JP2020022440A - Method for separating composite type sugar chain - Google Patents

Abstract

To provide a method for separating in a non-deformation condition, from a sugar protein, sugar peptide, or sugar chain, a composite type sugar chain comprising a two chain composite type sugar chain, a multi branched composite type sugar chain having three or more chains, or bisecting GlcNAc structure.SOLUTION: There is provided a method for separating a composite type sugar chain from sugar protein, sugar peptide or sugar chain, in which the composite type sugar chain is separated in a non-deformation condition using an endoglycosidase for recognizing difference of sugar on a non-reduction terminal side of the sugar chain. As the endoglycosidase, Endo-Tsp 1263 or Endo-Bno 1263 is used for separating the composite type sugar chain in which N-acetylglucosamine exists on the non-reduction terminal of the sugar chain, and Endo-Tsp 1006 or Endo-Bac 1008 is used for separating the composite type sugar chain in which α2,6 sialic acid or galactose exists on the non-reduction terminal of the sugar chain, thereby selectively separating the composite type sugar chain.SELECTED DRAWING: Figure 9

Description

  The present invention relates to a method for releasing a complex-type sugar chain from a glycoprotein, a glycopeptide, or a sugar chain under non-denaturing conditions, and a method for producing a glycoprotein, a glycopeptide, or a sugar chain in which the sugar chain has been cleaved. The N-linked sugar chain has a mannose (Man) linked to a diacetylchitobiose (GlcNAc-GlcNAc) unit on the reducing end side by β1-4 binding, and a mannose in a binding mode of α1-3 and α1-6. Are bound to form a core pentasaccharide. In the complex type sugar chain described below, N-acetylglucosamine (GlcNAc) is bound to both of these mannoses in a β1-2 binding manner. The multi-branched complex sugar chain of three or more chains refers to β1-4 or β1-6 in addition to N-acetylglucosamine bound to either or both of the above-mentioned mannoses in a β1-2 binding mode. Or a sugar chain to which N-acetylglucosamine is bound in both binding modes. The bisecting GlcNAc is N-acetylglucosamine bound to mannose bound to diacetylchitobiose unit by β1-4 in the binding mode of β1-4, and the sugar chain containing this is the bisecting GlcNAc sugar chain. . The non-denaturing condition is a condition in which a protein retains its original three-dimensional structure without adding a protein denaturant such as a surfactant, urea, and guanidine hydrochloride.

  The sugar chains of glycoproteins include an O-linked sugar chain in which a sugar chain is bonded via a hydroxyl group in a side chain portion of a serine or threonine residue and an N-linked sugar chain in which a sugar chain is bonded via an amide group of an asparagine residue. There is a sugar chain. N-linked sugar chains include high mannose sugar chains (sugar chains in which a mannose oligomer is linked to diacetylchitobiose); complex sugar chains (mannose and N-acetylglucosamine, galactose (Galactose to diacetylchitobiose). ), Sugar chains to which at least one of sialic acid (NeuAc) is bonded); and hybrid sugar chains (sugar chains in which high mannose sugar chains and complex sugar chains are mixed with diacetylchitobiose). It is roughly divided. Various types of sugar chains may be present depending on differences in constituent sugars, chain lengths, bonding modes, and the like.

  The bisecting GlcNAc sugar chain is formed in humans and mice by an enzyme called GnT-III, which is a kind of N-acetylglucosamine transferase. In addition, three or more multi-branched complex-type sugar chains are formed by enzymes such as GnT-IV, GnT-V, GnT-IX, which are a kind of N-acetylglucosamine transferase in humans and mice, and GnT-VI in chickens. Formed (FIG. 1). As used herein, the term “multi-branched sugar chain” means a complex sugar chain having three or more chains and a complex sugar chain having two or more bisecting GlcNAc.

  When bisecting GlcNAc is introduced into the N-linked sugar chain, the reaction of enzymes such as α-mannosidase II and N-acetylglucosamine transferase such as GnT-II, GnT-IV and GnT-V is inhibited, and Inhibition of formation and reduction of side chain elongation occur (Non-Patent Document 1). In cancer cells, when GlcNAc added to the N-linked sugar chain exposed on the cell surface by GnT-V is introduced by β1,6 bond, the metastatic ability increases. However, the overexpression of GnT-III causes the detecting GlcNAc to be detected. It has been reported that the inhibition of GlcNAc transfer by GnT-V suppresses the transfer of cancer cells (Non-patent Document 2). In addition, BACE1 protease involved in the production of amyloid β, one of the causative substances of Alzheimer's disease, has undergone modification of the bisecting GlcNAc sugar chain, and when this sugar chain modification is deleted, the intracellular localization of BACE1 protease changes. Therefore, there is a report that production of amyloid β is suppressed (Non-Patent Document 3).

