Sialyl transferase

Washiya, K., Furuike, T., Nakajima, F., Lee, Y. C. and Nishimura, S. I. (2000). Design of fluorogenic substrates for continuous assay of sialyl-transferase by resonance energy transfer. Anal. Biochem. 283, 39-48. 

Scheme 10.1 Glycopeptide synthesis and a-chymotrypsin catalyzed release from the solid support, a) 25% TFA (CH2CI2) b) 0-(N-Boc-Phe)glycolic acid (7 eq), BOP, HOBt, DIEA b) 25% TFA (CH2CI2) c) Boc-Gly-OH (7 eq), BOP, HOBt, DIEA c) 25% TFA (CH2CI2) d) Boc-Asn(GlcNAcb)-OH (3 eq), BOP, DIEA e) galactosyl transferase, sialyl transferase f) CT, H2O, pH 7.0 g) ultrafiltration h) a-l,3-fuco-syltransferase, GDP-Fuc (2.5 eq), 0.1 M HEPES (pH 7.0), 95%.

Scheme 10.2 Oligosaccharide synthesis and a-chymotrypsin catalyzed release from the solid support, a) Z-Phe-NH-(CH2)6-OH (7), CSA, (CHCljjz, 70°C b) Hj, Pd/C, MeOH, 50°C c) CH2 = CHCONH(CHjjsCOOH (8), EtOH-CeHe d) MeONa (cat.), MeOH/THF, CH2 = CHCONH2, TMEDA, APS, DMSO-H2O, SOT e) Galactosyl transferase, Sialyl transferase g) CT, Tris-HCI buffer, pH 7.8, 48 °C, 72% from (10).

N.m.r.-spectroscopic studies have furthennore shown that sialyl-transferase from bovine colostrum preferentially incorporates sialyl residues into a-(2— 6) linkages of glycopeptides from oq-acid glycoprotein.162... 

Substrate-specificity studies on microsomal, frog-liver sialyltrans-ferase revealed the presence of (2—>3) and (2—>6) activities.277 This enzyme system readily sialylates oligosaccharides, but is almost inactive with asialofetuin, which is in contrast to the sialylation of oligosaccharides, as well as asialofetuin, by rat-liver sialyltransferase.278 The conclusion from this observation is that acceptor specificity of sialyl-transferases isolated from liver of evolutionary distant animals is similar for substrates of low molecular weight, but differs for compounds of high molecular weight.279... 

McCaffrey, G. and Jamieson, J.C. (1993). Evidence for the role of a cathepsin D-like activity in the release of gal-beta-1 -4glcnac-alpha-2-6-sialyl transferase from rat and mouse-liver in whole-cell systems. Comp. Biochem. Physiol. B-Biochem. Mol. Biol., 104,... 

In this technique, the enzyme solution is put inside a dialysis bag which is then immersed in a solution of substrate, or cofactors. Small molecules can diffuse through the wall of the bag and react in the presence of the enzyme, while products, if also small molecules, diffuse into the outside solution, where they may be recovered. This technique has been used in syntheses with sialyl aldolase, Kdo-synthetase, the common aldolase, a mixture of hexokinase and pyruvate kinase, a-(2— 6) sialyl transferase,26 a mixture of pyruvate kinase and adenylate kinase,27 and CMP-Neu5Ac synthetase.28... 

In order to try to overcome some of the problems associated with chemical synthesis of oligosaccharides containing N-acetylneuraminic acid, Sabesan and Paulson [277] have used a combination of chemical and enzymatic methods using purified sialyl-transferases in the presence of CMP-iV-acetylneuraminic acid and synthetic acceptor molecules to give sialyl derivatives of oligosaccharides which were characterised by NMR. Thus, methyl P-D-galactopyranoside, methyl P-D-lactoside and iV-acetyl-... 

Exposure of HeLa cells to butyrate had no effect on the activity of GM3-sialidase when GM3 specifically labeled in the sialic acid residue was used as substrate (Fig. 3a). We were unable to detect any "ecto"-sialidase activity in either control or butyrate-treated cells (14) although others have postulated that such an enzyme is important in regulating plasma membrane gangliosides (15,16). In contrast, the activity of the specific sialyl transferase involved in GM3 biosynthesis increased over 20-fold following butyrate treatment (Fig. 3b). The effect was specific as activities of the other glycosphingolipid transferases that could be measured in HeLa cells were not altered in butyrate-treated cells (4,8,17). 

Increased sialyl transferase activity was dose and time dependent, and reversible (8). Maximal activity was obtained by exposing the cells to 5 mM butyrate for 24 h. Following removal of butyrate, the enzyme had a half-life of 7 h and activity reached control levels by 24 h. Of the numerous short chain fatty acids and derivatives tested, only butyrate, pentanoate and propionate were effective (8). 

