| Abstract
| - Sialic acids are key determinants in many carbohydrates involved in biological recognition.We studied the acceptor specificities of three cloned sialyltransferases (STs) [α2,3(N)ST, α2,3(O)ST,and α2,6(N)ST] and another α2,3(O)ST present in prostate cancer cell LNCaP toward mucin core 2tetrasaccharide [Galβ1,4GlcNAcβ1,6(Galβ1,3)GalNAcα-O-Bn] and Globo [Galβ1,3GalNAcβ1,3Galα-O-Me] structures containing sialyl, fucosyl, sulfo, methyl, or fluoro substituents by identifying the productsby electrospray ionization tandem mass spectral analysis and other biochemical methods. The Globoprecursor was an efficient acceptor for both α2,3(N)ST and α2,3(O)ST, whereas only α2,3(O)ST used itsdeoxy analogue (d-Fucβ1,3GalNAcβ1,3-Gal-α-O-Me); 2-O-MeGalβ1,3GlcNAc and 4-OMeGalβ1,4GlcNAc were specific acceptors for α2,3(N)ST. Other major findings of this study include: (i) α2,3sialylation of β1,3Gal in mucin core 2 can proceed even after α1,3 fucosylation of β1,6-linked LacNAc.(ii) Sialylation of β1,3Gal must precede the sialylation of β1,4Gal for favorable biosynthesis of mucincore 2 compounds. (iii) α2,3 sialylation of the 6-O-sulfoLacNAc moiety in mucin core 2 (e.g., GlyCAM-1) is facilitated when β1,3Gal has already been α2,3 sialylated. (iv) α2,6(N)ST was absolutely specificfor the β1,4Gal in mucin core 2. Either α1,3 fucosylation or 6-O-sulfation of the GlcNAc moiety reducedthe activity. Sialylation of β1,3Gal in addition to 6-O-sulfation of GlcNAc moiety abolished the activity.(v) Prior α2,3 sialylation or 3-O-sulfation of β1,3Gal would not affect α2,6 sialylation of Galβ1,4GlcNAcof mucin core 2. (vi) A 3- or 4-fluoro substituent in β1,4Gal resulted in poor acceptors for the clonedα2,6(N)ST and α2,3(N)ST, whereas 4-fluoro- or 4-OMe-Galβ1,3GalNAcα was a good acceptor for clonedα2,3(O)ST. (vii) 4-O-Methylation of β1,4Gal abolished the acceptor ability toward α2,6(N)ST but increasedthe acceptor efficiency considerably toward α2,3(N)ST. (viii) Just like LNCaPα1,2-FT and Gal-3-O-sulfotransferase T2, the cloned α2,3(N)ST which modifies terminal Gal in Galβ1,4GlcNAc also efficientlyutilizes the terminal β1,3Gal in the Globo backbone. Utilization of C-3 blocked compounds such as 3-O-sulfo-Galβ1,3GalNAcβ1,3Galα-OMe as acceptors by cloned α2,3(O)ST and analyses of the resultingproducts by lectin chromatography and mass spectrometry indicate that α2,3(O)ST is capable of attachingNeuAc to another position in C-3-substituted β1,3Gal.
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