  In order to analyze the structure of an N-linked sugar chain and prepare a sugar chain sample from a glycoprotein, it is necessary to release the sugar chain from the glycoprotein. As a chemical method for releasing a sugar chain from a glycoprotein, there is a method using a substance having a hydrazide group that specifically binds to an aldehyde group of the sugar chain. However, in this method, a protein portion is decomposed, and Deacetylation and epimerization in which the structure of the released sugar chain is partially changed occur (Patent Document 1).

  On the other hand, there is also a method of releasing a sugar chain from a glycoprotein using an enzyme such as Peptide-N-Glycosidase F (PNGase F) or Endo-β-N-Acetylglucosaminodase (ENGase). PNGase F specifically cleaves a binding site (GlcNAc-Asn bond) between an N-linked sugar chain (high mannose type sugar chain, mixed type sugar chain, complex type sugar chain) and a protein regardless of the structure of the sugar chain. I do. At this time, the Asn residue to which the sugar chain has been bound is converted into Asp, so that the amino acid sequence of the protein may change, which may affect the function of the protein. In addition, in the case of sugar chain cleavage by PNGase F, the protein may be denatured in order to efficiently remove the sugar chain, and thus may not be suitable for analyzing the function of the protein after sugar chain removal.

  ENGase specifically cleaves between diacetylchitobiose units on the reducing end side of the N-linked sugar chain, and this cleavage reaction depends on the substrate specificity of each ENGase. For example, EndoH derived from Streptomyces plicatus is an ENGase that can cleave high-mannose-type sugar chains and some mixed-type sugar chains, but cannot cleave complex-type sugar chains. After the cleavage reaction, GlcNAc of one residue remains at the Asn residue to which the sugar chain was bound, so that the conversion of the Asn residue to Asp observed during the sugar chain cleavage reaction by PNGase F did not occur. The amino acid sequence of the protein does not change. In addition, in the sugar chain cleavage by ENGase, sugar chains can be efficiently removed in many cases without denaturing the protein, and the functional analysis of the protein after sugar chain removal is possible.

  In recent years, sugar chain remodeling methods for replacing heterogeneous sugar chains bound to glycoproteins with sugar chains having a uniform structure have been developed (Non-Patent Documents 4, 5 and Patent Document 2). In this method, first, a sugar chain of a glycoprotein is cleaved with ENGase, and a uniform glycoprotein having only a GlcNAc residue (which may be bound with core fucose) and having no variation in sugar chain structure is obtained. Acceptor molecule. Next, using an ENGase mutant (glycosynthase) that suppresses the sugar chain cleavage activity of ENGase and exhibits glycosyltransferase activity, a uniform sugar chain prepared by chemical synthesis or the like is used to form a GlcNAc residue on the acceptor molecule. Transfer to Then, a glycoprotein having a uniform sugar chain structure can be prepared.

  As described above, the bisecting GlcNAc sugar chain has various roles, and various glycoproteins containing the sugar chain exist. In addition, there are various types of glycoproteins containing three or more multi-branched sugar chains, and the multi-branched sugar chains have characteristics such as a large occupied region and multivalent binding, and these are functions of the glycoprotein. , Structure, stability, and interaction via sugar chains. When releasing these sugar chains from the glycoprotein, PNGase F can be used, but in many cases, it is necessary to denature the glycoprotein. Further, in the protein from which the sugar chain has been removed using PNGase F, GlcNAc does not remain on the Asn residue which is a sugar chain addition site (which is converted into Asp after sugar chain cleavage). Not available as an acceptor molecule for Glycoproteins.

  On the other hand, in the existing ENGase, an enzyme capable of efficiently releasing a bisecting GlcNAc sugar chain from various glycoproteins has not been found. In addition, in the existing ENGase, an enzyme having an activity of releasing some hyperbranched sugar chains from a glycoprotein is present, but the structure of the sugar chain that can be released is limited by its substrate specificity. . Therefore, in the prior art, from various glycoproteins including bisecting GlcNAc sugar chains and hyperbranched sugar chains, it is possible to efficiently release the sugar chains without denaturing the protein, and to use the sugar chain remodeling method. It has been difficult to prepare acceptor molecules for glycoproteins.

