Recombinant (2-->3)-alpha-sialyltransferase immobilized on nickel-agarose for preparative synthesis of sialyl Lewis(x) and Lewis(a) precursor oligosaccharides.
The specificity of recombinant (2-->3)-alpha-sialyltransferase (ST3Gal-III), expressed in baculovirus-infected insect cells, has been determined with various oligosaccharide acceptors and sugar-nucleotide donors using a fluorescence based assay. Recombinant ST3Gal-III tagged with a polyhistidine tail was immobilized on Ni(2+)-NTA-Agarose as an active enzyme for use in the synthesis of three sialylated oligosaccharides: (i) the divalent molecule [alpha-Neu5Ac-(2-->3)-D-Galp-(1-->4)-beta-D-GlcpNAc-O-CH(2)](2)-C-(CH(2)OBn)(2) (12); (ii) the dansylated derivative, alpha-Neu5Ac-(2-->3)-D-Galp-(1-->3)-beta-D-GlcpNAc-O-(CH(2))(6)-NH-dansyl and; (iii) the tetrasacharide alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-O-CH(3). Compound 12 was itself prepared from the divalent N-acetyllactosamine molecule built on pentaerythritol by a chemo-enzymatic route.
Laboratoire de Chimie Organique Multifonctionnelle, UMR 8614, Université de Paris-Sud, F-91405, Orsay, France.
Tatiana
T
Ivannikova
Fabrice
F
Bintein
Annie
A
Malleron
Sylvie
S
Juliant
Martine
M
Cerutti
Anne
A
Harduin-Lepers
Philippe
P
Delannoy
Claudine
C
Augé
André
A
Lubineau
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002240:Carbohydrate Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007983:Lewis Blood Group Antigens; D016256:Lewis X Antigen; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009532:Nickel / Q000737:chemistry*; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D012685:Sepharose / Q000737:chemistry*; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera; D013379:Substrate Specificity; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D004800:Enzymes, Immobilized; D007983:Lewis Blood Group Antigens; D016256:Lewis X Antigen; D009844:Oligosaccharides; D011994:Recombinant Proteins; C111125:lewis(a)-lewis(x); D009532:Nickel; D012685:Sepharose; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase