No abstract

Sialic acids are key determinants in many carbohydrates involved in biological recognition. We studied the acceptor specificities of three cloned sialyltransferases (STs) [alpha2,3(N)ST, alpha2,3(O)ST, and alpha2,6(N)ST] and another alpha2,3(O)ST present in prostate cancer cell LNCaP toward mucin core 2 tetrasaccharide [Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-Bn] and Globo [Galbeta1,3GalNAcbeta1,3Galalpha-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 alpha2,3(N)ST and alpha2,3(O)ST, whereas only alpha2,3(O)ST used its deoxy analogue (d-Fucbeta1,3GalNAcbeta1,3-Gal-alpha-O-Me); 2-O-MeGalbeta1,3GlcNAc and 4-OMeGalbeta1,4GlcNAc were specific acceptors for alpha2,3(N)ST. Other major findings of this study include: (i) alpha2,3 sialylation of beta1,3Gal in mucin core 2 can proceed even after alpha1,3 fucosylation of beta1,6-linked LacNAc. (ii) Sialylation of beta1,3Gal must precede the sialylation of beta1,4Gal for favorable biosynthesis of mucin core 2 compounds. (iii) alpha2,3 sialylation of the 6-O-sulfoLacNAc moiety in mucin core 2 (e.g., GlyCAM-1) is facilitated when beta1,3Gal has already been alpha2,3 sialylated. (iv) alpha2,6(N)ST was absolutely specific for the beta1,4Gal in mucin core 2. Either alpha1,3 fucosylation or 6-O-sulfation of the GlcNAc moiety reduced the activity. Sialylation of beta1,3Gal in addition to 6-O-sulfation of GlcNAc moiety abolished the activity. (v) Prior alpha2,3 sialylation or 3-O-sulfation of beta1,3Gal would not affect alpha2,6 sialylation of Galbeta1,4GlcNAc of mucin core 2. (vi) A 3- or 4-fluoro substituent in beta1,4Gal resulted in poor acceptors for the cloned alpha2,6(N)ST and alpha2,3(N)ST, whereas 4-fluoro- or 4-OMe-Galbeta1,3GalNAcalpha was a good acceptor for cloned alpha2,3(O)ST. (vii) 4-O-Methylation of beta1,4Gal abolished the acceptor ability toward alpha2,6(N)ST but increased the acceptor efficiency considerably toward alpha2,3(N)ST. (viii) Just like LNCaPalpha1,2-FT and Gal-3-O-sulfotransferase T2, the cloned alpha2,3(N)ST which modifies terminal Gal in Galbeta1,4GlcNAc also efficiently utilizes the terminal beta1,3Gal in the Globo backbone. Utilization of C-3 blocked compounds such as 3-O-sulfo-Galbeta1,3GalNAcbeta1,3Galalpha-OMe as acceptors by cloned alpha2,3(O)ST and analyses of the resulting products by lectin chromatography and mass spectrometry indicate that alpha2,3(O)ST is capable of attaching NeuAc to another position in C-3-substituted beta1,3Gal.

l-[U-(14)C]Threonine is incorporated into N-(purin-6-ylcarbamoyl)-l-threonine riboside of rat liver and Escherichia coli tRNA. A pathway is suggested for the biosynthesis of this nucleoside.

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewis x (Neu5Acα2-3Galβ1-4(Fuc-α1-3)GlcNAc). Much effort has gone into the synthesis of sialylated and sulfated Lewis x analogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewis x as part of a branched Core 2 O-linked structure, we recently synthesized Galβ1-4(Fuc-α1-3)GlcNAcβ1-6(SE-3Galβ1-3)GalNAc1αOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al., Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al., Biochemistry, 31, 9126-9131, 1992; Imai et al., Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acα2-3Galβ1-3Gal-NAc might have the capability of interacting with L-and P-selectin. To examine this hypothesis, we synthesized Galβ1-4(Fucα1-3)GlcNAcβ1-6(Neu5Acα2-3Galβ1-3)-GalNAcα1-OB, which was found to be 2-to 3-fold better than sialyl Le x for P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcβ1-4(Fucα1-3)GlcNAcβ1-OMe (GalNAc-Lewis x -O-methyl glycoside), which also proved to be a better inhibitor of L-and P-selectin than sialyl Lewis x -OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5-to 6-fold better at inhibiting L-and P-selectin than sialyl Lewis x -OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (β1-3) arm, and a modified Le x unit on the branched (β1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLe x for both L-and P-selectin.