Proteins having glycosyltransferase activity

Details Proteins having glycosyltransferase activity Proteins having glycosyltransferase activity

1995-05-26

1997-06-24

Wax, Robert A.

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

4352542, 4353201, 536 232, 536 234, C12N 910, C12N 120, C12N 1500, C07H 2104

Patent

active

056416687

DESCRIPTION:

BRIEF SUMMARY
The invention relates to proteins having glycosyltransferase activity and to a recombinant process for the production of proteins having glycosyltransferase activity.
Glycosyltransferases transfer sugar residues from an activated donor substrate, usually a nucleotide sugar, to a specific acceptor sugar thus forming a glycosidic linkage. Based on the type of sugar transferred, these enzymes are grouped into families, e.g. galactosyltransferases, sialyltransferases and fucosyltransferases. Being resident membrane proteins primarily located in the Golgi apparatus, the glycosyltransferase share a common domain structure consisting of a short amino-terminal cytoplasmic tail, a signal-anchor domain, and an extended stem region which is followed by a large carboxy-terminal catalytic domain. The signal-anchor or membrane domain acts as both uncleavable signal peptide and as membrane spanning region and orients the catalytic domain of the glycosyltransferase within the lumen of the Golgi apparatus. The luminal stem or spacer region is supposed to serve as a flexible tether, allowing the catalytic domain to glycosylate carbohydrate groups of membrane-bound and soluble proteins of the secretory pathway enroute through the Golgi apparatus. Furthermore, the stem portion was discovered to function as retention signal to keep the enzyme bound to the Golgi membrane (PCT Application No. 91/06635). Soluble forms of glycosyltransferases are found in milk, serum and other body fluids. These soluble glycosyltransferases are supposed to result from proteolytic release from the corresponding membrane-bound forms of the enzymes by endogenous proteases.
Glycosyltransferases are valuable tools for the synthesis or modification of glycoproteins, glycolipids and oligosaccharides. Enzymatic synthesis of carbohydrate structures has the advantage of high stereo- and regioselectivity. In contrast to chemical methods the time-consuming introduction of protective groups is superfluous. However, enzymatic synthesis of carbohydrate structures has been a problem because glycosyltransferases are not readily available. Therefore, production using recombinant DNA technology has been worked on. For example, galactosyltransferases have been expressed in E. coli (PCT 90/07000) and Chinese hamster ovary (CHO) cells (Smith, D. F. et al. (1990) J. Biol. Chem. 265, 6225-34), sialyltransferases have been expressed in CHO cells (Lee, E. U. (1990) Diss. Abstr. Int. B. 50, 3453-4) and COS-1 cells (Paulson, J. C. et al. (1988) J. Cell. Biol. 107, 10A), and fucosyltransferases have been produced in COS-1 cells (Goelz, S. E. et al. (1990) Cell 63, 1349-1356; Larsen R. D. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6674-6678) and CHO cells (Potvin, B. (1990) J. Biol. Chem. 265, 1615-1622). Recently, Paulson et al. have disclosed a method for producing soluble glycosyltransferases (U.S. Pat. No. 5,032,519). However, there still is a need for proteins having favorable glycosylating properties and for advantageous methods for producing such proteins.
It is an object of the present invention to provide novel proteins having glycosyltransferase activity, recombinant DNA molecules encoding proteins having glycosyltransferase activity, hybrid vectors comprising such recombinant DNA molecules, transformed hosts suitable for the multiplication and/or expression of the recombinant DNA molecules, and processes for the preparation of the proteins, DNA molecules and hosts.
The present invention concerns a protein having glycosyltransferase activity and comprising identical or different catalytically active domains of glycosyltransferases, e.g. hybrid proteins.
Preferred is a protein of the invention which comprises two identical or two different catalytically active domains of glycosyltransferases.
Particularly preferred is such a protein exhibiting two different glycosyltransferase activities, i.e. a protein capable of transferring two different sugar residues.
Besides the catalytically active domains a protein of the invention may comprise additional amino acid sequence

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