JP2015080453A - Endoglycosidase derived from coprinus cinereus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an endoglycosidase which has an activity having a wide substrate specificity, cleaving and releasing the N-glycan from glycoprotein, and transferring the sugar chain, and to provide an enzyme which does not have an activity cleaving and releasing the N-glycan from glycoprotein, and only has an activity transferring the sugar chain.SOLUTION: The invention provides: an endoglycosidase derived from Coprinus cinereus consisting of a specific amino acid sequence which has an activity cleaving the N-glycan from glycoprotein to release the sugar chain, and transferring the sugar chain; and an enzyme derived from Coprinus cinereus consisting of a specific amino acid sequence which does not have an activity cleaving the N-glycan from glycoprotein to release the sugar chain, and only has an activity transferring the sugar chain.

Description

  The present invention relates to an endoglycosidase having an activity of cleaving and releasing an N-linked sugar chain possessed by a glycoprotein and transferring the sugar chain, and an N-linked type possessed by a glycoprotein which is a modified form of the endoglycosidase. The present invention relates to an enzyme having only an activity of transferring sugar chains without having an activity of cleaving and releasing sugar chains.

  Endo-β-N-acetylglucosaminidase (endoglycosidase) is used for glycosylation and transfer of glycoproteins.

  Examples of the endoglycosidase include Endo A (Non-patent document 1), Endo F (Non-patent document 2), Endo H (Non-patent document 3), Endo M (Non-patent document 4), and the like. These enzymes act only on sugar chains having a specific structure or have a strong effect on sugar chains having a specific structure. Moreover, the reactivity with complex type sugar chains was not so great.

  In addition, when these enzymes are produced using recombinant technology, there is a problem that they cannot be produced when E. coli is used.

Takegawa K. et al. Appl. Environ. Microbiol. 55, 3107-3112 (1989) Takegawa K. et al., Eur. J. Biochem, 202, 175-180 (1991) Terentino A.L. et al. J. Biol. Chem. 274, 811 (1974) Kadowaki K. et al., Agric, Biol. Chem. 1988, 54, 97

  The present invention relates to an endoglycosidase having an activity of cleaving and releasing an N-linked sugar chain possessed by a glycoprotein having a wide substrate specificity and transferring the sugar chain, and an N-linked sugar chain possessed by the glycoprotein. It is an object of the present invention to provide an enzyme having only an activity of transferring sugar chains without having an activity of cleaving and releasing.

  In light of the fact that the substrate specificity of the conventional endoglycosidase activity for sugar chains is not necessarily wide, the present inventors have intensively studied to obtain an endoglycosidase with a broader substrate specificity. As a result, it has been found that endoglycosidase derived from Coprinus cinereus has a broad substrate specificity that has a wide cleavage activity on not only high-mannose sugar chains but also complex sugar chains. This enzyme was named Endo-CC.

  The present inventor further studied the activity by adding mutations to amino acids in order to obtain an enzyme (glycosynthase) that has no transglycosylation activity but only transfer activity. As a result, it was found that by substituting one specific amino acid, an enzyme having no transglycosylation activity and having only translocation activity was obtained, and this enzyme was named modified Endo-CC.

That is, the present invention is as follows.
[1] Activity derived from the following (a) or (b) Coprinus cinereus, which cleaves and releases an N-linked sugar chain (asparagine-linked sugar chain) possessed by a glycoprotein and transfers the sugar chain Endoglycosidase having:
(a) an endoglycosidase consisting of the amino acid sequence represented by SEQ ID NO: 2;
(b) including an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and cleaving and releasing the N-linked sugar chain of the glycoprotein; And an endoglycosidase having an activity of transferring a sugar chain.
[2] The following (a) or (b) derived from Coprinus cinereus, which encodes an endoglycosidase having an activity of cleaving and releasing an N-linked sugar chain of a glycoprotein and transferring the sugar chain DNA:
(a) an endoglycosidase consisting of the amino acid sequence represented by SEQ ID NO: 2;
(b) including an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and cleaving and releasing the N-linked sugar chain of the glycoprotein; And an endoglycosidase having an activity of transferring a sugar chain.
[3] The following (c) or (d) derived from Coprinus cinereus, which encodes an endoglycosidase having an activity of cleaving and releasing an N-linked sugar chain of a glycoprotein and transferring the sugar chain DNA:
(c) DNA comprising the base sequence represented by SEQ ID NO: 1;
(d) hybridizing under stringent conditions with a DNA comprising a sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 1 to cleave and release the N-linked sugar chain of the glycoprotein; And a DNA encoding a protein having an activity of transferring a sugar chain.
[4] The following (e) or (f) derived from Coprinus cinereus, has no activity to cleave and release N-linked sugar chains possessed by glycoproteins, and only has an activity to transfer sugar chains enzyme:
(e) an enzyme consisting of the amino acid sequence represented by SEQ ID NO: 4;
(f) an N-linked sugar chain comprising an amino acid sequence in which one or several amino acids other than the 180th glutamine in the amino acid sequence represented by SEQ ID NO: 4 are deleted, substituted or added, and possessed by a glycoprotein An enzyme having only the activity of transferring sugar chains without having the activity of cleaving and releasing.
[5] It is derived from the following (e) or (f) Coprinus cinereus, has no activity to cleave and release N-linked sugar chains of glycoproteins, and only has an activity to transfer sugar chains DNA encoding the enzyme:
(e) an enzyme consisting of the amino acid sequence represented by SEQ ID NO: 4;
(f) an N-linked sugar chain comprising an amino acid sequence in which one or several amino acids other than the 180th glutamine in the amino acid sequence represented by SEQ ID NO: 4 are deleted, substituted or added, and possessed by a glycoprotein An enzyme having only the activity of transferring sugar chains without having the activity of cleaving and releasing.
[6] The following (g) or (h) derived from Coprinus cinereus, has no activity to cleave and release N-linked sugar chains possessed by glycoproteins, and only has an activity to transfer sugar chains DNA encoding the enzyme:
(g) DNA containing the base sequence represented by SEQ ID NO: 3;
(h) hybridizes under stringent conditions with a DNA comprising a sequence complementary to the DNA comprising the nucleotide sequence represented by SEQ ID NO: 3, wherein the 538th to 540th nucleotide sequence is CAA, and is a glycoprotein DNA encoding an enzyme that has no activity to cleave and release N-linked sugar chains, but only has an activity to transfer sugar chains.
[7] An N-linked saccharide derived from Coprinus cinereus of [1], which comprises introducing an expression vector containing the DNA of [2] or [3] into E. coli and culturing E. coli. A method for producing an endoglycosidase having an activity of cleaving and releasing a chain and transferring a sugar chain.
[8] An N-linked saccharide derived from Coprinus cinereus according to [4], which comprises introducing an expression vector containing the DNA according to [5] or [6] into Escherichia coli and culturing Escherichia coli. A method for producing an enzyme having only an activity of transferring a sugar chain without having an activity of cleaving and releasing a chain.
[9] A method for producing a free sugar chain, comprising contacting the endoglycosidase of [1] with a glycoprotein, cleaving the sugar chain to obtain a free sugar chain.
[10] A method for producing a glycoprotein, comprising bringing the enzyme according to [4] into contact with a protein and an oxazoline sugar chain, and transferring the sugar chain to the protein.

