Analysis of the specificity of sialyltransferases toward mucin core 2, Globo, and related structures.: Identification of the sialylation sequence and the effects of sulfate, fucose, methyl, and fluoro substituents of the carbohydrate chain in the biosynthesis of selectin and siglec ligands, and novel sialylation by cloned α2,3(O)sialyltransferase

Sialic acids are key determinants in many carbohydrates involved in biological recognition. We studied the acceptor specificities of three cloned sialyltransferases (STs) [alpha 2,3(N)ST, alpha 2,3(O)ST, and alpha 2,6(N)ST] and another alpha 2,3(O)ST present in prostate cancer cell LNCaP toward mucin core 2 tetrasaccharide [Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn] and Globo [Gal beta 1,3GalNAc beta 1,3Gal alpha-O-Me] structures containing sialyl, fucosyl, sulfo, methyl, or fluoro substituents by identifying the products by electrospray ionization tandem mass spectral analysis and other biochemical methods. The Globo precursor was an efficient acceptor for both alpha 2,3(N)ST and alpha 2,3(O)ST, whereas only alpha 2,3(O)ST used its deoxy analogue (D-Fuc beta 1,3GalNAc beta 1,3-Gal -alpha-O-Me); 2-O-MeGal beta 1,3GlcNAc and 4-OMeGal beta 1,4GlcNAc were specific acceptors for alpha 2,3(N)ST. Other major findings of this study include: (i) alpha 2,3 sialylation of beta 1,3Gal in mucin core 2 can proceed even after alpha 1,3 fucosylation of beta 1,6-linked LacNAc. (ii) Sialylation of beta 1,3Gal must precede the sialylation of beta 1,4Gal for favorable biosynthesis of mucin core 2 compounds. (iii) alpha 2,3 sialylation of the 6-O-sulfoLacNAc moiety in mucin core 2 (e.g., GlyCAM-1) is facilitated when beta 1,3Gal has already been alpha 2,3 sialylated. (iv) alpha 2,6(N)ST was absolutely specific for the beta 1,4Gal in mucin core 2. Either alpha 1,3 fucosylation or 6-O-sulfation of the GlcNAc moiety reduced the activity. Sialylation of 1,3Gal in addition to 6-O-sulfation of GlcNAc moiety abolished the activity. (v) Prior alpha 2,3 sialylation or 3-O-sulfation of beta 1,3Gal would not affect alpha 2,6 sialylation of Gal beta 1,4GlcNAc of mucin core 2. (vi) A 3- or 4-fluoro substituent in 1,4Gal resulted in poor acceptors for the cloned alpha 2,6(N)ST and alpha 2,3(N)ST, whereas 4-fluoro- or 4-OMe-Gal beta 1,3GalNAc alpha was a good acceptor for cloned alpha 2,3(O)ST. (vii) 4-O-Methylation of beta 1,4Gal abolished the acceptor ability toward alpha 2,6(N)ST but increased the acceptor efficiency considerably toward alpha 2,3(N)ST. (viii) Just like LNCaP alpha 1,2-FT and Gal-3-O-sulfotransferase T2, the cloned alpha 2,3(N)ST which modifies terminal Gal in Gal beta 1,4GlcNAc also efficiently utilizes the terminal beta 1,3Gal in the Globo backbone. Utilization of C-3 blocked compounds such as 3-O-sulfo-Gal beta 1,3GalNAc beta 1,3Gal alpha-OMe as acceptors by cloned alpha 2,3(O)ST and analyses of the resulting products by lectin chromatography and mass spectrometry indicate that alpha 2,3(O)ST is capable of attaching NeuAc to another position in C-3-substituted beta 1,3Gal.