期刊
GLYCOCONJUGATE JOURNAL
卷 25, 期 1, 页码 59-68出版社
SPRINGER
DOI: 10.1007/s10719-007-9062-z
关键词
carbohydrate; sialyltransferase; specificity; enzyme; glycan array
资金
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [U54GM062116] Funding Source: NIH RePORTER
- NIGMS NIH HHS [GM62116] Funding Source: Medline
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 alpha 2,6sialyltransferase-I (hST6Gal-I) also sialylates chitobiose structures (GlcNAc beta 1-4GlcNAc)(n) including N-glycans, rat alpha 2,3sialyltransferase (rST3Gal-III) tolerates fucosylated acceptors such as Lewis(a), human alpha 2,3sialyltransferase-IV (hST3Gal-IV) broadly sialylates oligosaccharides of types 1 4 and porcine alpha 2,3sialyltransferase-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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