  Endo-β-N-acetylglucosaminidase (endoglycosidase) Endo-CC of the present invention is obtained by hydrolyzing a diacetylchitobiose part (GlcNAc-GlcNAc) present in the root part of an N-linked sugar chain of a glycoprotein. It has the activity of cleaving and releasing, and transferring sugar chains. This enzyme has wide cleavage activity and transglycosylation activity not only for high mannose type sugar chains but also for complex type sugar chains. Therefore, various sugar chains can be cleaved, and the cleaved sugar chain can be transferred to a recombinant protein to which a monosaccharide such as GlcNAc or D-Glucose is added (hereinafter referred to as “GlcNAc-protein”). Further, the modified Endo-CC, which is an enzyme that has only the activity of transferring a sugar chain without cleaving and releasing the N-linked sugar chain of the glycoprotein of the present invention, has a free sugar chain. GlcNAc-protein can be transferred to produce glycoproteins. Sugar chains cleaved by Endo-CC can be transferred to GlcNAc-proteins by modified Endo-CC, and uniform sugar chains can be added to therapeutic protein drugs such as antibody drugs.

It is a figure which shows the structure of various sugar chains. It is a figure which shows the substrate specificity of Endo-CC of this invention (the 1). It is a figure which shows the substrate specificity of Endo-CC of this invention (the 2). It is a figure which shows the structure of SGP (sialylglycopeptide) and SG (sialylated sugar chain) produced by hydrolyzing SGP. It is a figure which shows the transglycosylation activity of Endo-CC of this invention. It is a figure which shows a transglycosylation reaction. It is a figure which shows the detection result of the transfer activity of modified Endo-CC.

  Hereinafter, the present invention will be described in detail.

1. Endoglycosidase having activity to cleave and release diacetylchitobiose part (GlcNAc-GlcNAc) present in the root part of N-linked sugar chain of glycoprotein and transfer sugar chain. Endo-β, which has the activity of cleaving and releasing the diacetylchitobiose moiety (GlcNAc-GlcNAc) present in the root of the N-linked sugar chain (asparagine-linked sugar chain) by hydrolysis. -N-acetylglucosaminidase (endoglycosidase). The N-linked sugar chain binds to an asparagine residue of the sugar chain binding sequence represented by Asn-any amino acid-Ser / Thr in the amino acid sequence of the protein.

  The endoglycosidase of the present invention is derived from Coprinus cinereus (Nenagahi Toyotake), which belongs to the genus Basidiomycetes Agaricidae, and can be isolated from Coprinus cinereus.

  SEQ ID NO: 1 shows the base sequence of DNA encoding the endoglycosidase of the present invention. The amino acid sequence of the enzyme of the present invention is shown in SEQ ID NO: 2.

  An endoglycosidase having an activity of cleaving and releasing an N-linked sugar chain of the glycoprotein of the present invention and transferring the sugar chain is referred to as Endo-CC.

  The enzyme of the present invention has at least one, preferably one or several amino acids in the amino acid sequence as long as the protein comprising the amino acid sequence has the activity to cleave and release N-linked sugar chains and transfer sugar chains. Mutations such as deletion, substitution and addition may occur in the amino acids.

