Enzymatic transfer of a beta1,6-linked N-acetylglucosamine to the alpha-galactose of globo-N-tetraose: in vitro synthesis of a novel hybrid pentasaccharide of lacto-globo type.
A novel saccharide was synthesized by incubating globo-N-tetraose, GalNAc beta1-3Gal alpha1-4Gal beta1-4Glc, and UDP[3H]GlcNAc with hog gastric mucosal microsomes, known to contain beta1,6-N-acetylglucosaminyltransferase activity of a broad acceptor specificity. Chromatography and MALDI-TOF mass spectrometry of the product, as well as the amount of incorporated radioactivity indicated that one [3H]GlcNAc residue was transferred to the acceptor saccharide. One- and two-dimensional 1H NMR-spectroscopic analysis of the product and ESI-CID mass spectrometry of the pentasaccharide in permethylated form established its structure as GalNAc beta1-3([3H]GlcNAc beta1-6)Gal alpha1-4Gal beta1-4Glc. The new enzyme activity possesses substrate specificity features common to a purified beta1,6-GlcNAc-transferase from bovine tracheal epithelium, which forms branches at the subterminal beta1,3-substituted galactose and accepts both GlcNAc- and Gal-configuration at the terminal residue of the acceptor (Ropp et al. (1991) J. Biol. Chem., 266, 23863-23871). The new beta1,6-GlcNAc-branch was readily galactosylated by bovine milk beta1,4-galactosyltransferase, revealing a pathway to novel hybrid type glycans with N-acetyllactosamine chains on globotype saccharides. This pathway may lead to the rare IP blood-group antigen and to globoside-like molecules mediating cell adhesion.
Natunen
J
J
Institute of Biotechnology and Department of Biosciences (Division of Biochemistry), University of Helsinki, Finland.
Seppo
A
A
Helin
J
J
Reinhold
B B
BB
Rabina
J
J
Costello
C E
CE
Renkonen
O
O
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D004848:Epithelium / Q000201:enzymology; D005753:Gastric Mucosa / Q000201:enzymology*; D005915:Globosides / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes / Q000201:enzymology*; D008892:Milk / Q000201:enzymology; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013552:Swine; D014132:Trachea / Q000201:enzymology; D014316:Tritium; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D005915:Globosides; D009844:Oligosaccharides; D014316:Tritium; D014537:Uridine Diphosphate N-Acetylglucosamine; C035968:globotetraose; D017351:N-Acetylglucosaminyltransferases; C066287:UDP-GlcNAc-Gb(5)Cer beta1-6N-acetylglucosaminyltransferase