Sialylation, with transferases

Notes and discussion. A major drawback of enzymatic sialylation with sialyl-transferases is the strict acceptor specificity of these enzymes. This synthesis addresses this limitation by showing a widely applicable transfer of sialic acid (NeuAc) from a donor substrate of the sequence NeuAca-(2 —3)Gal-ORi to virtually any galactose acceptor (Gal-OR2). This method uses the less substrate specific glycosidase from Trypanosoma cruzi. The problem with this method is that the product is made at the expense of another sialoside, used as the donor substrate, and as NeuAc transfer is a reversible process, it is difficult to drive the equilibrium in favour of the desired sialoside. For this reason the sialoside donor substrate is regenerated in situ by an a-(2 3)-sialytransferase enzyme, thus enhancing the production of the desired product. The specificity of the sialyltransferase ensures that only the galactose byproduct, formed from the sialyl donor, is re-sialyated as the Gal-OR2 acceptor substrate is a poor substrate. Due to the broad specificity of the trans-sialidase, many a-NeuAc-(2 3)-Gal-OR sequences can be synthesised by... 

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... 

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... 

Sialyl residues in oligosaccharides are introduced by the reaction of cyti-dine monophosphate-V-acetylneuraminic acid (49) as the sugar donor with the appropriate substrate, in the presence of specific transferases. Three of these have been utilized in syntheses which may be considered to be preparative. None are readily available. The most common, which we have called STA (see Table I), catalyzes the transfer of a 5-acetamido-3,5-di-deoxy-D-g/ycm>-a -D-ga/arfo-2-hexulopyranosonic acid unit (the a-D-pyra-nose form of JV-acetylneuraminic acid) to the primary position of D-galactose in a JV-acetyllactosamine residue.86 This enzyme also transfers vV-acetyl-9-O-acetylneuraminic acid (20) and V-glycolylneuraminic acid (12) from the corresponding cytidine monophosphate derivatives.16 The commercial enzyme is rather expensive, but pork liver from a butcher is a... 

Most sialylations so far reported have been achieved with soluble transferases, and seldom on a more than 20-/imol scale (see Table IX), with the intention to prepare and describe sequences present in glycoproteins and glycolipids. Trisaccharide < -D-Neu5Ac-(2— 3)-/J-D-Galp-(l — 3)-/J-d-... 

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-... 

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). 

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-... 

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 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. 

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