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Glycoconj J. 2008 Jan;25(1):59-68. Epub 2007 Oct 4.

Glycan microarrays for screening sialyltransferase specificities.

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Glycan Array Synthesis Core-D, Consortium for Functional Glycomics, Department of Molecular Biology, CB216, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.


Here we demonstrate that glycan microarrays can be used for high-throughput acceptor specificity screening of various recombinant sialyltransferases. Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) was biotinylated at position 9 of N-acetylneuraminic acid (Neu5Ac) by chemoenzymatic synthesis generating CMP-9Biot-Neu5Ac. The activated sugar nucleotide was used as donor substrate for various mammalian sialyltranferases which transferred biotinylated sialic acids simultaneously onto glycan acceptors immobilized onto a microarray glass slide. Biotinylated glycans detected with fluorescein-streptavidin conjugate to generate a specificity profile for each enzyme both confirming previously known specificities and reveal additional specificity information. Human alpha2,6sialyltransferase-I (hST6Gal-I) also sialylates chitobiose structures (GlcNAcbeta1-4GlcNAc)(n) including N-glycans, rat alpha2,3sialyltransferase (rST3Gal-III) tolerates fucosylated acceptors such as Lewis(a), human alpha2,3sialyltransferase-IV (hST3Gal-IV) broadly sialylates oligosaccharides of types 1-4 and porcine alpha2,3sialyltransferase-I (pST3Gal-I) sialylates ganglio-oligosaccharides and core 2 O-glycans in our array system. Several of these sialyltransferases perform a substitution reaction and exchange a sialylated acceptor with a biotinylated sialic acid but are restricted to the most specific acceptor substrates. Thus, this method allows for a rapid generation of enzyme specificity information and can be used towards synthesis of new carbohydrate compounds and expand the glycan array compound library.

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