Complete enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin.
Sialyl Lewis x (sLe(x)) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-alpha-D-galactosaminide (GalNAc(alpha1-pNp as core substrate, the sLe(x)-oligosaccharide Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)[Gal(bet a1-3)]GalNAc(alpha1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(beta1-3)GalNAc(alpha1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of beta3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 beta6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc(alpha1-pNp in a yield of >85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk beta4-galactosyltransferase 1 (EC 2.4.1.38) (yield of >85%), then sialylated using CMP-Neu5Ac and purified recombinant alpha3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human alpha3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 micromol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure.
Zeng
S
S
Physiologisches Institut, Universität Zürich, Switzerland.
Gallego
R G
RG
Dinter
A
A
Malissard
M
M
Kamerling
J P
JP
Vliegenthart
J F
JF
Berger
E G
EG
D000818:Animals; D002240:Carbohydrate Sequence; D000939:Epitopes / Q000737:chemistry*; D016695:Glycosyltransferases / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry*; D009844:Oligosaccharides / Q000138:chemical synthesis*; D019007:P-Selectin / Q000737:chemistry; D051381:Rats; D000080506:Sialyl Lewis X Antigen; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000939:Epitopes; D008562:Membrane Glycoproteins; D009077:Mucins; D009844:Oligosaccharides; D019007:P-Selectin; C084882:P-selectin ligand protein; D000080506:Sialyl Lewis X Antigen; C409211:mucin-type sialyl Lewis x epitope; D016695:Glycosyltransferases