Biosynthesis of sialyl-oligomeric-Lewisx and VIM-2 epitopes: site specificity of human milk fucosyltransferase

4GlcNAc (LacNAc) linked to a synthetic hydrophobic aglycon, (LacNAc)3-[(trifluoroacetamido)phenyl]ethyl, by a partially purified alpha 3-fucosyltransferase preparation from normal human milk [De Vries, Th., Norberg, T., Lonn, H., & van den Eijnden, D. H. (1993) Eur. J. Biochem. 216, 769-777]. Using the same fucosyltransferase preparation, we have now studied the fucosylation of the oligosaccharide NeuAc alpha 2-->3(LacNAc)3-Me. This compound was generated from the asialo analogue by use of an alpha 3-sialyltransferase preparation from human placenta. The location of the fucose residues in the monofucosylated and difucosylated intermediate products was determined by analyzing digests obtained after endo-beta-galactosidase treatment using HPLC on amino-bonded silica. In addition, the fucosylated NeuAc alpha 2-->3(LacNAc)3-Me structures were characterized by high-pH anion-exchange chromatography with pulsed amperometric detection and were identified by 400-MHz 1H-NMR spectroscopy. Intermediate products included oligosaccharides that contained the VIM-2, sialyl-LewisX, and sialyl-dimeric-LewisX epitopes. The final product was identified as the sialyl-trimeric-LewisX oligosaccharide. Kinetic analysis of the fucosylation reaction indicated that there is a significant difference in the rate of transfer of the first, second, and third fucose residues onto the acceptor molecule. Transfer of the first fucose occurred to either of the three GlcNAc residues in NeuAc alpha 2-->3(LacNAc)3-Me with only a modest preference for the proximal and medial residues. A similar slight preference for these GlcNAc residues was found for the attachment of the second fucose residue.(ABSTRACT TRUNCATED AT 250 WORDS) -->
ISSN: 0006-2960
Přístupová URL adresa: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e3d91a935236276d10f876ecb0712435
https://pubmed.ncbi.nlm.nih.gov/8061002
Přírůstkové číslo: edsair.doi.dedup.....e3d91a935236276d10f876ecb0712435
Autor: de Vries T, van den Eijnden Dh
Rok vydání: 1994
Předmět:
Zdroj: Biochemistry. 33(33)
ISSN: 0006-2960
Popis: In a previous study we have established the order of fucosylation of a trimer of Gal beta 1-->4GlcNAc (LacNAc) linked to a synthetic hydrophobic aglycon, (LacNAc)3-[(trifluoroacetamido)phenyl]ethyl, by a partially purified alpha 3-fucosyltransferase preparation from normal human milk [De Vries, Th., Norberg, T., Lonn, H., & van den Eijnden, D. H. (1993) Eur. J. Biochem. 216, 769-777]. Using the same fucosyltransferase preparation, we have now studied the fucosylation of the oligosaccharide NeuAc alpha 2-->3(LacNAc)3-Me. This compound was generated from the asialo analogue by use of an alpha 3-sialyltransferase preparation from human placenta. The location of the fucose residues in the monofucosylated and difucosylated intermediate products was determined by analyzing digests obtained after endo-beta-galactosidase treatment using HPLC on amino-bonded silica. In addition, the fucosylated NeuAc alpha 2-->3(LacNAc)3-Me structures were characterized by high-pH anion-exchange chromatography with pulsed amperometric detection and were identified by 400-MHz 1H-NMR spectroscopy. Intermediate products included oligosaccharides that contained the VIM-2, sialyl-LewisX, and sialyl-dimeric-LewisX epitopes. The final product was identified as the sialyl-trimeric-LewisX oligosaccharide. Kinetic analysis of the fucosylation reaction indicated that there is a significant difference in the rate of transfer of the first, second, and third fucose residues onto the acceptor molecule. Transfer of the first fucose occurred to either of the three GlcNAc residues in NeuAc alpha 2-->3(LacNAc)3-Me with only a modest preference for the proximal and medial residues. A similar slight preference for these GlcNAc residues was found for the attachment of the second fucose residue.(ABSTRACT TRUNCATED AT 250 WORDS)
Databáze: OpenAIRE
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