Mucin synthesis. UDP-GlcNAc:GalNAc-R .beta.3-N-acetylglucosaminyltransferase and UDP-GlcNAc:GlcNAc.beta.1-3GalNAc-R (GlcNAc to GalNAc) .beta.6-N-acetylglucosaminyltransferase from pig and rat colon mucosa

Abstract

Pig and rat colon mucosal membrane preparations catalyze the in vitro transfer of N-acetyl-D-glucosamine (GlcNAc) from UDP-GlcNAc to GalNAc-ovine submaxillary mucin to form GlcNAc.beta.1-3GalNAc-mucin. Rat colon also catalyzes the in vitro transfer of GlcNAc from UDP-GlcNAc to GlcNAc.beta.1-3GalNAc-mucin to form GlcNAc.beta.1-3(GlcNAc.beta.1-6)GalNAc-mucin. This is the 1st demonstration of in vitro synthesis of the GlcNAc.beta.1-3GalNAc disaccharide and of the GlcNAc.beta.1-3-(GlcNAc.beta.1-6)GalNAc trisaccharide, 2 of the 4 major core types found in mammalian glycoproteins of the mucin type, i.e., those containing oligosaccharides with GalNAc-.alpha.-serine (threonine) linkages. The activity catalyzing synthesis of the disaccharide was named UDP-GlcNAc:GalNAc-R .beta.3-N-acetyl-glucosaminyltransferase (mucin core 3 .beta.3-GlcNAc-transferase), while the activity responsible for synthesizing the trisaccharide was named UDP-GlcNAc:GlcNAc.beta.1-3GalNAc-R (GlcNAc to GalNAc) .beta.6-N-acetylglucosaminyltransferase (mucin core 4 .beta.6-GlcNAc-transferase). The .beta.3-GlcNAc-transferase from pig colon is activated by Triton X-100, has an absolute requirement for Mn2+, and transfers GlcNAc to GalNAc-.alpha.-phenyl, GalNAc-.alpha.-benzyl and GalNAc-ovine submaxillary mucin with apparent Km values of 5, 2, and 3 mM and Vmax values of 59, 62, and 37 nmol/h (mg of protein), respectively. The rat colon .beta.6-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 GlcNAc.beta.1-3GalNAc-.alpha.-benzyl with an apparent Km of 0.6 mM and a Vmax of 714 nmol/h (mg of protein). Mucin products were prepared on a large scale (0.26-0.42 .mu.mol) 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 GlcNAc.beta.1-3GalNAcOH, while the rat colon preparation formed 2 radioactive oligosaccharide products in a 1:1 ratio, identified as GlcNAc.beta.1-3GalNAcOH and GlcNAc.beta.1-3(GlcNAc.beta.1-6)GalNAcOH. Products were identified by proton NMR spectroscopy and methylation analysis. The .beta.3-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 .beta.6-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 .beta.3-GlcNAc-transferase. Competition experiments suggest that, in the rat colon mucosa, core 4 .beta.6-GlcNAc-transferase may be the same enzyme as UDP-GlcNAc:Gal.beta.1-3GalNAc-R (GlcNAc to GalNAc) .beta.6-GlcNAc-transferase, the enzyme responsible for core 2 synthesis.