title: Biosynthesis of hyaluronan: direction of chain elongation.
abstract: The mechanism of hyaluronan biosynthesis in vertebrates had been proposed to occur at the reducing end of growing chains. This mechanism was questioned because a recombinant synthase appeared to add new monosaccharides to the non-reducing end. I reinvestigated this problem with membranes from the eukaryotic B6 cell line. The membranes were incubated with UDP-[3H]GlcNAc and UDP-[14C]GlcA to yield differentially labelled reducing terminal and non-reducing terminal domains. Digestion of the product with a mixture of the exoglycosidases beta-glucuronidase and beta-N-acetylglucosaminidase truncated the hyaluronan chain strictly from the non-reducing end. The change in 3H/14C ratio of the remaining hyaluronan fraction, during the course of exoglycosidase digestion, confirmed the original results that the native eukaryotic synthase extended hyaluronan at the reducing end. This mechanism demands that the UDP-hyaluronan terminus is bound to the active site within the synthase and should compete with the substrates for binding. Accordingly, increasing substrate concentrations enhanced hyaluronan release from the synthase. A model is proposed that explains the direction of chain elongation at the reducing end by the native synthase and at the non-reducing end by the recombinant synthase based on a loss of binding affinity of the synthase towards the growing UDP-hyaluronan chain.
authors.affiliation: Münster University Hospital, Institute of Physiological Chemistry and Pathobiochemistry, Waldeyerstrasse 15, D-48149 Münster, Germany. prehm@uni-muenster.de
authors.forename: Peter
authors.initials: P
authors.lastname: Prehm
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D006224:Cricetinae; D005966:Glucuronidase / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism*; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis* / Q000737:chemistry; D051379:Mice
chemical_list: D006820:Hyaluronic Acid; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases; D005966:Glucuronidase; D000118:Acetylglucosaminidase