JP 2009-142238 A International Patent Publication No. WO2013 / 120066

Ellen W. Easton et al. , J. et al. Biol. Chem. 266, 21742-21680 (1991) Masafumi Yoshimura et al. , Proc. Natl. Acad. Sci. USA 92, 8754-8758 (1995). Yasushiko Kizuka et al. , EMBO Molecular Medicine 7, 175-189 (2015) Wei Huang et al. , J. et al. Am. Chem. Soc. 134, 12308-12318 (2012) Masaki Kurogochi et al. , PLoS One 10, e0132848 (2015) Daotian Fu et al. , Carbohydrate Res. 261, 173-186 (1994) Michael J. et al. Treuheit et al. , Biochem. J. 283, 105-112 (1992) Shinji Hashimoto et al. , Cancer 101, 2825-2836 (2004). Krista Y. White et al. , J. et al. Proteome Res. 8, 620-630 (2009) Katsuko Yamashita et al. , J. et al. Biol. Chem. 258, 3099-3106 (1983) Midori Umekawa et al. , J. et al. Biol. Chem. 285, 511-521 (2010)

  The present invention has been made in view of the above background art, and its object is to solve the above-mentioned problems and to provide a complex double-chain sugar chain, or a multi-branched complex sugar chain of three or more chains, or a bisecting GlcNAc. An object of the present invention is to provide a method for releasing a complex type sugar chain containing a structure from a glycoprotein, glycopeptide or sugar chain containing the sugar chain using an endoglycosidase under non-denaturing conditions.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, surprisingly, endo-β-N-acetylglucosaminidase Endo-Tsp1006 derived from Tannerella bacterium and endobacterium derived from Muribaculum bacterium were surprisingly obtained. Endo-Bac1008 which is -β-N-acetylglucosaminidase is a double-stranded complex sugar chain having α2,6 sialic acid or galactose at the non-reducing end of the sugar chain, or a multi-branched complex sugar having three or more chains It has been found that a complex type sugar chain containing a chain or a bisecting GlcNAc structure has an activity to be released from a glycoprotein under non-denaturing conditions. Furthermore, Endo-Tsp1263 which is an endo-β-N-acetylglucosaminidase derived from a bacterium belonging to the genus Tannerella and Endo-Bno1263 which is an endo-β-N-acetylglucosaminidase derived from a bacterium belonging to the genus Bacteroides have N- at the non-reducing end of the sugar chain. The activity of releasing a complex double-chain sugar chain in which acetylglucosamine is present, a multi-chain complex sugar chain having three or more chains, or a complex sugar chain containing a bisecting GlcNAc structure from a glycoprotein under non-denaturing conditions. And found that the present invention was completed.

In the present invention, Endo-Tsp1006, which is an endo-β-N-acetylglucosaminidase derived from a bacterium of the genus Tannerella, is a recombinant protein of the following (a), (b) or (c).
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 3 in the Sequence Listing;
(B) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 3 in the Sequence Listing, and having Endo-Tsp1006 enzyme activity;
(C) A protein comprising an amino acid sequence having 90% or more homology with the amino acid sequence described in SEQ ID NO: 3 in the Sequence Listing, and having an Endo-Tsp1006 enzyme activity.

In the present invention, Endo-Bac1008, which is endo-β-N-acetylglucosaminidase derived from a bacterium of the genus Muribaculum, is a recombinant protein of the following (a), (b) or (c).
(A) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 7 in the sequence listing,
(B) A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 7 in the Sequence Listing, and having Endo-Bac1008 enzyme activity.
(C) a protein consisting of an amino acid sequence having 90% or more homology with the amino acid sequence described in SEQ ID NO: 7 in the Sequence Listing, and having an Endo-Bac1008 enzyme activity;

In the present invention, Endo-Tsp1263, which is an endo-β-N-acetylglucosaminidase derived from a bacterium of the genus Tannerella, is a recombinant protein of the following (a), (b) or (c).
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in the Sequence Listing;
(B) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 in the Sequence Listing, and having Endo-Tsp1263 enzyme activity;
(C) a protein consisting of an amino acid sequence having 90% or more homology with the amino acid sequence described in SEQ ID NO: 1 in the Sequence Listing, and having an Endo-Tsp1263 enzyme activity;

In the present invention, Endo-Bno1263, which is an endo-β-N-acetylglucosaminidase derived from Bacteroides bacteria, is a recombinant protein of the following (a), (b) or (c).
(A) a protein comprising the amino acid sequence of SEQ ID NO: 9;
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 9 in the Sequence Listing, and having Endo-Bno1263 enzyme activity;
(C) a protein consisting of an amino acid sequence having 90% or more homology with the amino acid sequence described in SEQ ID NO: 9 in the Sequence Listing, and having Endo-Bno1263 enzyme activity;

  The complex type sugar chain in which α2,6 sialic acid, galactose, or N-acetylglucosamine is mixed at the non-reducing end of the sugar chain also has an Endo-Tsp1006, depending on the number of each sugar chain component, the bonding position, the bonding mode and the like. Endo-Bac1008, Endo-Tsp1263, or Endo-Bno1263, or all of these enzymes, or by using these enzymes in combination, is considered to be cleaved.

