric mucosal UDP-GlcNAc:Galβ1-3(R1)GalNAc-R2 (GlcNAc to Gal) β3-N-acetylglucosaminyltransferase on high molecular weight Substrates

Abstract

Membrane preparations from pig gastric mucosa were shown to transfer [¹⁴C]GlcNAc from UDP-[¹⁴C]GlcNAc to blood group A-negative porcine submaxillary mucin previously subjected to mild acid hydrolysis to remove terminal sialyl and fucosyl residues. O-Glycosyl oligosaccharides were removed from enzyme product by alkaline borohydride treatment and, after purification, were subjected to high resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Two trisaccharide products were detected: [¹⁴C]GlcNAcβ1-3Galβ1-3GalNAcOH and Galβ1-3([¹⁴C]GlcNAcβ1-6)GalNAcOH. We have previously reported the in vitro synthesis of the latter compound, a branched trisaccharide, by UDP-GlcNAc:Galβ1-3GalNAc-R (GlcNAc to GalNAc) β6-N-acetylglucosaminyltransferase from canine submaxillary glands. However, this is the first report of the in vitro synthesis of the linear trisaccharide GlcNAcβ1-3Galβ1-3GalNAc. Pig gastric mucosal β3-N-acetylglucosaminyltransferase catalyzed the formation of this trisaccharide by incorporation of GlcNAc into the terminal Gal of Galβ1-3GalNAc-α-R when R was a polypeptide from either mucin or antifreeze glycoprotein, but not when R was o-nitrophenyl. We have previously reported the in vitro synthesis by pig gastric mucosa of the tetrasaccharide GlcNAcβ1-3Galβ1-3(GlcNAcβ1-6)GalNAc-α-R when R was o-nitrophenyl or benzyl. We show in this report that pig gastric mucosa can synthesize this tetrasaccharide in vitro when R is a polypeptide from either porcine submaxillary mucin or antifreeze glycoprotein. Pig gastric mucosa therefore contains a β6-N-acetylglucosaminyltransferase capable of converting Galβ1-3GalNAc-α-R to Galβ1-3(GlcNAcβ1-6)GalNAc-α-R and one or more β3-N-acetylglucosaminyltransferases which can add GlcNAc in β1-3 linkage to a terminal Gal residue to form either GlcNAcβ1-3Galβ1-3GalNAc-α-R or GlcNAcβ1-3Galβ1-3(GlcNAcβ1-6)GalNAc-α-R where R is the polypeptide backbone of either mucin or antifreeze glycoprotein.