N-acetylglucosamine-6-O-sulfotransferase-1: production in the baculovirus system and its applications to the synthesis of a sulfated oligosaccharide and to the modification of oligosaccharides in fibrinogen.
N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the C-6 position of non-reducing GlcNAc. Human GlcNAc6ST-1 was expressed as a fusion protein with protein A in an insect cell line (Tn 5 cells) using the baculovirus system. The recombinant enzyme was purified to homogeneity by IgG Sepharose column chromatography. The substrate specificity and the kinetic properties of the enzyme were similar to those of the enzyme expressed in the mammalian system. The purified recombinant enzyme was used to synthesize 6-sulfo GlcNAcbeta1-3Galbeta1-4Glc, which was identified by time of flight mass spectrometry. This sulfated trisaccharide served as a better substrate for microsomal galactosyltransferase from the mouse colon compared to 6-sulfo GlcNAc. The purified recombinant enzyme was also used to sulfate oligosaccharide chains on fibrinogen after enzymatic desialylation and degalactosylation to expose nonreducing GlcNAc residues. It is known that desialylation greatly increases the rate of clotting of fibrinogen after the addition of thrombin. Subsequent sulfation of desialylated and degalactosylated fibrinogen slightly decreased the rate of clotting. The recombinant GlcNAc6ST-1 is a useful reagent for 6-sulfate exposed GlcNAc residues both in oligosaccharides and in glycoproteins.
El-Fasakhany
Fathy M
FM
Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
Ichihara-Tanaka
Keiko
K
Uchimura
Kenji
K
Muramatsu
Takashi
T
D000818:Animals; D016367:Baculoviridae / Q000737:chemistry / Q000201:enzymology*; D002417:Cattle; D005340:Fibrinogen / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis*; D013431:Sulfates / Q000032:analysis / Q000378:metabolism; D015238:Sulfotransferases / Q000032:analysis / Q000096:biosynthesis*; D000097811:Carbohydrate Sulfotransferases
D005340:Fibrinogen; D009844:Oligosaccharides; D011994:Recombinant Proteins; D013431:Sulfates; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases