Method of producing .alpha.2,3-sialyltransferase

Details Method of producing .alpha.2,3-sialyltransferase Method of producing .alpha.2,3-sialyltransferase

1998-08-31

2000-10-24

Achutamurthy, Ponnathapu

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

435183, 536 691, C12N 910, C08B 306

Patent

active

061365793

DESCRIPTION:

BRIEF SUMMARY
This invention relates to a method of producing .alpha.2,3 sialyltransferase.
This invention has particular but not exclusive application to production of an enzyme having eukaryotic .alpha.2,3-Gal.beta.1,3(4) GlcNAc sialyltransferase activity, and for illustrative purposes reference will be made to such application. However, it is to be understood that this invention could be used in other applications, such as production of assays for enzymes having eukaryotic .alpha.2,3-Gal.beta.1,3(4) GlcNAc sialyltransferase activity.
The glycosyltranferases are a highly polymorphic group of membrane-bound enzymes of endoplasmic reticulum and Golgi bodies that catalyze the transfer of a single monosaccharide unit from a nucleotide donor to the hydroxyl group of an acceptor saccharide in the biosynthesis of N-glycan (Asn-GlcNAc N-glycosidic linkage; GlcNAc, N-acetylglucosamine) and O-glycan (Ser/Thr-GalNAc, O-glycosidic linkage; GalNAc, N-acetylgalactosamine) moieties of glycoproteins and glycolipids.
The eukaryotic sialyltransferases comprise a family of glycosyltransferases that catalyze the transfer of N-acetylneuraminic acid (NeuAc), a sialic acid (SA), from CMP-SA to oligosaccharide chains of glycoconjugates. The addition of the SA normally terminates oligosaccharide chain elongation except for polysialic chains found on neural cell adhesion molecule and gangliosides.
Known eukaryotic sialyltransferases involved in the synthesis of N- and O-glycan derivatives of glycoprotein and glycolipid are summarized in Table 1, adapted from Palcic (1994 Methods in Enzymology, 230:300). In the table, the transferred sugar residue is in bold, and R represents the remainder of the acceptor glycoprotein, glycolipid or oligosaccharide chain.


TABLE 1 ______________________________________ Sialyltransferase (ST) EC number Linkage synthesised ______________________________________ Gal(2-6)-ST(ST6N) 2.4.99.1 NeuAc.alpha.2->6Gal.beta.1->4GlcNAc-R GalNAc.alpha.(2-6)-ST 2.4.99.4 NeuAc.alpha.2->6GalNAc.alpha.-R (ST6OI) Gal(2-3)-ST(ST3O) 2.4.99.4 NeuAc.alpha.2->3Gal.beta.1->4GalNAc.alpha.-R Gal(2-3)-ST(ST3N) 2.4.99.6 NeuAc.alpha.2->3Gal.beta.1->3/4GlcNAc-R ______________________________________ NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc-R N-Ac-neuramide .alpha.(2-8)-2.4.99.8 NeuAc.alpha.2.fwdarw.8NeuAc.alpha.2.fwdarw.Gal.beta.-R sialyltransferase
Proteins produced by prokaryotes rarely possess N-terminal sugar chains, whereas eukaryotes exhibit many examples of such glycoproteins. .alpha.2,3-sialyltransferases are useful eukaryotic enzymes for in vitro synthesis of N-linked and O-linked sialyl derivatives of glycoproteins, for determinations of acceptors, and other qualitative and quantitative research of glycoproteins.
Only three glycosyltransferases are commercially available. These are the .beta.1,4-galactosyltransferase (EC 2.4.1.38/2.4.1.90) isolated from bovine and human milk, the .alpha.2,6 Gal.beta.1,4 GlcNAc-sialyltransferase (ST6N, EC 2.4.99.1) isolated from rat liver, and the .alpha.2,3 Gal.beta.1,3 GalNAc-sialyltrasferase (ST30, EC 2.4.99.4) isolated from porcine liver. The ST30 enzyme has been withdrawn from the market by its erstwhile suppliers. No commercial source of .alpha.2,3 Gal.beta.1,3(4) GalNAc-sialyltrasferase (ST3N) or other glycosyltransferases currently exist.
No high expression vectors for cloned transferases are widely available, and accordingly most researchers isolate the required glycosyltransferases from mammalian tissues. Isolations from mammalian tissues are complicated by the low natural abundance of the glycosyltransferases and the need for chromatography for partial purification. These factors combine to render the isolates prone to contamination with other glycosyltransferases as well as enzyme inhibitors, resulting in poor and inconsistent results.
A method for partial purification of ST3N from rat liver is described by Palicic (1994, Methods in Enzymology 230:300) following the procedure of Weistein et al (1982, J.Biol.Chem. 257:13835).