  That is, the present invention relates to a glycoprotein containing a double-stranded complex-type sugar chain, or a complex sugar chain having three or more multi-branched complex-type sugar chains or a bisecting GlcNAc structure under non-denaturing conditions. Endo-Tsp1006, Endo-Bac1008, Endo-Tsp1263 or Endo-Bsp1263, which are endoglycosidases that recognize the difference in the sugar at the non-reducing terminal side from a glycopeptide or a sugar chain, or a combination of these enzymes The present invention provides a method of releasing by use.

  The present invention also relates to Endo-Tsp1457 or Endo-Tsp1603, which is an endo-β-N-acetylglucosaminidase derived from a bacterium belonging to the genus Tannerella having an activity of cleaving a high mannose-type sugar chain, Endo-Tsp1003, Endo-Tsp1006, Endo-Bac1008, and Endo. A method for releasing a sugar chain having a specific structure from a glycoprotein, a glycopeptide, or a sugar chain under non-denaturing conditions by using a combination of an endoglycosidase such as Tsp1263, and the cleavage of a sugar chain having a specific structure in such a manner. It is intended to provide a method for producing a glycoprotein, a glycopeptide or a sugar chain, or a method for producing a glycoprotein, a glycopeptide or a sugar chain in which only a sugar chain having a specific structure remains.

  According to the present invention, the above-mentioned problems are solved, and a simple step of enzymatic treatment without denaturing a sample enables efficient and low-cost production of a sugar chain having a specific structure from a glycoprotein, glycopeptide, or sugar chain. Can be released. The sugar chains thus released can be used as a sample for sugar chain structure analysis or as a raw material for a sugar chain having a specific structure. Furthermore, glycoproteins and glycopeptides whose sugar chains have been cleaved can be used as acceptor substrates for sugar chain remodeling methods using ENGase and glycosynthase.

1 shows biosynthetic pathways of various branched N-acetylglucosamine transferases of a hyperbranched sugar chain including a representative bisecting GlcNAc sugar chain (GOB sugar chain). The migration pattern by SDS-PAGE of each purified enzyme of Endo-Tsp1603, Endo-Tsp1407, Endo-Tsp1263, Endo-Tsp1006, Endo-Bac1008 and Endo-Bno1263 is shown. The results of performing a sugar chain cleavage reaction using various glycoproteins as substrates using purified enzymes and subjecting the reaction products to SDS-PAGE are shown. The protein in which the sugar chain has been cleaved is indicated by "Deglycosylated". The result of performing a sugar chain cleavage reaction using ovomucoid or ribonuclease B (RNase B) as a substrate using a purified enzyme and subjecting the reaction product to SDS-PAGE is shown. The protein in which the sugar chain has been cleaved is indicated by "Deglycosylated". It is the figure which showed the result of the mass spectrometry analysis of the sugar chain of RNase B cleaved by Endo-Tsp1457. It is the figure which showed the result of the mass spectrometry analysis of the sugar chain of ovomucoid cleaved by Endo-Tsp1263. It is the figure which showed the result of the mass spectrometry analysis of the sugar chain of (alpha) 1-acid glycoprotein ((alpha) 1-AGP) cleaved by Endo-Tsp1006. It is the figure which showed the result of the mass spectrometry analysis of the sugar chain of (alpha) 1-AGP cleaved by Endo-Bac1008. A method for preparing various glycopeptides used for examining the substrate specificity of Endo-Tsp1263, Endo-Tsp1006 and Endo-Bac1008 will be described. It is a figure which summarized Endo-Tsp1263, Endo-Tsp1006 and Endo-Bac1008 cleavage activity with respect to various glycopeptides. Using RNase B, Prostate specific antigen (PSA), ovomucoid, and α1-AGP as substrates, sugar chain cleavage reactions with various ENGases were performed, and the results of subjecting the reaction products to SDS-PAGE are shown. The protein in which the sugar chain has been cleaved is indicated by "Deglycosylated". An asterisk (*) is attached to the band derived from the enzyme solution. Using ovomucoid and galactosylated ovomucoid as substrates, a sugar chain cleavage reaction was carried out by each enzyme of Endo-Tsp1263, Endo-Tsp1006 and Endo-Bac1008, and the result of subjecting the reaction product to SDS-PAGE is shown. The protein in which the sugar chain has been cleaved is indicated by "Deglycosylated". A mixed solution of RNase B, α1-AGP, and ovomucoid was reacted with each of Endo-Tsp1407, Endo-Tsp1263, Endo-Tsp1006 and Endo-Bac1008 enzymes alone or in combination to carry out a sugar chain cleavage reaction. The result of subjecting the reaction product to SDS-PAGE is shown. The protein in which the sugar chain has been cleaved is indicated by "Deglycosylated". It is the figure which put together the result of having measured the transglycosylation activity of the wild-type Endo-Tsp1006 and the Endo-Tsp1006 N220Q mutant.