  For example, at least one, preferably one or several (for example, 1 to 10, more preferably 1 to 5, particularly preferably 1 or 2) amino acids of the amino acid sequence represented by SEQ ID NO: 2 have been deleted. At least 1, preferably 1 or several (eg, 1 to 9, more preferably 1 to 5, particularly preferably 1 or 2) amino acids are added to the amino acid sequence represented by SEQ ID NO: 2. Alternatively, at least 1, preferably 1 or several (for example, 1 to 9, more preferably 1 to 5, particularly preferably 1 or 2) of the amino acid sequence represented by SEQ ID NO: 2. An amino acid may be substituted with another amino acid.

  As an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 2 and the BLAST (Basic Local Alignment Search Tool at the National Center for Biological) Information (the US National Biological Information Center Basic Local Alignment Search Tool)) etc. (eg default or default parameters), at least 85% or more, preferably 90% or more, more preferably 95 %, Particularly preferably 97% or more of sequence identity.

  A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 is substantially the same as a protein having the amino acid sequence of SEQ ID NO: 2.

Further, it is a DNA that can hybridize with a DNA comprising a nucleotide sequence represented by SEQ ID NO: 1 and a complementary sequence under the following stringent conditions, and cleaves an N-linked sugar chain. DNA encoding a protein having an activity of releasing a sugar chain and transferring the sugar chain is also included in the DNA encoding the endoglycosidase of the present invention. Specifically, hybridization was performed at 68 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA was immobilized, and then a 0.1 to 2 fold concentration of SSC solution (1 fold concentration of SSC is 150 mM NaCl, 15 mM) This is a condition that can be identified by washing at 68 ° C. using sodium citrate. Alternatively, after transferring and immobilizing DNA on a nitrocellulose membrane by the Southern blotting method, hybridization buffer [50% formamide, 4 × SSC, 50 mM HEPES (pH 7.0), 10 × Denhardt ^, s) solution, 100 μg / ml salmon sperm DNA], which can form a hybrid by reacting overnight at 42 ° C.

  In addition, DNA consisting of the base sequence represented by SEQ ID NO: 1 and BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) etc. DNA having a sequence identity of at least 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more when calculated using the default parameters) DNA encoding a protein having an activity of cleaving and releasing an N-linked sugar chain and transferring the sugar chain is also included in the DNA encoding the endoglycosidase of the present invention.

  Furthermore, an RNA for the above DNA, or an RNA that can hybridize with the RNA under stringent conditions, is a protein having an activity of cleaving and releasing an N-linked sugar chain and transferring the sugar chain. Encoding RNAs are also included in the present invention.

  An endoglycosidase having an activity of cleaving and releasing an N-linked sugar chain of the glycoprotein of the present invention and transferring the sugar chain is a diacetylchitobiose (GlcNAc-GlcNAc) as a sugar chain that can be cleaved and transferred. A high mannose sugar chain having a structure in which an oligomer of mannose (Man) is bound to disaccharide, diacetylchitobiose, a complex sugar chain in which at least one of Man and GlcNAc, galactose (Gal), sialic acid (Neu5Ac) is bound, diacetyl There is a hybrid sugar chain having a sugar chain structure in which high mannose type and complex type are mixed with chitobiose. The structures of these sugar chains are shown in FIG. Examples of the high mannose type sugar chain include sugar chains called Man3 type, Man5 type, Man6 type, Man8 type and Man9 type in which 3, 5, 6, 8, and 9 mannoses are bound, respectively. . In addition, as a complex type sugar chain, for example, a sugar chain having a structure shown in FIG. There is.

  The endoglycosidase having the activity of cleaving and releasing the N-linked sugar chain of the glycoprotein of the present invention and transferring the sugar chain cleaves the sugar chain between diacetylchitobiose present at the root of the sugar chain. . The enzyme of the present invention reacts with any of a high mannose sugar chain, a hybrid sugar chain, and a complex sugar chain. For example, among the sugar chains shown in FIG. 1, the 4-chain type and Bisecting double-chain type sugar chains are not cleaved, but other sugar chains including complex type sugar chains, Man3 type, Man5 type, Man6 type, Man8 type. , Man9 type, Asialo double chain type, Sialo double chain type, Agalacto double chain type. Three-chain sugar chains are slightly cleaved.

  Endo A (Takegawa K. et al. Appl. Environ. Microbiol. 55, 3107-3112 (1989)), Endo F (Takegawa) have been known as endoglycosidase enzymes having endo-β-N-acetylglucosaminidase activity. K. et al., Eur. J. Biochem, 202, 175-180 (1991)), Endo H (Terentino AL et al. J. Biol. Chem. 274, 811 (1974), Endo M (Kadowaki K. et al., Agric, Biol. Chem. 1988, 54, 97) etc. These enzymes act only on sugar chains having a specific structure or have a strong effect on sugar chains having a specific structure. The enzyme of the present invention acts broadly and strongly on high mannose type sugar chains represented by Man3 type, Man5 type, Man6 type, Man8 type, Man9 type, and further, Asialo double chain type, Sialo double chain type, It also reacts with the complex type sugar chain represented by Agalacto double chain type and cleaves by hydrolysis, Fig. 2 shows the difference in substrate specificity between Endo-CC of the present invention and the above-mentioned End-M. The results are the results of examining the substrate specificity using pyridylaminated sugar chains to which various sugar chains are bound, as shown in Fig. 2. Endo-CC of the present invention is a Man3 type, Man5 type, Man6 type. , Asialo double chain type, Sialo double chain type, and Agalacto double chain type have higher reactivity (hydrolysis activity) than Endo-M, especially complex type sugar chains Asialo double chain type, Sialo double chain type and Agalacto double chain Since the reactivity to the chain type, particularly the reactivity to the Agalacto double chain type, is large, the Enodo-CC of the present invention can effectively cleave and transfer complex type sugar chains. The difference between sugar chains reactive to Man3 type, Man5 type, Man6 type and Man8 type is 3 times or more in Endo-M, but within 2 times in Endo-CC of the present invention. The Endo-CC of the present invention has a strong cleavage and transfer action on a wide range of sugar chains, and also has a Man8 type (Man8-GlcNAc2). When the reactivity with respect to 100 is set to 100, the reactivity of the Endo-CC of the present invention to Man3 type, Man5 type, Man6 type is 80 to 90 or more and 150 to 160 or less, whereas in End-M, It is 10-20 or more and 70-80 or less. In addition, when the reactivity to Man8 type is 100, the reactivity of the Endo-CC of the present invention to Man3 type, Man5 type, Man6 type, Man9 type is 40-50 or more and 150-160 or less. In End-M, it is 10-20 or more and 70-80 or less. Moreover, the reactivity with respect to Asialo double-stranded type, Sialo triple-stranded type, and Agalact double-stranded type is higher than Endo-M. For example, the reactivity of the Endo-CC of the present invention to the Agalacto duplex type is 10 or more, preferably 15 or more, more preferably 20 or more, when the reactivity to Man8 type (Man8-GlcNAc2) is 100. On the other hand, in End-M, it is 5 or less. This indicates that the Endo-CC of the present invention has a broader substrate specificity than End-M and performs sugar chain cleavage and sugar chain transfer on a wide variety of sugar chains.

2. An enzyme having only the activity of transferring a sugar chain without cleaving and releasing the N-linked sugar chain of the glycoprotein. The present invention further relates to the 180th asparagine of the amino acid sequence represented by SEQ ID NO: 2. An enzyme having an amino acid sequence in which an acid (N) is substituted with glutamine (Q), has no activity to cleave and release N-linked sugar chains of glycoproteins, and has only an activity to transfer sugar chains Including.

  The amino acid sequence of the enzyme having only the activity of transferring the sugar chain of the present invention is shown in SEQ ID NO: 4, and the base sequence of the DNA encoding the enzyme is shown in SEQ ID NO: 3. The base sequence shown in SEQ ID NO: 3 is a base sequence encoding Endo-CC shown in SEQ ID NO: 1, and the 538th to 540th AAC is mutated to CAA.

  The enzyme having only the activity of transferring a sugar chain without cleaving and releasing the N-linked sugar chain of the present invention, as long as the protein comprising the amino acid sequence has the activity of transferring the sugar chain, In the amino acid except amino acid sequence 180th glutamine (Q), mutation such as deletion, substitution, addition, etc. may occur in at least 1, preferably 1 or several amino acids.

  For example, at least one, preferably 1 or several (for example, 1 to 10, more preferably 1 to 5, more preferably 1 to 5) of amino acids other than the 180th glutamine (Q) in the amino acid sequence represented by SEQ ID NO: 4 1 or 2 amino acids may be deleted, and at least one, preferably 1 or several (for example, 1 to 10) of amino acids except the 180th glutamine (Q) in the amino acid sequence represented by SEQ ID NO: 4 , More preferably 1 to 5, particularly preferably 1 or 2, amino acids may be added, or at least amino acids other than the 180th glutamine (Q) of the amino acid sequence represented by SEQ ID NO: 4 One, preferably 1 or several (for example, 1 to 10, more preferably 1 to 5, particularly preferably 1 or 2) amino acids may be substituted with other amino acids.

  As an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 4 and the BLAST (Basic Local Alignment Search Tool at the National Center for Biological) Information (the US National Biological Information Center Basic Local Alignment Search Tool)) etc. (eg default or default parameters), at least 85% or more, preferably 90% or more, more preferably 95 %, Particularly preferably 97% or more of sequence identity.

  A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4 is substantially the same as a protein having the amino acid sequence of SEQ ID NO: 2.

A DNA that can hybridize under the following stringent conditions with a DNA consisting of a sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 3, wherein the base sequence is from the 538th to the 540th base sequence. Is a CAA, and does not have the activity of cleaving and releasing an N-linked sugar chain, and the DNA encoding a protein having only the activity of transferring a sugar chain is also included in the DNA encoding the enzyme of the present invention. Specifically, hybridization was performed at 68 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA was immobilized, and then a 0.1 to 2 fold concentration of SSC solution (1 fold concentration of SSC is 150 mM NaCl, 15 mM) This is a condition that can be identified by washing at 68 ° C. using sodium citrate. Alternatively, after transferring and immobilizing DNA on a nitrocellulose membrane by the Southern blotting method, hybridization buffer [50% formamide, 4 × SSC, 50 mM HEPES (pH 7.0), 10 × Denhardt ^, s) solution, 100 μg / ml salmon sperm DNA], which can form a hybrid by reacting overnight at 42 ° C.

  Also, DNA consisting of the base sequence represented by SEQ ID NO: 3 and BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) etc. At least 85%, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, from the 538th position. A DNA having the 540th base sequence of CAA, which encodes a protein having an enzyme that has no activity to cleave N-linked sugar chains and release sugar chains, but only to transfer sugar chains Such DNA is also included in the DNA encoding the enzyme of the present invention.

  Furthermore, RNA for the above DNA, or RNA that can hybridize with the RNA under stringent conditions, has no activity to cleave and release N-linked sugar chains, and activity to transfer sugar chains RNA that encodes a protein having an enzyme having only the phenotype is also included in the present invention.

  An enzyme that does not have the activity of cleaving and releasing the N-linked sugar chain of the glycoprotein of the present invention and has only the activity of transferring the sugar chain is referred to as modified Endo-CC. The modified Endo-CC has a transglycosylation activity of at least 5 times, preferably about 7 times that of wild-type Endo-CC.

  Modified Endo-CC can catalyze the transfer of sugar chains that can be cleaved and transferred by Endo-CC. The modified Endo-CC has sugar chain transfer activity such as the above-mentioned Man3 type, Man5 type, Man6 type, Man8 type, Man9 type, Asialo double chain type, Sialo double chain type, and Agalacto double chain type.

  Glycotransfer is a sugar chain cleaved using Endo-CC of the present invention, and a sugar chain having GlcNAc at the reducing end is converted to an oxazolineized sugar chain using an oxazolinating agent, and the oxazolineated sugar chain is used as a substrate. The sugar chain may be transferred to GlcNAc-protein by the modified End-CC of the present invention. Examples of the oxazoline agent include CDMBI (2-Chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride), DMC (2-Chloro-1,3-dimethyl-imidazolinium chloride) and the like.

3. Production of the enzyme of the present invention Endo-CC, an endoglycosidase having an activity of cleaving and releasing the N-linked sugar chain of the glycoprotein of the present invention and transferring the sugar chain, cultivates Coprinus cinereus, It can be produced and can be isolated from a culture such as a culture solution of Coprinus cinereus using a known method. An endoglycosidase having an activity of cleaving and releasing an N-linked sugar chain of the glycoprotein of the present invention and transferring the sugar chain introduces the DNA encoding the enzyme into a host microorganism, It can be produced as a recombinant enzyme by culturing. For example, an expression vector may be prepared by ligating (inserting) the DNA of the present invention into an appropriate vector, the expression vector is introduced into the host microorganism, and the host microorganism is transformed.

  Similarly, the modified Endo-CC, which is an enzyme that does not have the activity of cleaving and releasing the N-linked sugar chain of the glycoprotein of the present invention and has only the activity of transferring the sugar chain, encodes the enzyme. DNA can be produced as a recombinant enzyme by introducing the DNA into a host microorganism and culturing the microorganism. For example, an expression vector may be prepared by ligating (inserting) the DNA of the present invention into an appropriate vector, the expression vector is introduced into the host microorganism, and the host microorganism is transformed.

  The vector for inserting the DNA of the present invention is not particularly limited as long as it can replicate in a host cell such as a bacterium such as Escherichia coli, a yeast, or an animal cell, and examples thereof include plasmid DNA and phage DNA. . As the vector DNA used for the construction of the expression vector, a widely spread and easily available DNA is used. For example, pET vector, pQE vector, pCold vector, pUC19 vector and the like can be mentioned.

The method for constructing the expression vector of the present invention is not particularly limited, and can be performed by a conventional method.
The host cell transformed with the expression vector of the present invention is not particularly limited as long as it can express the DNA of the present invention. For example, Escherichia coli and Bacillus subtilis are used as bacteria, and Saccharomyces cerevisiae and the like are used as yeasts. However, examples of animal cells include Chinese hamster ovary (CHO) cells, monkey COS cells, mouse fibroblasts, and the like.

  At this time, it is preferably produced using E. coli. The conventionally known endo-β-N-acetylglucosaminidase becomes insoluble when trying to produce recombinant proteins using E. coli, and the active enzyme cannot be efficiently produced using E. coli. (Fujita et al., Archives of Biochemistry and Biophsics 432 (2004) 41-49), but the enzyme of the present invention does not cause the above-mentioned problems even if it is produced using E. coli. An enzyme (endoglycosidase) can be produced.

  The present invention provides an endoglycosidase having an activity of culturing host cells containing the above DNA under conditions capable of expressing the DNA, cleaving and releasing the N-linked sugar chain of the glycoprotein, and transferring the sugar chain. And a method for producing an endoglycosidase having an activity of cleaving and releasing an N-linked sugar chain possessed by the glycoprotein and transferring the sugar chain.

  Furthermore, the present invention provides a method for cultivating a host cell containing the above DNA under conditions capable of expressing the DNA, and has no activity to cleave and release the N-linked sugar chain possessed by the glycoprotein. A method for producing an enzyme, which comprises producing an enzyme having only the activity to be recovered and recovering the enzyme.

  Enzymes produced by host cells are known, for example, gel filtration chromatography, ultrafiltration, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, chromatofocusing, isoelectric focusing, gel electrophoresis, etc. These purification methods can be purified alone or in combination.

4). Use of the enzyme of the present invention The endoglycosidase Endo-CC of the present invention has an activity of cleaving and releasing an N-linked sugar chain of a glycoprotein. For example, by allowing the Endo-CC of the present invention to act on a uniform glycopeptide or glycoprotein, the sugar chain is cleaved and a uniform free sugar chain can be obtained.

  The obtained sugar chain is converted into an oxazolineized sugar chain by an oxazolinating agent, and has no activity of cleaving and releasing the N-linked sugar chain of the glycoprotein of the present invention, and only having an activity of transferring the sugar chain The modified Endo-CC can be transferred to GlcNAc-protein to produce a glycoprotein.

