CMP-dependent sialidase activity
AssigneeRoche Diagnostics Operations, Inc.
InventorsHarald Sobek, Michael Greif, Marco Thomann, Sebastian Malik
Life of this patent
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Abstract
The properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions are disclosed. Particularly, mutants that exhibit α-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as co-substrate, and in the presence of a suitable acceptor site, are disclosed. A fundamental finding documented in the present disclosure is that enzymes are not only capable of catalyzing transfer of a sialidyl moiety but they are also capable of catalyzing hydrolytic cleavage of terminally bound sialic acid from a glycan.
Claims7
A method of producing in vitro a sialylated target molecule comprising:
- (a) providing an aqueous composition comprising
- (i) a soluble human β3-galatoside-α-2,6-sialyltransferase I comprising an amino acid motif from position 90 to position 108 in SEQ ID NO:1;
- (ii) cytidine-5′-monophospho-N-acetylneuraminic acid;
- (iii) a glycosylated target molecule selected from the group consisting of a glycoprotein and a glycolipid, the target molecule comprising one or more antenna(e), at least one antenna having as a terminal structure a β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moiety with a hydroxyl group at the C6 position in the galactosyl residue;
- (iv) an aqueous solution permitting glycosyltransferase enzymatic activity comprising a buffer salt;
- wherein the aqueous composition further comprises an enzyme selected from the group consisting of an alkaline phosphatase, an acid phosphatase and a 5′ nucleotidase that hydrolyzes the phosphoester bone in 5′-cytidine-monophosphate under conditions permitting glycosyltransferase enzymatic activity;
- (b) forming one or more terminal antennal N-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine residue(s) by incubating the aqueous composition of step (a), thereby reacting cytidine-5′-monophospho-N-acetylneuraminic acid as co-substrate, thereby forming 5′-cytidine-monophosphate;
- and
- (c) hydrolyzing the phosphoester bond of the 5′-cytidine-monophosphate formed in step (b), thereby reducing 5′-cytidine-monophosphate-mediated inhibition, thereby maintaining the activity of the soluble human β-galactoside-α-2,6-sialyltransferase I;
- thereby producing in vitro the sialylated target molecule.
Dependencies are inferred from the claim text.