  Hereinafter, embodiments and an implementation method of the present invention will be described in detail. However, the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the scope of the technical idea.

  FIG. 1 shows a multi-branched sugar chain containing a representative bisecting GlcNAc sugar chain (G0B sugar chain). In the present specification, the term “bisecting GlcNAc sugar chain” refers to a G0B-type sugar chain further containing galactose or sialic acid. In the present specification, the term “multi-branched sugar chain” refers to various sugar chains shown in FIG. 1 and further includes N-acetylglucosamine, N-acetylgalactosamine (GalNAc), and galactose. , Fucose, and sugar chains modified with sialic acid and the like. Regarding these sugar chains, differences in constituent sugars, chain lengths, and bonding modes are not limited with respect to the entire sugar chain structure unless otherwise specified.

  The method of the present invention for releasing a complex type glycan containing a multi-branched glycan under non-denaturing conditions comprises the steps of: The sugar chain is released using an endoglycosidase that recognizes the difference between In the present invention, among endoglycosidases, Endo-β-N-Acetylglucosaminodase (ENGase) classified into enzyme number EC 3.2.1.96 is used. Preferably, the ENGase used in the present invention is an enzyme derived from a microorganism. The microorganism is not particularly limited, but includes the genus Tannerella, the genus Prevotella, the genus Bacteroides, the genus Muribaculum, the genus Copropacter, the genus Alloprevotella, the genus Porphyromonadiasaum, the genus Barnesiula, the genus Barneciura, the genus Barunibacteria, the genus Municipalum, the genus Baruibacterium Microorganisms that live in the oral cavity and intestine of vertebrates such as Bacillus, Lactobacillus, Paenibacillus, and Virgibacillus are preferred, and Tannerella, Bacteroides, and Muribaculum bacteria are particularly preferred.

  Examples of ENGase derived from a bacterium of the genus Tannerella include an enzyme having the amino acid sequence of SEQ ID NO: 1, 3, 5, or 11, which is encoded by a gene having the nucleotide sequence of SEQ ID NO: 2, 4, 6, or 12, respectively. Examples of ENGase derived from bacteria of the genus Muribaculum include an enzyme having the amino acid sequence of SEQ ID NO: 7 encoded by a gene having the nucleotide sequence of SEQ ID NO: 8. Examples of ENGase derived from Bacteroides genus bacteria include an enzyme having the amino acid sequence of SEQ ID NO: 9 encoded by a gene having the nucleotide sequence of SEQ ID NO: 10. In the present invention, the enzyme having the amino acid sequence of SEQ ID NO: 1 is called Endo-Tsp1263. The enzyme having the amino acid sequence of SEQ ID NO: 3 is called Endo-Tsp1006. Further, the enzyme having the amino acid sequence of SEQ ID NO: 5 is called Endo-Tsp1457. The enzyme having the amino acid sequence of SEQ ID NO: 11 is called Endo-Tsp1603. Further, an enzyme having the amino acid sequence of SEQ ID NO: 7 is called Endo-Bac1008. The enzyme having the amino acid sequence of SEQ ID NO: 9 is called Endo-Bno1263. These enzymes have a total amino acid sequence of 1263, 1006, 1457, 1603, 1008 and 1263 residues, respectively.

  Alternatively, among the ENGases used in the present invention, ENGase used for releasing a complex type sugar chain having α2,6 sialic acid or galactose at the non-reducing terminal of the sugar chain from the glycoprotein, glycopeptide or sugar chain is Has an amino acid sequence having at least 50%, preferably at least 60%, more preferably at least 70% homology with any one of the amino acid sequences of SEQ ID NO: 3 or SEQ ID NO: 7, and has a total amino acid length of 1000 It is an enzyme having a motif called an NxE motif in the active site, which belongs to the GH85 family according to the classification of CAZy (Carbohydrate-Active enZymes Database; www.caszy.org) database and has about amino acid residues. Here, "homology" means homology calculated using BLAST analysis provided by the National Center for Biotechnology Information (NCBI) in the United States with standard settings.

  Further, among the ENGases used in the present invention, ENGase used for releasing a complex type sugar chain having N-acetylglucosamine at the non-reducing end of the sugar chain from the glycoprotein, glycopeptide, or sugar chain is SEQ ID NO: Has an amino acid sequence having homology of at least 50%, preferably at least 60%, more preferably at least 70% with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, and has a total amino acid length of about 1200 to 1300 amino acid residues. And an enzyme having a motif called an NxE motif in the active site, which belongs to the GH85 family according to the classification of the CAZy database.