  By using Endo-CC and modified Endo-CC of the present invention, a glycoprotein having a uniform sugar chain can be produced by binding a sugar chain to GlcNAc-protein. The presence of sugar chains plays a very important role in the activity and pharmacokinetics of glycoprotein drugs such as antibody drugs. By using the Endo-CC and modified Endo-CC of the present invention, a uniform antibody drug Etc. can be produced.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Preparation of Enzyme Expression Vector Coprinus cinerea (Coprinopsis cinerea) from which the gene was isolated was purchased from ATCC (American Type Culture Collection) (ATCC No. MYA-4618).

  Coprinopsis cinerea was shake-cultured in 250 ml liquid medium (25 ° C., 100 rpm, 7 days) in Matsutake medium (Ebios tablets 5 g, Glucose 20 g, total 1.0 L). The culture broth was collected by filtration, and the cells were placed in a 2 ml microtube with a cap together with a metal cone and frozen at -80 ° C. for 1 hour. 1 ml of RNAiso was added to the frozen sample and crushed with a MULTI-BEADS SHOCKER manufactured by Yasui Kikai (1500 rpm, 10 sec). When disruption was complete, it was removed from the machine and incubated at room temperature for 5 minutes. During this time, the metal cone was taken out. 200 μl of chloroform was added to the sample, mixed by inverting 30 to 40 times, and then centrifuged (13200 g, 5 min). The supernatant was transferred to another tube, an equal volume of isopropanol was added and mixed by inversion, and then centrifuged (13200 g, 5 min).

  The supernatant was removed, 100 μl of 100% ethanol was added, and the mixture was centrifuged again (13200 g, 5 min). The supernatant was removed and the precipitate was dried at room temperature. 55 μl of DEPC-treated sterilized water was added to the dried sample to obtain an RNA solution.

  The resulting RNA solution was reverse-transcribed using ReverTra Ace qPCR RT Master Mix manufactured by TOYOBO to prepare cDNA.

Reaction conditions: 5 × RT Master Mix 2μl, RNA template 1μg, Nuclease-free Water, Total 10μl
Before the reverse transcription reaction, the absorbance of the RNA solution ABS260 was measured to determine the RNA concentration in advance, and the RNA solution was incubated at 65 ° C. for 5 minutes and then rapidly cooled on ice. The reverse transcription reaction was performed by incubation at 37 ° C., 15 min; 50 ° C., 5 min; 98 ° C., 5 min; 4 ° C., hold in this order.

  The Endo-CC gene was then amplified from the obtained cDNA. At that time, the primer was designed to have an Nde site on the N-terminal side and an Xho site on the C-terminal side. PrimeSTAR HS manufactured by Takara Bio Inc. was used as the DNA polymerase.

  The reaction was carried out by incubating at a temperature of 98 ° C., 10 sec; 61 ° C., 15 sec; 72 ° C., 3 min (30 cycles of this);

  In addition, inverse PCR was performed on the pET-23b vector starting from the Nde site of the multicloning site and ending at the Xho site, and the PCR product was obtained. This PCR also used PrimeSTAR HS manufactured by Takara Bio Inc. as a DNA polymerase.

  The reaction was carried out by incubating at a temperature of 98 ° C., 10 sec; 50 ° C., 15 sec; 72 ° C., 7 min (30 cycles of this);

  Finally, the Endo-CC gene fragment with the restriction enzyme site and the pET-23b vector obtained by inverse PCR were joined together using Clontech's In-Fusion HD Enzyme Premix.

Reaction conditions: Endo-CC gene PCR product 2 μl, pET-23b inverse PCR product 1 μl, In-Fusion Premix 0.5 μl, ddw 1.5 μl
After incubation at 50 ° C. for 15 minutes, E. coli XL1-Blue was transformed immediately after completion of the reaction to obtain an E. coli expression vector.

  The obtained Escherichia coli expression vector was introduced into Escherichia coli, and Escherichia coli was cultured to produce an enzyme (Endo-CC).

Example 2 Purification of recombinant enzyme
5 ml of MMI medium + Amp (ampicillin) + Cm (chloramphenicol) medium was inoculated and cultured at 37 ° C. for 12 hours. This culture solution was used as a preculture solution. Next, 250 ml of LB medium + Amp + Cm liquid medium was inoculated with the preculture solution so that OD600 = 0.05, and cultured at 15 ° C. for 48 hours.

  The resulting culture was collected by centrifugation at 6000 rpm for 7 minutes, the supernatant was discarded, and 5 ml of disruption buffer was added to suspend. The solution crushed by ultrasonic crushing (output = 5, Duty = 70, 10 times × 4 sets) was centrifuged at 15000 rpm for 10 minutes, and the supernatant was collected. The protein solution was purified using a His-Trap column.

Example 3 Examination of substrate specificity The substrate specificity was measured using the Endo-CC prepared in Example 2. At this time, Endo-M (Tokyo Chemical Industry Co., Ltd.) was used for comparison.

