Sialyltransferases cloned

Haraguchi, M., Yamashiro, S., Yamamoto, A., Fumkawa, K., Takamiya, K., Lloyd, K. O., Shiku, H., and Fumkawa, K., 1994, Isolation ofGD3 synthasegene by expression cloning ofGM3 a-2,8-sialyltransferase cDNA using anti-GD2 monoclonal antibody. Proc. Natl. Acad. Sci. USA 91 10455-10459. 

Kono M, Takashima S, Liu H, Inoue M, Kojima N, Lee YC, Hamamoto T, Tsuji S. Molecular cloning and functional expression of a fifth-type alpha 2,3-sialyltransferase (mST3Gal V GM3 synthase). Biochem. Biophys. Res. Commun. 1998 253-175. Daniotti JL, Martina JA, Giraudo CG, Zurita AR, Maccioni HJ. GM3 alpha2,8-sialyltransferase (GD3 synthase) protein characterization and sub-golgi location in CHO-Kl cells. J. Neurochem. 2000 74 1711-1720. 

Livingston BD, Paulson JC (1993) Polymerase chain reaction cloning of a developmentally regulated member of the sialyltransferase gene family. J Biol Chem 268 11504-11507... 

Lee YC, Kurosawa N, Hamamoto T, Nakaoka T, Tsuji S (1993) Molecular cloning and expression of Gal/il,3GalNAc a2,3-sialyltransferase from mouse brain. Eur J Biochem 216 377-385... 

K, Nagai Y, Sanai Y (1994) Expression cloning of a CMP-NeuAc NeuAc 0 2-3Gal jSl-4Glc jll-l Cer 0 2,8-sialyltransferase (GD3 synthase) from human melanoma cells. Proc Natl Acad Sci USA 91 7952-7956... 

Kono M, Yoshida Y, Kojima N, Tsuji S (1996) Molecular cloning and expression of a fiffii type of 0 2,8-sialyltransferase (ST8Sia V) Its substrate specificity is similar to that of SAT-V/III, which synthesize GDlc, GTla, GQlb and GT3. J Biol Chem 271 29366-29371... 

Yoshida Y, Kojima N, Kurosawa N, Hamamo-to T, Tsuji S (1995) Molecular cloning of Sia o 2,3Gal l,4GlcNAc a2,8-sialyltransferase from mouse brain. J Biol Chem 270 14628-14633... 

Kurosawa N, Kojima N, Inoue M, Hamamo-to T, Tsuji S (1994) Cloning and expression of Gal l,3GalNAc-sp ecific GalNAc a2,6-sialyltransferase. J Biol Chem 269 19048-19053... 

Fukumoto S, Miyazaki H, Goto G, Urano T, Furukawa K, Furukawa K (1999) Expression cloning of mouse cDNA of CMP-NeuAc Lactosylceramide a2,3-sialyltransferase, an enzyme that initiates the synthesis of ganglio-sides. J Biol Chem 274 9271-9276... 

Photobacterium damsela (x2,6-siaiyltransferase (Pd2,6ST) was the first bacterial sialyltransferase which has been cloned and purified by the Yamamoto group (33, 34). This enzyme has a relaxed acceptor specificity (35, 36). For example, it has been applied for the enzymatic sialylation of Tn glycopeptides with GalNAc a-linked to either serine or threonine residue) (37). It was also shown to be able to transfer sialic acid to both N- and 0-linkM glycoproteins (38). 

Characterization of a bifunctional bacterial sialyltransferase, Cst-11 cloned from C. jejuni having both a2,3- and a2,8-sialyltransferase activities, has been reported. This sialyltransferase has been used for the synthesis of GD3 oligosaccharides Neu5Aco2,8Neu5Ac(x2,3LacOR and other gangliosides (53, 54). 

Sialic acid biosynthesis in bacteria is less complex than in mammals. There are only three enzymes involved in NeuSAc biosynthesis they are the NeuSAc-synthase, CMP-Neu5Ac-synthetase, and sialyltransferase. Although these enzymes were cloned only within the past 10 years, their straightforward overexpression in bacteria has allowed extensive studies to be performed. 

In terms of preparative chemistry, the use of CMP- 3-Neu5Ac as a glycosyl donor and (cloned) a-2,3/6-sialyltransferases as biocatalysts have achieved a permanent position in the planning of synthetic routes for sialo-oligosaccharide chains see e.g. refs. [314,342, 345,351,472,473,535-544]. Especially in the field of the preparation of sialyl Le frag-... 

So far, the acceptor specificity of nearly 20 eu- and prokaryotic sialyltransferases has been elucidated and 15 different cDNA clones of these enzymes have been obtained (Table 14 and references therein). This list demonstrates the rapid progress in this field and also includes the first cloning of mammalian polysialyltransferase (polysialyltransferase-1 from hamster ovary cells) [620]. The same group has recently published the molecular analysis of the biosynthetic pathway of the a-2,8-polysialic acid capsule by Neisseria meningitidis serogroup B [658]. 

Efficient, stereoselective sialylations are still a cumbersome challenge for synthetic carbohydrate chemists due to the lack of neighboring group participation of the sialic acid [29]. Transferase-catalyzed sialylations therefore offer a welcome synthetic alternative. To date, eight different sialic acid linkage types have been identified. Out of the more than a dozen different sialyltransferases - SiaT - that have been found and cloned [99] a rat liver a(2-6)SiaT (E.C.2.4.99.1) and porcine a(2-3)SiaT (E.C.2.4.99.4) are commercially available. The synthesis of the natural CMP-sialic acid donor and that of various derivatives have been described [39, 48, 100, 101]. The capability for transfer of donor analogs by a(2-6)SiaT and rat liver a(2-3)SiaT (E.C.2.4.99.6) has recently been compiled [28,48]. 

Fig. 8 Double staining of wild type CHO cells (A) and CHO clones stably transfected with three different sialyltransferase minigenes, STa (B), STb (C) and STc (D). SNA-FITC labeling is in light grey [green online] and nuclear staining with DAPI in medium grey [blue online].

Fig. 9 Mass spectrometry analysis of protein membrane of CHO clones engineered with different sialyltransferases. Black bars were obtained with STa, grey bars with STb and white bars with STc enzymes.

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