  ENGase used in the present invention has an activity of releasing a sugar chain of a glycoprotein or a glycopeptide. Endo-Tsp1263 and Endo-Bno1263 are particularly preferred as enzymes used when releasing complex-type sugar chains including hyperbranched sugar chains having N-acetylglucosamine at the non-reducing terminal of the sugar chains under non-denaturing conditions. Enzymes used for releasing a complex type sugar chain containing a hyperbranched sugar chain having α2,6 sialic acid or galactose at the non-reducing end of the sugar chain under non-denaturing conditions include Endo-Tsp1006 and Endo- Bac1008 is particularly preferred, and Endo-Tsp1457 and Endo-Tsp1603 are particularly preferred as enzymes used for releasing a high-mannose type sugar chain under non-denaturing conditions, but other enzymes having the same activity may be used. Endoglycosidase may be used.

  Examples of such other endoglycosidases include ENGase derived from microorganisms and animals and plants, enzymes obtained by modifying the enzyme by genetic recombination technology, and enzymes obtained by adding various tags to the enzyme. Are all included in the scope of the present invention.

  Enzymes modified by genetic recombination techniques include enzymes that have modified a part of the amino acid sequence of the wild-type enzyme to improve the function and stability of the wild-type enzyme, and those other than glycosylation activities such as glycosyltransferase activity. An enzyme having the activity of (1) is included. Among them, the enzyme imparted with glycosyltransferase activity is particularly called glycosynthase.

  The enzyme used in the present invention is an enzyme purified from a culture supernatant or cells of a microorganism producing the enzyme by a combination of a protein extraction method, a fractionation method, and various chromatographic operations used in a general enzyme purification method. The enzyme may be used, or an enzyme similarly purified from a culture obtained by culturing a host cell transformed with a recombinant vector containing a gene encoding the enzyme may be used.

  Various chromatography operations include anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) sepharose, cation exchange chromatography using a resin such as sulfopropyl (SP) sepharose, butyl sepharose, phenyl sepharose Such as hydrophobic chromatography using resin, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, etc. Alternatively, they can be used in combination to obtain a final purified enzyme preparation.

  By using the enzyme thus purified alone or in combination, a sugar chain having a specific structure including a multi-branched sugar chain can be released from a glycoprotein or glycopeptide under non-denaturing conditions. If the released sugar chains are collected and fractionated and purified, they can be used as raw materials for sugar chains having various structures including hyperbranched sugar chains. Furthermore, glycoproteins and glycopeptides in which sugar chains having a specific structure are released from glycoproteins and glycopeptides under non-denaturing conditions, or glycoproteins and glycopeptides in which all sugar chains are released, are subjected to various chromatographic operations described above to obtain purified samples. It can be used as an acceptor substrate for the sugar chain remodeling method.

  The present invention will be described in more detail with reference to examples described below, but the present invention is not limited to these examples unless it exceeds the gist.

<Example 1> Method for preparing endoglycosidase which releases complex-type sugar chains containing hyperbranched sugar chains from glycoproteins under non-denaturing conditions and measurement of its activity bisecting GlcNAc sugar chain is an enzyme called GnT-III in humans and mice. Is a sugar chain having a characteristic structure as shown in FIG. 1 and present on various glycoproteins. In recent years, a sugar chain remodeling method utilizing ENGase or a mutant thereof to replace a glycoprotein having a heterogeneous sugar chain with a glycoprotein having a uniform sugar chain has been developed (Non Patent Literatures 4, 5 and 5). In order to apply this method 2), it has become necessary to release various sugar chains from glycoproteins under non-denaturing conditions. On the other hand, there has been no report on ENGase which can efficiently release a bisecting GlcNAc sugar chain from various glycoproteins, and it has been difficult to release the sugar chain from various glycoproteins under non-denaturing conditions. In order to overcome this problem, the present invention has been working on a search for ENGase that can efficiently release a bisecting GlcNAc sugar chain from various glycoproteins.

  First, CAZy which is a database of carbohydrate-related enzymes, UniProt (www.uniprot.org) which is a database of various proteins, and a database (http: //www.ncbi.nim.nih.) Of the National Center for Biotechnology Information (NCBI) in the United States. .Gov) and the like, three enzyme groups formed by microorganism-derived ENGase belonging to the GH85 family in the classification of the CAZy database were found.

