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Process for modification of glycoprotein using Beta-(1,4)-n-acetylgalactosaminyl transferase or mutant thereof

A technology of acetylgalactosamine and glycoprotein, applied in the field of enzymatic modification of glycoprotein

Pending Publication Date: 2017-08-29
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] In conclusion, it is not surprising that there are no reports of in vitro methods for modifying glycoproteins by means of GalNAc transferases of non-natural GalNAc derivatives such as 2-keto or 2-azidoacetyl derivatives.

Method used

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  • Process for modification of glycoprotein using Beta-(1,4)-n-acetylgalactosaminyl transferase or mutant thereof
  • Process for modification of glycoprotein using Beta-(1,4)-n-acetylgalactosaminyl transferase or mutant thereof
  • Process for modification of glycoprotein using Beta-(1,4)-n-acetylgalactosaminyl transferase or mutant thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0392] Selection and design of embodiment 1.GalNAc transferase

[0393] Five specific sequences were selected for initial evaluation, specifically the Uniprot accession numbers: Q9GUM2 (C. elegans; represented herein as SEQ ID NO:2), U1MEV9 (Ascaris suum; represented herein as SEQ ID NO:3) , Q6J4T9 (Telina, represented herein as SEQ ID NO: 4), Q7KN92 (Drosophila melanogaster; represented herein as SEQ ID NO: 5), and Q6L9W6 (Homo sapiens).

[0394] The following polypeptides were constructed based on the predicted deletion of the cytoplasmic and transmembrane domains. These peptides contain predicted:

[0395] Caenorhabditis elegans (CeGalNAcT [30-383] represented by SEQ ID NO: 6)

[0396] KIPSLYENLTIGSSTLIADVDAMEAVLGNTASTSDDLLDTWNSTFSPISEVNQTSFMEDIRPILFPDNQTLQFCNQTPPHLVGPIRVFLDEPDFKTLEKIYPDTHAGGHGMPKDCVARHRVAIIVPYRDREAHLRIMLHNLHSLLAKQQLDYAIFIVEQVANQTFNRGKLMNVGYDVASRLYPWQCFIFHDVDLLPEDDRNLYTCPIQPRHMSVAIDKFNYKLPYSAIFGGISALTKDHLKKINGFSNDFWGWGGEDDDLATRTSMAGLKVSRYPTQIARYKMIKHSTEATNP...

Embodiment 2

[0406] Example 2. Design of the Caenorhabditis elegans GalNAcT mutant of I257

[0407] Based on the sequence alignment of CeGalNAcT and GalT, the isoleucine 257 Three active site mutants were designed with mutations to leucine, methionine or alanine (underlined).

[0408] CeGalNacT(30-383; I257L) represented by SEQ ID NO: 10

[0409] KIPSLYENLTIGSSTLIADVDAMEAVLGNTASTSDDLDTWNSTFSPISEVNQTSFMEDIRPILFPDNQTLQFCNQTPPHLVGPIRVFLDEPDFKTLEKIYPDTHAGGHGMPKDCVARHRVAIIVPYRDREAHLRIMLHNLHSLLAKQQLDYAIFIVEQVANQTFNRGKLMVASRLYPWQCFIFHDDVDLPEDDRNLK L FGGISALTKDHLKKINGFSNDFWGWGGEDDDLATRTSMAGLKVSRYPTQLARYKMIKHSTEATNPVNKCRYKIMGQTKRRWTRDGLSNLKYKLVNLELKPLYTRAVVDLLEKDCRRELRRDFPTCF

[0410] CeGalNAcT(30-383; I257M) represented by SEQ ID NO: 11

[0411] KIPSLYENLTIGSSTLIADVDAMEAVLGNTASTSDDLDTWNSTFSPISEVNQTSFMEDIRPILFPDNQTLQFCNQTPPHLVGPIRVFLDEPDFKTLEKIYPDTHAGGHGMPKDCVARHRVAIIVPYRDREAHLRIMLHNLHSLLAKQQLDYAIFIVEQVANQTFNRGKLMVASRLYPWQCFIFHDDVDLPEDDRNLK M FGGISALTKDHLKKINGFSNDFWGWGGEDDDLATRTSMAGLKVSRYPTQIAR...

Embodiment 3

[0414] Example 3. Design of the Caenorhabditis elegans GalNAcT mutant of M312

[0415] by adding methionine 312 CeGalNAcT mutants were designed by mutation to histidine.

[0416] CeGalNacT (30-383; M312H) represented by SEQ ID NO: 13

[0417] KIPSLYENLTIGSSTLIADVDAMEAVLGNTASTSDDLLDTWNSTFSPISEVNQTSFMEDIRPILFPDNQTLQFCNQTPPHLVGPIRVFLDEPDFKTLEKIYPDTHAGGHGMPKDCVARHRVAIIVPYRDREAHLRIMLHNLHSLLAKQQLDYAIFIVEQVANQTFNRGKLMNVGYDVASRLYPWQCFIFHDVDLLPEDDRNLYTCPIQPRHMSVAIDKFNYKLPYSAIFGGISALTKDHLKKINGFSNDFWGWGGEDDDLATRTSMAGLKVSRYPTQIARYK H IKHSTEATNPVNKCRYKIMGQTKRRWTRDGLSNLKYKLVNLELKPLYTRAVVDLLEKDCRRELRRDFPTCF

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Abstract

The present invention relates to a process for the modification of a glycoprotein, using a Beta-(1,4)-N-acetylgalactosaminyltransferase or a mutant thereof.The process comprisesthe step of contacting a glycoprotein comprising a glycan comprising a terminal GlcNAc-moiety, in the presence of a Beta-(1,4)-N-acetylgalactosaminyl-transferase or a mutant thereof, with anon-natural sugar-derivative nucleotide.The non-natural sugar-derivative nucleotideis according to formula (3), wherein A is selected from the group consisting of-N3;-C(0)R3;-C=C-R4;-SH;-SC(0)R8;-SC(V)OR8, wherein V is O or S;-X wherein X is selected from the group consisting of F, CI, Br and I;-OS(0)2R5; an optionally substituted C2-C24 alkyl group; an optionally substituted terminal C2-C24 alkenyl group; and an optionally substituted terminal C3-C24 allenyl group.

Description

technical field [0001] The present invention relates to methods for enzymatically modifying glycoproteins. More specifically, the present invention relates to methods for modifying glycoproteins with sugar derivative nucleotides using β-(1,4)-N-acetylgalactosamine transferase or mutants thereof, and to β-(1,4) - N-acetylgalactosamine transferase mutants. Background technique [0002] Glycosyltransferases constitute a superfamily of enzymes involved in the synthesis of complex carbohydrates present on glycoproteins and glycolipids. The basic role of glycosyltransferases is to transfer the sugar moieties of nucleotide derivatives to specific sugar acceptors. β-1,4-Galactosyltransferase (β4Gal-T) (EC 2.4.1.38) constitutes one of the subfamilies of the glycosyltransferase superfamily, which contains at least seven Gal-T1 to Gal-T7 A member that catalyzes the transfer of galactose (Gal) from UDP-Gal to different sugar acceptors. A consensus motif generated by galactosyltransf...

Claims

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Application Information

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IPC IPC(8): C12P21/00
CPCC12P21/005C07H19/10C07K16/32C07K2317/24C07K2317/41C12N9/1051C12Y204/01C12Y204/01038C12Y204/01092
Inventor A·A·瓦西尔F·L·范代尔夫特S·S·范博凯尔
Owner SYNAFFIX
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