Sialylation increase

The binding potency of the native sialyl-Lewis X can be increased with three orders of magnitudes by conjugating this saccharide to BSA. This is probably due to the clustering of saccharides that may favour binding to E-selectin [80]. 

Another factor influencing nonspecific tissue distribution is the carbohydrate portion of the IgG molecule, which is attached to the therapeutic protein via an N-linked glycan in the constant domain (Fc). Loss of terminal sialylated residues on the carbohydrate of IgG exposes galactose and promotes receptor-mediated binding of IgG to hepatocytes. Consequently this results in an increase in nonspecific distribution to the liver. Details of desialyation of IgG and its consequences are discussed in Chapter 10. Other glycoproteins may exert similar mechanisms of nonspecific distribution. 

CMP, CDP, CTP, and synthetic derivatives of these nueleotides have been found to inhibit sialyltransferase activity.301" 02 Interest in such inhibitors is increasing, as they may be expected to serve as anticancer agents.269 901,303 Therefore, regulation of Golgi sialyltransferase activity appears possible by nucleotides as products of sialyl- and other glycosyl-transferase activities.1" 2 Interestingly, Epstein-Barr virus infection of human B, lymphoblastoid cell-lines leads to a diminution of sialyltransferase activity.304... 

Immobilization has other advantages it can slow enzyme deactivation by inhibiting protease attack and minimizing shear, interfacial, temperature, or solvent denaturation. As for the scarcity of some potentially very useful enzymes, it may be only a temporary problem. The development of cloning techniques, and probably the very increase in demand will result in lower prices. One spectacular instance is sialyl aldolase (see Table I). Industrial production of this enzyme by the gene-cloned strain of Escherichia coli has been reported.1,2 Sialylaldolase is now available from Toyobo at a moderate price. 

When HeLa cells were.cultured in medium supplemented with 5 mM sodium butyrate, their content of GM3 increased (Fig.2a). Increases varied from 3.5 to 5-fold depending on the experiment (4,8,12,13). When the butyrate was removed and the cells were cultured in normal medium for 24 h, the GM3 levels returned to those found in untreated cells (Fig. 2a). Similar results were observed when N-[acetyl-3H]-D-mannosamine, a precursor of sialic acid, was also included in the culture medium. In the butyrate-treated cells, radioactivity associated with GM3 increased 6.5-fold and 24 h after butyrate was removed, the amount of labeling returned to control values (Fig. 2b). We also were able to label the GM3 by means of a cell surface labeling technique. Control and butyrate-treated cells were exposed to 10 mM sodium periodate and the oxidized sialyl residues were reduced with NaBSfy. There was 5.5-fold more 3h associated with the GM3 recovered from the butyrate-... 

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). 

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-... 

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. 

Konings, P. N., Philipsen, R. L., Veeneman, G. H., and Ruigt, G. S. (1994b). Alpha-sialyl cholesterol increases laminin in Schwann cell cultures and attenuates cytostatic drug-induced reduction of... 

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