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CMP-dependent sialidase activity

A technology for sialyltransferase and enzymatic activity, which is applied in the field of controlled hydrolysis of α2,6 glycosidic bonds, and can solve the problems of low expression yield, poor activity and limited availability of hST6Gal-I, etc.

Active Publication Date: 2017-12-01
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to recombinant expression in different hosts (methylotrophic yeast Pichia pastoris, cultured Spodoptera frugiperda cells, E. coli-based expression system) Low expression yield and / or poor activity of expressed hST6Gal-I, the availability of recombinant hST6Gal-I for such applications is still limited

Method used

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  • CMP-dependent sialidase activity
  • CMP-dependent sialidase activity
  • CMP-dependent sialidase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0172] Tests for sialyltransferase enzyme activity

[0173] Asialo-fetuin (desialylated fetuin, Roche Applied Science) was used as acceptor and CMP-9-fluoro-NANA (CMP-9-fluoresceinyl-NeuAc) was used as donor. Enzymol substrates (Brossmer, R. and GrossH. J. (1994) Meth. Enzymol. 247, 177-193). The enzymatic activity of sialyltransferase was determined by measuring the transfer of sialic acid from the donor compound to asialo-fetuin. The reaction mixture (35 mM MES, pH 6.0, 0.035% Triton X-100, 0.07% BSA) contained 2.5 µg of enzyme sample, 5 µL of asialo-fetuin (20 mg / mL) and 2 µL of CMP-9-fluoro-NANA (1.0 mg / mL). The reaction mixture was incubated at 37°C for 30 min. Stop the reaction by adding 10 µL of the inhibitor CTP (10 mM). Load the reaction mixture onto a PD10 desalting column equilibrated with 0.1 M Tris / HCl (pH 8.5). Fetuin was eluted from the column using equilibration buffer. Fraction size is 1 mL. Determine the concentration of fetuin formed using a spectrofl...

Embodiment 2

[0175] SDS gel electrophoresis

[0176] Analytical SDS gel electrophoresis was performed using NuPAGE gels (4-12%, Invitrogen). Samples (36 µL) were diluted with 12 µL of NuPAGE LDS sample buffer (Invitrogen) and incubated at 85°C for 2 min. Load an aliquot (typically containing 5 µg protein) onto the gel. Gels were stained using SimplyBlue SafeStain (Invitrogen).

Embodiment 3

[0178] N-terminal sequencing by Edman degradation

[0179] The N-terminal sequences of the expressed variants of human ST6Gal-I were analyzed by Edman degradation using reagents and devices from Life Technologies. Sample preparation was performed as described in the instruction manual for the Life Technologies ProSorb Sample Preparation cartridge (Cat. No. 401950) and the Life Technologies ProBlott Mini PK / 10 Membrane (Cat. No. 01194). For sequencing, the Procise Protein Sequencing Platform (Procise Protein Sequencing Platform) was used.

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Abstract

The present disclosure is directed to the properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions. Any of the mutants disclosed in here exhibit alpha-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. A fundamental finding documented in the present disclosure is that such enzyme 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. Particularly it was found that in the presence of cytidine 5'-monophosphate (CMP) glycosyltransferase activity is inhibited, and sialidase activity is stimulated. Sialidase activity was found to be dependent on the presence of a particular stretch of amino acids (position 90 to 108) in the polypeptide sequence of the reference (wild type) hST6Gal-I polypeptide. Deletion of this sequence portion in an N-terminal truncation variant was found to abolish sialidase activity, notably both in the presence and in the absence of CMP. Thus, disclosed are compositions, uses and methods employing the CMP-mediated feed-back regulation documented herein.

Description

[0001] The present invention relates to the properties of certain glycosyltransferase variants with N-terminal truncating deletions or internal deletions. Any of the mutants disclosed herein exhibit alpha-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as a co-substrate and in the presence of an appropriate acceptor site. A fundamental finding documented in the present invention is that such enzymes are not only able to catalyze the transfer of sialidyl moieties, but they are also able to catalyze the hydrolytic cleavage of terminally bound sialic acids from glycans. Specifically, it was found that in the presence of cytidine-5'-monophosphate (CMP), glycosyltransferase activity is inhibited and sialidase activity is stimulated. Sialidase activity was found to be dependent on the presence of a specific stretch of amino acids (positions 90-108) in the polypeptide sequence of the reference (wild-type) hST6Gal-I polypeptide. It was found that d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12P19/18
CPCC12N9/1081C12P19/18C12P19/44C12Y301/03C12P21/005C12Y204/99001C07K16/00C07K2317/41
Inventor H.佐贝克M.格赖夫M.托曼S.马利克
Owner F HOFFMANN LA ROCHE & CO AG
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