  Group 1 belongs to an enzyme having about 1000 amino acids, group 2 belongs to an enzyme having about 1250 amino acids, and group 3 belongs to an enzyme having 1300 or more amino acids. Belonged. In order to examine the substrate specificity of the enzymes belonging to each group, from group 1, an enzyme of 1006 amino acid residues derived from bacteria belonging to the genus Tannerella (SEQ ID NO: 3, Endo-Tsp1006) and an enzyme having 1008 amino acid residues derived from the bacteria belonging to the genus Muribaculum ( SEQ ID NO: 7, Endo-Bac1008), an enzyme of 1263 amino acid residues derived from bacteria belonging to the genus Tannerella (SEQ ID NO: 1, Endo-Tsp1263) and an enzyme having 1263 amino acid residues derived from bacteria belonging to the genus Bacteroides (SEQ ID NO: 9, Endo-Bno 1263), an enzyme of 1457 amino acid residues derived from bacteria belonging to the genus Tannerella (SEQ ID NO: 5, Endo-Tsp1457) and an enzyme of 1603 amino acid residues (SEQ ID NO: 11, Endo-T p1603) I was to be analyzed for.

  Endo-Tsp1006 gene (SEQ ID NO: 4), Endo-Bac1008 gene (SEQ ID NO: 8), Endo-Tsp1263 gene (SEQ ID NO: 2), Endo-Bno1263 gene (SEQ ID NO: 10), Endo-Tsp1457 gene (SEQ ID NO: 10) SEQ ID NO: 6) and Endo-Tsp1603 gene (SEQ ID NO: 12) were artificially synthesized, respectively, inserted into pGEX6P-1 vector (GE Healthcare), and each enzyme was ligated with glutathione S-transferase (GST). An expression vector for expression in Escherichia coli as a fusion protein was constructed. At this time, a tag sequence consisting of histidine 6 residues was added to the C-terminal of each enzyme.

  Escherichia coli BL21 (DE3) strain was transformed using each expression vector, and these transformants were cultured at 37 ° C. for 3 hours in 100 mL LB medium supplemented with 50 μg / mL ampicillin. Next, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a concentration of 0.1 mM, and the cells were cultured at 25 ° C. for 3 hours. Thereafter, the cells were collected by centrifugation, and 5 mL of phosphate buffered saline (PBS) was added thereto to suspend the cells, and the cells were crushed by an ultrasonic crusher. Triton X-100, a surfactant, was added to the mixture so as to have a concentration of 1%. After shaking at room temperature for 30 minutes, the supernatant was collected by centrifugation. An appropriate amount of glutathione sepharose (GE Healthcare) was added to the supernatant, followed by stirring at 4 ° C. for 16 hours to adsorb the GST fusion protein to glutathione sepharose.

  After collecting the above glutathione sepharose by centrifugation, it is sufficiently washed with PBS, and 0.6 mL of a reaction solution for PreScission Protease (GE Healthcare) (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7). .0), and 2 units (1 μL) of PreScission Protease were added thereto and reacted at 4 ° C. for 16 hours. This reaction cleaves and removes GST from the GST fusion protein. In addition, PreScission Protease added to the reaction solution is adsorbed on glutathione sepharose, and can be removed together with glutathione sepharose by centrifugation.

  After GST was cleaved and removed from the GST fusion protein by the above reaction, the reaction solution was recovered by centrifugation. The reaction solution was ultrafiltrated and concentrated using VIVASPIN500 (fraction molecular weight: 3,000, Sartorius) to obtain a purified enzyme preparation. FIG. 2 shows the migration pattern of each purified enzyme by SDS-polyacrylamide electrophoresis (PAGE).

  In order to examine the substrate specificity of each enzyme, 0.4 μg of the enzyme and 1 to 3 μg of various unmodified glycoproteins were mixed with 50 mM citrate buffer (pH 5.0 or pH 6.0 [Endo-Bno1263 and Endo-Tsp1603]. ]) (Reaction volume: 10 μL) and reacted at 40 ° C. (Endo-Tsp1603) or 45 ° C. for 20 hours, and then subjected to SDS-PAGE to analyze the state of cleavage of glycoprotein sugar chains. As a result (FIG. 3A), Endo-Tsp1457 was able to cleave the sugar chain of ribonuclease B (RNase B, Non-Patent Document 6) to which a high mannose sugar chain was bound, but was able to cut α1-acid glycoprotein to which a complex sugar chain was bound. (Α1-AGP, mainly bound by 2-4 complex glycans; Non-Patent Documents 7 and 8), and prostate specific antigen (PSA, mainly bound by 2 to 3 complex glycans; Non-Patent Document 9) Since the sugar chains of ovomucoid and ovomucoid (mainly a complexing sugar chain containing bisecting GlcNAc bound; Non-Patent Document 10) could not be cleaved, it was considered to be an enzyme using a high mannose sugar chain as a good substrate.