  The enzyme reaction was performed by adding 2 μl of 30 μg / ml enzyme to a solution in which 2 μl of 1 pM PA (pyridylaminated) sugar chain (Masuda Chemical Co., Ltd.), 1 M phosphate buffer pH = 6.0 μl, and 5 μl of ultrapure water were added. The reaction was carried out at 15 ° C. for 15 minutes. The sugar chains of PA sugar chains are Man3 type, Man5 type, Man6 type, Man8 type, Man9 type, Asialo double chain type, Sialo double chain type, triple chain type, Agalacto double chain type, quadruple chain type and Bisecting 2 It was a single chain type. The structures of these sugar chains are shown in FIG. After the reaction, the reaction was stopped by heating at 100 ° C. for 3 minutes to deactivate the enzyme. The obtained reaction solution was quantified for the enzyme product (PA-GlcNAc) by HPLC, and the degradation amount of each pyridylaminated sugar chain was measured. The HPLC conditions were the HPLC system of GL Science, the column was TOS-GEL Amide-80 manufactured by TOSOH, the flow rate was 0.5 ml / min, and 100% acetonitrile and 50 mM formate buffer (pH 4.5) were used. Elution by gradient. The fluorescence was analyzed using a fluorescence detector with excitation 320 nm and emission 400 nm.

  Figures 2-1 and 2-2 show the degradation of pyridylaminated sugar chains to which each sugar chain is bound, assuming that the amount of Man8-GlcNAc2-PA that is a pyridylaminated sugar chain to which Man8 type is bound is 100 Amount indicated. In FIG. 2A, it is represented by a bar graph, and in FIG. 2B, it is represented by a numerical value (relative value).

  As shown in FIGS. 2-1 and 2-2, Endo-M showed a strong hydrolysis activity for a specific small number of sugar chains, while the hydrolysis activity for other sugar chains was low. Endo-CC also had different hydrolytic activity depending on the sugar chain, but it had higher hydrolytic activity on more sugar chains than Endo-M, and its substrate specificity was broader than End-M.

Example 4 Glycosyltransferase activity of Endo-CC Endo-CC produced in Example 2 and SGP (sialylglycopeptide, top of FIG. 3) (Fushimi Pharmaceutical) and sugar chain acceptors GlcNAc and D-Glucose (glucose) SLC is hydrolyzed and SG (sialylated sugar chain: bottom of Fig. 3) is transferred to GlcNAc and D-Glcose to determine whether SG-GlcNAc and SG-D-Glucose are produced. (Merck Millipore silica gel 60) was confirmed. The structure of SGP and SG is shown in FIG.

  Endo-CC 5 μl, SGP 10 μg / ml, and 0.5 M GlcNAc or D-Glucose 4 μl were reacted in a final volume of 20 μL of 50 mM acetate buffer (pH 6.0). For GlcNAc, the reaction was monitored over time up to 75 minutes every 15 minutes, and for D-Glucose, the reaction after 3 hours was developed by TLC and examined.

From the left, TLC has Blank (BLANK reacted for 3 hours without adding GlcNAc or D-Glucose), 0 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, reacted with GlcNAc, Blank (BLANK was reacted for 3 hours without adding GlcNAc or D-Glucose), there was a spot reacted with D-Glucose for 3 hours.
The developing solvent was propanol; acetic acid; water = 3: 2: 2 and color was developed by the orcinol-sulfuric acid method.

The results are shown in FIG. The origin indicates SGP. Regarding GlcNAc, as time passes, an increase in hydrolyzate is observed, and at the same time, an increase in glycosylated product (SG-GlcNAc) is observed. Similarly for D-Glucose, hydrolyzate and glycosylated product (SG-D-Glucose) were confirmed after 3 hours.
This result indicates that the Endo-CC of the present invention has transglycosylation activity.

Example 5 Activity of Modified Endo-CC The transglycosylation activity of modified Endo-CC (N180Q strain) was measured.

(1) Preparation of oxazolineized SG Oxazolineated SG was performed according to the method of Noguchi et al. (Helvetica Chimica Acta, 2012).
SG 12.3 mg was dissolved in 0.5 M CDMBI 61 μl and cooled to 4 ° C. Thereafter, 61 μl of 1.5 M potassium phosphate buffer was added and stirred at 4 ° C. for 2 hours. After centrifugation at 4 ° C. and 14000 rpm for 15 minutes, the mixture was filtered through a 0.2 μm filter and subjected to high performance liquid chromatography under the following conditions to collect the fraction of interest.

Column: Inertsil ODS-3V (4.6 x 250 mm) GL Science solvent: 100% distilled water
Temperature: 30 ° C
Flow rate: 1.0 ml / min
Detector: UV (214 nm)
Equipment: Pump HITACHI L-2130
Column heater Sugaikemi U-620
UV GL Science UV702

  The obtained fraction was freeze-dried to obtain 6.8 mg of oxazolineated SG. The recovery rate was 55%.

(2) Confirmation of transglycosylation of wild-type and N180Q strain modified Endo-CC
Confirmation of the transglycosylation activity of Endo-CC was performed as follows. Final concentration 100 mM sodium phosphate buffer (pH 6.0), 10 mM oxazolinated SG, 4 mM acceptor molecule (p-nitrophenyl N-acetylglucosamine), and wild strain or N180Q strain Endo-CC enzyme solution (enzyme) The reaction solution (a total of 200 μl) containing 24.5 μg) was incubated at 30 ° C. for 3 hours, and heated at 99 ° C. for 5 minutes to stop the enzyme reaction. The total amount of the reaction solution was subjected to high performance liquid chromatography under the following conditions.