REFERENCES:
patent: 4727136 (1988-02-01), Jennings et al.
patent: 5017487 (1991-05-01), Stunnenberg et al.
patent: 5079353 (1992-01-01), Ratcliffe et al.
patent: 5352670 (1994-10-01), Venot et al.
patent: 5374655 (1994-12-01), Kashem et al.
patent: 5384249 (1995-01-01), Sasaki et al.
patent: 5461143 (1995-10-01), Wong et al.
Gillespie W. et al., (1992) "Cloning and Expression of the Gal.beta.1,3 GalNAc .alpha.2,3 sialyltransferase" J. Biol. Chem. 267(29):21004-21010.
Sasaki et al., (1993) "Expression Cloning of a novel Gal.beta.(1-3/1-4) GlcNac .alpha.2,3-sialyltransferase using lectin resistance selection" J. Biol. Chem. 268(30):22782-22787.
Lee Y-C et al. (1994) "Cloning and Expression of cDNA for a new type of Gal.beta. 1,3 GalNAc .alpha.2,3 sialyltransferase", J. Biol. Chem. 269(13):10028-10033.
Goebel S.J. et al., (1990), "The complete DNA sequence of Vaccinia virus" Virology 179(1):247-266.
Palcic (1994) Methods in Enzymology 230:300-316.
Weinstein et al., "Purification of a Gal.beta.1.fwdarw.4GlcNAc .alpha.2.fwdarw.6 Sialyltransferase and a Gal.beta.1.fwdarw.3(4)GlcNAc .alpha.2.fwdarw.3 Sialyltransferase to Homogeneity from Rat Liver" J. Biol. Chem., 257(22):13835-13844 (1982).
Esposito (1991) "Properties of the Virus Particle", Archives of Virology (Suppl) 2:91-102.
Gross et al. (1990) "A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor" Analytical Biochem 186:127-134.
McFadden G. (1988) "Poxviruses of Rabbits" In "Virus Diseases in laboratory and captive animals" Ed. G. Darai, Martinus Nijhoff Publishers Boston 36-62.
Russell, J. R. and Robbins, S.J. (1989) "Cloning and Molecular Characterization of the Myxoma Virus Genome", Virology 170:147-159.
Mykytowycz (1953) "An attentuated strain of the myxomatosis virus recovered from the field", Nature 172:448-449.
Stamenkovic et al., (1990) "The B cell antigen CD75 is a cell surface sialyltransferase" J. Exp. Med. 172:641-643.
Bast et al., (1992) "The HB-6, Cdw75 and CD76 differentiation antigens are unique cell-surface carbohydrate determinants generated by the .beta.-galactoside .alpha.2,6-sialyltransferase" J. Cell Biol. 116:423-435.
Bouvier (1954) Quelques remarques sur la myxomatose Bulletin de L'Office International des Epizooites 46:76-77.
Jackson, R.J. and Bults, H.G. (1992) "A myxoma virus intergenic transient dominant selection vector" J. Gen Virol. 73:3241-3245.
Sticher, U. et al., (1988) "Purification of .alpha.2,6-sialyltransferase from rat liver by dye chromatography" Biochem J. 253:577-580.
Paulson et al., "Purification of a Sialyltransferase from Bovine Colostrum by Affinity Chromatography on CDP-agarose" J. Biol. Chem., 252(7):2356-2362 (1977).
Niemela et al., "Complementary Acceptor and Site Specificities of Fuc-TIV and Fuc-TVII Allow Effective Biosynthesis of Sialyl-TriLex and Related Polylactosamines Present on Glycoprotein Counterreceptors of Selectins", J. Biol. Chem., 273(7):4021-4026 (1998).
Abbas et al., "Tumor-Associated Oligosaccharides 1: Synthesis of Sialyl-Lewis.sup.a Antigenic Determinant" Proc. Japanese-German Symp. Berlin, pp. 22-23 (1988).
Baisch et al., "Enzymatic Fucosylations with Purine-Diphosphate-Fucoses (PDP-Fucosos)" Bioorganic and Medicinal Chemistry Letters 6(24):2953-2956 (1996).
Barsoum et al., "Production of Autoantibodies by Immunization with Rabbit Transferrin Modified at its Glycosidic Moiety" Mol. Immunol., 18(5):367-372 (1981).
Baumberger et al., "Synthesis of N-Acetyl-4-deoxyneuraminic Acid" Helv. Chim. Acta, 69:1535-1541 (1986).
Beau et al., "Metabolism of 4-O-Methyl-N-acetylneuraminic Acid a Synthetic Sialic Acid" Eur. J. Biochem., 106:531-540 (1980).
Bergh et al., "Aglycon specificity of fetal calf liver and ovine and porcine submaxillary gland .alpha.- N-acetylgalactosaminide .alpha.2.fwdarw.6 sialyltransferase" Eur. J. Biochem., 136:113-118 (1983).
Beyer et al., "Glycosyltransferases and Their Use in Assessing Oligosaccharide Structure and Structure-Function Relationships" Adv. Enzymol., 5

Affiliated with

Also associated with

Rating

Method of producing .alpha.2,3-sialyltransferase does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method of producing .alpha.2,3-sialyltransferase, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of producing .alpha.2,3-sialyltransferase will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-1962377