Synthesis and biological evaluation of acceptor substrates for alpha-1,3-fucosyltransferase

Polysaccharides;

Smith, Shona L. ; Field, Rob ; Field, Rob

University:University of St Andrews

Department:Biology (School of)

关键词: Polysaccharides;   

Others  :  https://research-repository.st-andrews.ac.uk/bitstream/handle/10023/14221/ShonaSmithPhDThesis.pdf?sequence=2&isAllowed=y

来源: DR-NTU

【 摘 要 】

The chemical synthesis of sulfate and phosphate derivatives of galactose-α-1,4-N-acetylglucosamine-OR (octyl N-acetyllactosamine) and galactose-α-1,3-N- acetylglucosamine-OR [where R= -(CH2)7CH3] are reported here using N-acetylglucosamine and galactose as starting materials. Sialylation of octyl N- acetyllactosamine derivatives was achieved using trans-sialidase. These compounds were evaluated as potential acceptor substrates for five recombinant α-1,3- fucosyltransferases (α-1,3-FucT) and a semi-pure α-1,3/4-FucT (human milk). The kinetic data showed a wide range of acceptor specificities between different recombinant enzymes. Octyl N-acetyllactosamine 6-O-sulfate proved to be an excellent substrate for α-1,3-FucT VI, with a KM of 0.85 ?M. This substrate has a lower KM than any reported substrate for any α-1,3-FucT. An unusual result was observed for octyl N- acetyllactosamine derivatives containing a sulfate or phosphate group at the site of glycosylation. These compounds were found to be good acceptor substrates for α-1,3-FucT VI and milk α-1,3/4-FucT with KM and Vmax values similar to those of the parent compound, octyl N-acetyllactosamine. Preliminary studies show that the product of such a reaction could contain a sulfate or phosphate diester linkage between fucose and octyl LacNAc. If the anionic substituent at the site of glycosylation is being fucosylated, current models proposed for a mechanism involving an enzyme active site base mechanism cannot explain this result. An alternative mechanism has been suggested involving Mn2+ coordination to the hydroxyl group of the acceptor substrate being glycosylated. This mechanism can also be used to explain the unusual kinetic results obtained for substrates containing a sulfate or phosphate group at the site of glycosylation.