Column: Inertsil ODS-3V (4.6 x 250 mm) GL Science solvent:
Liquid A: 0.1% TFA
Liquid B: acetonitrile containing 0.1% TFA
Liquid A: Liquid B = 90:10, elution temperature: 30 ° C
Flow rate: 1.0 ml / min
Detector: UV (300 nm)
Equipment: Pump HITACHI L-2130
Column heater Sugaikemi U-620
UV GL Science UV702

  Fractions that appeared to be glycosyltransferases were collected, and mass spectrometry was performed by MALDI-TOFMS (autoflex II, Bruker Daltonics) to identify glycosyltransferases.

FIG. 6 shows the result of detecting the transfer activity of the modified Endo-CC. FIG. 5 shows a reaction diagram of the transglycosylation reaction. A peak (9.47 min) that appears to be a transglycosylation product was detected. As a result of fractionating this peak and performing MS analysis, a molecular ion peak corresponding to the predicted molecular weight of the glycosyltransferase was detected (m / z = 2366.683 [M + Na] ⁺ ). In addition, the peak area of the transfer reaction product showed that the N180Q strain modified Endo-CC had about 7 times the sugar chain transfer activity compared to the wild strain Endo-CC.

  Using the Endo-CC and modified Endo-CC of the present invention, a recombinant glycoprotein for therapeutic use such as an antibody drug having a uniform sugar chain can be produced. Furthermore, the Endo-CC and modified Endo-CC of the present invention can be used as reagents for studying sugar chains.

Claims (10)

The following (a) or (b) Coprinus cinereus-derived endoglycosidase having an activity of cleaving and releasing an N-linked sugar chain of a glycoprotein and transferring the sugar chain:
(a) an endoglycosidase consisting of the amino acid sequence represented by SEQ ID NO: 2;
(b) including an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and cleaving and releasing the N-linked sugar chain of the glycoprotein; And an endoglycosidase having an activity of transferring a sugar chain. The following (a) or (b) derived from Coprinus cinereus, a DNA encoding an endoglycosidase having an activity of cleaving and releasing an N-linked sugar chain of a glycoprotein and transferring the sugar chain:
(a) an endoglycosidase consisting of the amino acid sequence represented by SEQ ID NO: 2;
(b) including an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and cleaving and releasing the N-linked sugar chain of the glycoprotein; And an endoglycosidase having an activity of transferring a sugar chain. The following (c) or (d) derived from Coprinus cinereus, a DNA encoding an endoglycosidase having an activity of cleaving and releasing an N-linked sugar chain of a glycoprotein and transferring the sugar chain:
(c) DNA containing the nucleotide sequence represented by SEQ ID NO: 1
(d) hybridizing under stringent conditions with a DNA comprising a sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 1 to cleave and release the N-linked sugar chain of the glycoprotein; And a DNA encoding a protein having an activity of transferring a sugar chain. The following enzyme (e) or (f) derived from Coprinus cinereus, having no activity of cleaving and releasing an N-linked sugar chain of a glycoprotein and having only an activity of transferring a sugar chain:
(e) an enzyme consisting of the amino acid sequence represented by SEQ ID NO: 4;
(f) an N-linked sugar chain comprising an amino acid sequence in which one or several amino acids other than the 180th glutamine in the amino acid sequence represented by SEQ ID NO: 4 are deleted, substituted or added, and possessed by a glycoprotein An enzyme having only the activity of transferring sugar chains without having the activity of cleaving and releasing. The following (e) or (f) derived from Coprinus cinereus, which encodes an enzyme that does not have the activity of cleaving and releasing the N-linked sugar chain possessed by the glycoprotein but has only the activity of transferring the sugar chain DNA to:
(e) an enzyme consisting of the amino acid sequence represented by SEQ ID NO: 4;
(f) an N-linked sugar chain comprising an amino acid sequence in which one or several amino acids other than the 180th glutamine in the amino acid sequence represented by SEQ ID NO: 4 are deleted, substituted or added, and possessed by a glycoprotein An enzyme having only the activity of transferring sugar chains without having the activity of cleaving and releasing. The following (g) or (h) derived from Coprinus cinereus, which encodes an enzyme that does not have the activity of cleaving and releasing the N-linked sugar chain of the glycoprotein but only has the activity of transferring the sugar chain DNA to:
(g) DNA containing the base sequence represented by SEQ ID NO: 3
(h) hybridizes under stringent conditions with a DNA comprising a sequence complementary to the DNA comprising the nucleotide sequence represented by SEQ ID NO: 3, wherein the 538th to 540th nucleotide sequence is CAA, and is a glycoprotein DNA encoding an enzyme that has no activity to cleave and release N-linked sugar chains, but only has an activity to transfer sugar chains.   An N-linked sugar chain derived from Coprinus cinereus according to claim 1, which comprises introducing the expression vector containing the DNA according to claim 2 or 3 into E. coli and culturing the E. coli. A method for producing an endoglycosidase having an activity of cleaving and releasing and transferring a sugar chain.   An N-linked sugar chain derived from Coprinus cinereus according to claim 4, which comprises introducing the expression vector containing the DNA according to claim 5 or 6 into E. coli and culturing the E. coli. A method for producing an enzyme having only the activity of transferring sugar chains without having the activity of cleaving and releasing.   A method for producing a free sugar chain, comprising contacting the endoglycosidase according to claim 1 with a glycoprotein, cleaving the sugar chain to obtain a free sugar chain.   A method for producing a glycoprotein, comprising bringing the enzyme according to claim 4 into contact with a protein and an oxazolinized sugar chain, and transferring the sugar chain to the protein.

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