Glycan microarrays for screening sialyltransferase specificities.
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.
Blixt
Ola
O
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. olablixt@scripps.edu
Allin
Kirk
K
Bohorov
Ognian
O
Liu
Xiaofei
X
Andersson-Sand
Hillevi
H
Hoffmann
Julia
J
Razi
Nahid
N
D000818:Animals; D002460:Cell Line; D054794:Glycomics / Q000379:methods; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis* / Q000378:metabolism*; D051381:Rats; D012799:Sialyltransferases / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D011134:Polysaccharides; D012799:Sialyltransferases