Butyrate appears to induce sialyl transferase activity as addition of actinomycin D or cycloheximide to the medium along with butyrate blocked the increase in activity (4,8). Specific cell cycle inhibitors such as thymidine and colcemid did not cause an increase in activity in control cells or prevent induction in butyrate-treated cells (8). Induction of sialyl transferase activity also occurred in serum-free medium (8). When homogenates of control and butyrate-treated cells were admixed and assayed for sialyl transferase activity, there was no evidence of an inhibitor in the former or activator in the latter cells (8). 

In recent work on CHO cells, it had been suggested that the effects of butyrate are mediated by cyclic AMP (18). We found, however, that cyclic AMP (2 mM), its mono- (1 mM and dibutyryl (0.5 mM) derivatives, theophylline and prostaglandins did not cause an elevation in si alyl transferase activity (1). Choleragen, which is a potent and persistant activator of adenylate cyclase (see below), also did not elevate sialyl transferase activity in HeLa cells (Table I) or alter cell morphology (unpublished observations). Thus, it is unlikely that these effects of butyrate are mediated by elevation of cyclic AMP levels. 

Increased GM3 content was also observed in another strain of HeLa exposed to butyrate but not in butyrate-treated normal human fibroblasts (experiments in collaboration with E. Stanbridge, University of California at Irvine and R. 0. Brady, NINCDS). Butyrate appeared to have similar effects on GM3 biosynthesis in KB cells, another human carcinoma-derived cell line (20). Butyrate-treated KB cells had 9-fold elevated levels of sialyl transferase activity. In contrast, butyrate as well as dibutyryl-... 

Induction of Sialyl transferase Activity in HeLa Cells 1... 

Experiment Number Treatment Time (h) Sialyl transferase Activity Control Butyrate Choleragen ... 

Butyrate appears to have its most profound effects on neoplastic cells such as HeLa in addition to morphological and biochemical differentiation, the fatty acid inhibits cell growth (2). Previous studies have established a correlation between decreased ganglioside synthesis and malignant transformation (43-46). Transformed baby hamster kidney and newborn rat kidney cells exhibited a loss of GM3 and sialyl transferase activity (43,44). 

Kinetic Properties of Sialyltransferases. The sialyl-transferase activities with the endogenous glycoprotein and glycolipid acceptors in the standard assays (15) were linear with time for at least 60 min, while those with the exogenously added GMi and DS-fetuin were linear with time only for about 30 min (Figure 1). Activities were directly proportional to the amount of enzyme added up to 0.75 mg protein/assay (Figure 2). 

The increase in incorporation into polypeptides of lower molecular weights may be due to their faster diffusion rate in membrane, thus allowing them to undergo faster sialylation than the higher molecular weight species. This proposition is based on the assumption that there is only one species of glycoprotein sialyl-transferase, an assumption, which is evident later, may not be true. 

The basis for the multiplicity of the sialyltransferase activities remains to be elucidated. We plan to purify these enzyme species to homogeneity, using isoelectric focusing columns of smaller pH ranges in conjunction with affinity chromatography which has been successfully used to purify the soluble sialyl-transferases from bovine colostrum (57). Possibility exists that the heterogeneity of sialyltransferase activities as observed is due to differences in polypeptide sequences, carbohydrate content, or non-covalent interactions with other membrane components, and these possibilities can be clarified only with highly purified enzyme preparations. 

Enzymatic glycosylation on SP was first reported by Schuster et al. (157) and since then has been used to perform multiple glycosylations. Two examples are reported in Fig. 2.28. The synthesis of oligosaccharides 2.94 and 2.95 related to the sialyl Lewis X antigen was carried out via glycosylations mediated by P-l,4-galactosyltransferase and a-2,3-sialyltransferase (171) and by p-l,4-galactosyltransferase, a-2,3-sialyl-transferase, and fucosyltransferase (172), respectively. The use of unprotected saccha-... 

Figure 3 Structure of the major acidic GSLs (gangiiosides). Lactosylceramide, GM3, GD3, and GT3 are substrates for the same series of glycosyl transferases reactions, nameiy, GaiNac transferase, gaiactosyl transferase II, and sialyl transferase V.

Following the enzymatic synthesis of CMP-Neu5Ac, isolated a2-6-sialyl-transferases could be used to introduce sialic acid to the N-acetyl lactosamine derivates a-f in position 6. This afforded a series of trisaccharide derivates Neu5Aca2-6 Gal l-4GlcNAc l-R. The unblocked trisaccharide a [55], the methyl glycoside b [56], the -Asn derivative c [57], the pentapeptide derivative d [58], the allyl e [59], and the pent-4-enyl glycoside f [48] can be prepared in convincing yields (Fig. 6). 

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