Use of glycosyltransferases to restore pertussis toxin receptor activity to asialoagalactofetuin.
Fetuin derivatives with enzymatically altered oligosaccharide units were tested for their ability to inhibit pertussis toxin-mediated agglutination of goose erythrocytes and the binding of 125I-labeled fetuin to pertussis toxin-coated polystyrene tubes. Fetuin oligosaccharides were sequentially degraded by treatment with: neuraminidase (asialofetuin) followed by beta-galactosidase (asialoagalactofetuin) and, lastly, with beta-N-acetylhexosaminidase (asialoagalacto-a[N-acetylglucosamino]fetuin). Asialofetuin retained only 19 and 53% of the inhibitory activity of native fetuin in the hemagglutination and 125I-fetuin binding assays, respectively. Asialoagalactofetuin showed no further reduction of inhibition in the hemagglutination system and, instead, resulted in partial recovery of inhibition in the 125I-fetuin-pertussis toxin binding assay. Asialoagalacto-a[N-acetylhexosamino]fetuin showed a further decrease in ability to inhibit pertussis toxin binding in both assays. The inhibitory activity of asialoagalactofetuin could be restored to that of native fetuin by adding back D-galactose with UDP-Gal:D-glucosyl-1,4-beta-galactosyltransferase, followed by the addition of terminal sialic acid residues with CMP-N-acetylneuraminic acid:beta-D-galactosyl-1,4-N-acetyl-beta-D-glucosamine-alpha-2,6-N- acetylneuraminyltransferase. The data suggested that a requirement for pertussis toxin binding to fetuin may be the presence of acetamido-containing sugar groups in the nonreducing terminal position of fetuin's oligosaccharides.
Armstrong
G D
GD
Department of Medical Microbiology and Infectious Diseases, University of Alberta, Edmonton, Canada.
Howard
L A
LA
Peppler
M S
MS
D000818:Animals; D001212:Asialoglycoproteins; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002849:Chromatography, Gas; D060748:Fetuins; D005777:Geese; D005964:Glucosyltransferases; D006385:Hemagglutination Inhibition Tests; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis; D037342:Pertussis Toxin; D011975:Receptors, Mitogen / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000378:metabolism*; D000509:alpha-Fetoproteins / Q000378:metabolism*
D001212:Asialoglycoproteins; D002241:Carbohydrates; D060748:Fetuins; D009844:Oligosaccharides; D011975:Receptors, Mitogen; D010566:Virulence Factors, Bordetella; D000509:alpha-Fetoproteins; C017029:asialofetuin; D005964:Glucosyltransferases; D037342:Pertussis Toxin