HUMAN SERUM CONTAINS A NOVEL BETA-1,6-N-ACETYLGLUCOSAMINYLTRANSFERASE ACTIVITY THAT IS INVOLVED IN MIDCHAIN BRANCHING OF OLIGO(N-ACETYLLACTOSAMINOGLYCANS)

Incubation of UDP-GlcNAc and radiolabeled GlcNAc-beta-1-3Gal-beta-1-4GlcNAc-beta-1-3Gal-beta-1-4GlcNAc (1) with human serum resulted in the formation of the branched hexasaccharide GlcNAc-beta-1-3Gal-beta-1-4GlcNAc-beta-1-3(GlcNAc-beta-1-6)Gal-beta-1-4GlcNAc (2) in yields of up to 22.2%. The novel reaction represents midchain branching of the linear acceptor; the previously known branching reactions of oligo-(N-acetyllactosaminoglycans) involve the nonreducing end of the growing saccharide chains. The structure of 2 was established by use of appropriate isotopic isomers of it for degradative experiments. The hexasaccharide 2 was cleaved by an exhaustive treatment with jack bean beta-N-acetylhexosaminidase, liberating two GlcNAc units and the tetrasaccharide Gal-beta-1-4GlcNAc-beta-1-3Gal-beta-1-4GlcNAc (3). Endo-beta-galactosidase from Bacteroides fragilis cleaved 2 at one site only, yielding the disaccharide GlcNAc-beta-1-3Gal (4) and the branched tetrasaccharide GlcNAc-beta-1-3(GlcNAc-beta-1-6)Gal-beta-1-4GlcNAc (5). The structure of 5 was established by partial acid hydrolysis and subsequent identification of the disaccharide GlcNAc-beta-1-6Gal (6), together with the trisaccharides GlcNAc-beta-1-6Gal-beta-1-4GlcNAc (7) and GlcNAc-beta-1-3(GlcNAc-beta-1-6)Gal (8) among the cleavage products. Galactosylation of 2 with bovine milk beta-1,4-galactosyltransferase and UDP-[6-H-3]Gal gave the octasaccharide [6-H-3]Gal-beta-1-4GlcNAc-beta-1-3Gal-beta-1-4GlcNAc-beta-1-3([6-H-3]Gal-beta-1-4GlcNAc-beta-1-6)[U-C-14]Gal-beta-1-4GlcNAc (17), which could be cleaved with endo-beta-galactosidase into the trisaccharide [6-H-3]Gal-beta-1-4GlcNAc-beta-1-3Gal (18) and the branched pentasaccharide GlcNAc-beta-1-3([6-H-3]Gal-beta-1-4GlcNAc-beta-1-6)[U-C-14]Gal-beta-1-4GlcNAc (19). Partial hydrolysis of 2 with jack-bean beta-N-acetylhexosaminidase gave the linear pentasaccharide 1 and the branched pentasaccharide Gal-beta-1-4GlcNAc-beta-1-3 (GlcNAc-beta-1-6)Gal-beta-1-4GlcNAc (20). The serum beta-1,6-GlcNAc transferase catalyzed also the formation of GlcNAc-beta-1-3Gal-beta-1-4GlcNAc-beta-1-3(GlcNAc-beta-1-6)Gal-beta-1-4Glc (11) from UDP-GlcNAc and GlcNAc-beta-1-3Gal-beta-1-4GlcNAc-beta-1-3Gal-beta-1-4Glc (10). The pentasaccharide Gal-alpha-1-3Gal-beta-1-4GlcNAc-beta-1-3Gal-beta-1-4GlcNAc (16), too, served as an acceptor for the enzyme. The trisaccharide GlcNAc-beta-1-3Gal-beta-1-4GlcNAc (9), and the tetrasaccharide GlcNAc-beta-1-3Gal-beta-1-4GlcNAc-beta-1-3Gal (15) were poor acceptors, confirming that the enzyme does not cause branching at the nonreducing area of the acceptors. The poor acceptor properties of the tetrasaccharide 15 imply that the GlcNAc residue at the reducing end side of the branching galactose in the pentasaccharide 1 plays a role in the reaction.