Enzyme assay of sialyltransferases for oligosaccharides

Enzyme assay of sialyltransferases for oligosaccharides

comments for reagent

1) CMP-Sia 

If the acceptor substrate is labeled with isotope or some fluorescent materials, you can use cold CMP-Sia. If not, you should use isotope labeled CMP-Sia, such as CMP-[6-¹⁴C]NeuAc or CMP-[6-³H]NeuAc from Amasham. In this assay, you should use the highest purified CMP-Sia, such as Cytidine-5'-monophospho-N-acetylneuraminic Acid Disodium Salt (Nacalai Tesque code no: 10432-24).

2) Detergent

The sialyltransferase itself does not require detergent for the enzymatic activity. If the solubility of acceptor substrate is poor, you should use detergent, such as Triton CF-54. Although Triton CF-54 was commonly used as a detergent for the enzyme assay, unfortunately, it is now hardly obtainable. As far as seen our data, Triton CF-74 is well substituted for Triton CF-54.

3) Other reagents used in this assay should be of the highest purity obtainable.

comments for procedure

This assay protocol is mainly for mammalian enzyme. Though it will be essentially adaptable for other enzymes, if you try to assay other enzymes, please check the following references.

1) Mammalian sialyltransferases (Takashima et al, 2002)

From the vertebrate, 20 sialyltransferase genes and their essential genomic structure have been identified, respectively. Their cDNAs have been cloned and their enzymatic properties have been character­ized (Table 1).

The cloned sialyltransferases have a type II transmembrane topology and contain highly conserved domains called sialylmotifs L (Long), S (Short), VS (Very Short) and III. These motifs are highly conserved regions comprising about 20% of the total protein sequence. The L- and S-sialylmotifs were shown to bind to the donor CMP-NeuAc and acceptor saccharide substrates. The VS-sialylmotif is considered to be participated in the catalytic center though the strict data has not been published. Each sialyltransferase exhibits strict specificity for acceptor substrates and link­ages they synthesize.

The cloned sialyltransferases can be classified into four families according to the carbohydrate linkages they synthesize, i.e., the b-galactoside α2,3-sialyltransferase family (ST3Gal-I-VI), the β-galactoside α2,6-sialyltransferase family (ST6Gal-I,II), the GalNAc α2,6-sialyltransferase family (ST6GalNAc-I-VI), and the α2,8-sialyltransferase family (ST8Sia-I-VI).

2) Bacterial sialyltransferases

Several bacterial sialyltransferases (ST3Gal, ST6Gal and ST8Sia) have been cloned and characterized (Yamamoto et al., 1998). Interestingly, the sialylmotif has not been found in the amino acid sequences of these bacterial sialyltransferases even though they transfer NeuAc from CMP-NeuAc to oligosaccharides in analogous to the mammalian enzymes. Generally, bacterial enzymes have broader acceptor tolerances compared to mammalian enzymes. For example, Neisseria meningitidis ST3Gal can utilize both α- and (3-linked terminal galactoses as acceptors (Gilbert et al., 1997).

3) Viral sialyltransferases

A novel myxoma virus early gene, MST3N, is a member of the eukaryotic sialyltransferase gene family. Myxoma virus is a member of the poxvirus family of double stranded DNA viruses, and it is known to infect Old World or European rabbits causing highly lethal myxomatosis. mammalian cells infected by Myxoma virus produce viral α2,3-sialyltransferase (v-ST3Gal-I) in the cells. v-ST3Gal-I is closely related to mammalian ST3Gal-IV, though it has a very broad acceptor specificity that is not found among the mammalian or bacterial α2,3-sialyltransferases. Acceptors include not only type I to III disaccharides but also fucosylated Lewis^x and Lewis^a (Sujino, K. et al., 2000)

4) Plant sialyltransferases (Takashima et al, 2006)

Sialic acids are widely distributed among living crea­tures, from bacteria to mammals, but it has been commonly accepted that they do not exist in plants, although the possibility remains that sialylated glycoconjugates may exist in plant cells. Interestingly, however, putative gene homologs for mammalian sialyltransferases and CMP-Sia transporters have been detected in the genome and/or expressed sequence tag (EST) data­bases of some plants, such as Arabidopsis thaliana (thale-cress) and Oryza sativa (Japanese rice). This suggests that plants have potential ability of sialylation despite the absence of sialic acids in them. Takashima et al. cloned three genes from 0. sativa, each encoding a protein having sialyl motif-like sequences, and analyzed the enzymatic activity of the proteins. One of them, called OsSTLP1, really transferred sialic acid from CMP-NeuAc to galactose of Galβ1,4GlcNAc through α2,6-linkage.

Figure & Legends

Table 1. Acceptor specificities of mouse and human sialyltransferases, and chromosomal localization of human genes

Asterisk means preferential but not specific substratre.

This table was originally published in J Biol Chem. Takashima S, Tsuji S. et al. "Characterization of the second type of human beta-galactoside alpha 2,6-sialyltransferase (ST6Gal II), which sialylates Galbeta 1,4GlcNAc structures on oligosaccharides preferentially. Genomic analysis of human sialyltransferase genes" 2002, 277(48):45719-28, and reused in "Experimental Glycoscience -Glycobiology" edited by Taniguchi N. et al. Springer Japan KK. 2008, pp.42-46 (Tsuji S. Part1: Section I).