Mucin Synthesis. UDP-GlcNAc:GalNAc-R β3-N-Acetylglucosaminyltransferase and UDP-GlcNAc:GlcNAcβ1-3GalNAc-R (GlcNAc to GalNAc) β6-N-Acetylglucosaminyltransferase from Pig and Rat Colon Mucosa

Pig and rat colon mucosal membrane preparations catalyze the in vitro transfer of N-acetyl-d-glucosamine (GlcNAc) from UDP-GlcNAc to GalN Ac-ovine submaxillary mucin to form GlcNAcβ1-3GalNAc-mucin. Rat colon also catalyzes the in vitro transfer of GlcNAc from UDP-GlcNAc to GlcNAcβ1-3GalNAc-mucin to form GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-mucin. This is the first demonstration of in vitro synthesis of the GlcNAcβ1-3GalNAc disaccharide and of the GlcNAcβ1-3-(GlcNAcβ1-6)GalNAc trisaccharide, two of the four major core types found in mammalian glycoproteins of the mucin type, i.e., those containing oligosaccharides with GalNAc-α-serine (threonine) linkages. The activity catalyzing synthesis of the disaccharide has been named UDP-GlcNAc:GalNAc-R β3-N-acetyl-glucosaminyltransferase (mucin core 3 β3-GlcNAc-transferase), while the activity responsible for synthesizing the trisaccharide has been named UDP-GlcNAc:GlcNAcβl-3GalNAc-R (GlcNAc to GalNAc) β6-N-acetylglucosaminyltransferase (mucin core 4 β6-GlcNAc-transferase). The β3-GlcNAc-transferase from pig colon is activated by Triton X-100, has an absolute requirement for Mn2+, and transfers GlcNAc to GalNAc-α-phenyl, GalNAc-α-benzyl, and GalNAc-ovine submaxillary mucin with apparent Km values of 5, 2, and 3 mM and Fmax values of 59, 62, and 37 nmol h-1 (mg of protein)-1, respectively. The rat colon β6-GlcNAc-transferase is active in the absence of detergent, is stimulated 17% by addition of detergent, does not require addition of divalent cation, and transfers GlcNAc to GlcNAcβ1-3GalNAc-α-benzyl with an apparent Km of 0.6 mM and a Kmax of 714 nmol h-1 (mg of protein)-1. Mucin products were prepared on a large scale (0.26-0.43 nmol) and were treated with alkaline borohydride to release oligosaccharides; these were analyzed by gel filtration and high-performance liquid chromatography. The pig colon preparation produced a single radioactive oligosaccharide product, identified as GlcNAcβ l-3GalNAcOH, while the rat colon preparation formed two radioactive oligosaccharide products in a 1:1 ratio, identified as GlcNAcβ1-3GalNAcOH and GlcNAcβl-3(GlcNAcβ1-6)GalNAcOH. Products were identified by proton nuclear magnetic resonance spectroscopy and methylation analysis. The β3-GlcNAc-transferase is therefore present in pig and rat colon and was also detected in rat, human, and monkey colon. Low levels were found in pig, rat, monkey, and sheep stomach. There was no detectable activity in pig, rat, and dog submaxillary gland. The β6-GlcNAc-transferase is present in rat colon and was also detected in pig, human, and monkey colon, in pig, rat, monkey, and sheep stomach, and in rat and dog submaxillary gland. It thus has a wider distribution than the β3-GlcNAc-transferase. Competition experiments suggest that, in the rat colon mucosa, core 4 β6-GlcNAc-transferase mav be the same enzyme as UDP-GlcNAc:Galβ1-3GalNAc-R (GlcNAc to GalNAc) β6-GlcNAc-transferase, the enzyme responsible for core 2 synthesis.