  On the other hand, Endo-Tsp1263 was able to cleave only a part of the sugar chain of RNase B or PSA, but was almost able to cleave the ovomucoid sugar chain. Was done. Endo-Tsp1006 and Endo-Bac1008 were able to cleave most of the α1-AGP and PSA sugar chains, but hardly cleaved ovomucoid or RNase B sugar chains. It was considered that the enzyme used a complex type sugar chain containing as a good substrate.

  In addition, Endo-Bno1263 belonging to the same group 2 as Endo-Tsp1263 was able to almost cleave the ovomucoid sugar chain similarly to Endo-Tsp1263 (FIG. 3B). Furthermore, Endo-Tsp1603 belonging to the same group 3 as Endo-Tsp1457 was able to cleave the RNase B sugar chain in the same manner as Endo-Tsp1457.

  The results of mass spectrometry analysis of RNase B sugar chains cleaved by Endo-Tsp1457, ovomucoid sugar chains cleaved by Endo-Tsp1263, and α1-AGP sugar chains cleaved by Endo-Tsp1006 or Endo-Bac1008 are shown in FIGS. D. In the reaction, 7 μg of the enzyme and 60 μg of various glycoproteins not subjected to denaturation treatment were mixed in a 50 mM citrate buffer (pH 5.0) (reaction volume: 10 μL) and reacted at 45 ° C. for 18 hours.

  FIG. 4A shows a high-mannose type sugar chain from M5 type (containing five mannoses) to M9 type (containing nine mannoses) released by Endo-Tsp1457 among the sugar chains bound to RNase B. Possible sugar chain-derived signals have been detected. In addition, FIG. 4B shows, among the sugar chains bound to ovomucoid, a signal derived from a sugar chain that is considered to be a complex type of five to five saccharide chains containing a bisecting GlcNAc structure released by Endo-Tsp1263. Has been detected. This suggests that Endo-Tsp1263 has activity of cleaving a bisecting GlcNAc-containing multibranched sugar chain. In these sugar chains, N-acetylglucosamine exposed at the non-reducing end of the sugar chain exists. In addition, FIGS. 4C and 4D show that, of the sugar chains bound to α1-AGP, four out of the two chains, which are hyperbranched sugar chains containing sialic acid released by Endo-Tsp1006 or Endo-Bac1008. Signals derived from sugar chains considered to be complex sugar chains of chains have been detected.

<Example 2> Method for investigating the substrate specificity of endoglycosidase of Endo-Tsp1263, Endo-Tsp1006 and Endo-Bac1008 using various glycopeptides as substrates Substrate specificity for Endo-Tsp1263, Endo-Tsp1006 and Endo-Bac1008 In order to examine in more detail, using various glycopeptides as substrates, their sugar chain cleavage activities were measured. In addition, various glycopeptides were prepared as follows (FIG. 5). First, a sialylglycopeptide (Sialylglycopeptide, SGP, α2,6A2-peptide; Fushimi Pharmaceutical Co., Ltd.) derived from chicken eggs is used as a raw material, and is subjected to an acid treatment to remove sialic acid to form G2-peptide, followed by the action of galactosidase. To prepare G0-peptide. On the other hand, human N-acetylglucosamine transferase GnT-IV or GnT-V recombinant enzyme produced in HEK293 cells was allowed to act to prepare GN3b-peptide and GN3a-peptide, respectively. G3GN3b-peptide and G3GN3a-peptide were prepared by reacting these with human galactose transferase B4GalT1 produced in HEK293 cells.

GnT-IV was allowed to act on GN3a-peptide to prepare GN4-peptide. Further, B4GalT1 was allowed to act on GN4-peptide to prepare G4GN4-peptide. For this G4GN4-peptide, by the action of recombinant enzyme α2,3 sialyltransferase ST3Gal-IV or α2,6-sialyltransferase ST6Gal-I human were produced in HEK293 cells, respectively Arufa2,3A _{2 -4} G4-peptide, α2,6A _2-4 G4-peptide were prepared.

  Further, G2-peptide was prepared by allowing B4GalT1 to act on G0-peptide. Here, α2,3A-peptide was prepared by reacting α2,3 sialyltransferase ST3Gal-IV. Furthermore, G0B-peptide was prepared by allowing G0-peptide to react with human N-acetylglucosamine transferase GnT-III produced in HEK293 cells. B4GalT1 was allowed to act on this to prepare G2B-peptide. Also, M3-peptide was prepared by allowing N-acetylglucosaminidase S (New England Biolabs) to act on G0-peptide.

  Further, G0F-peptide was prepared by allowing human α1,6 fucose transferase FUT8 produced in HEK293 cells to act on G0-peptide. G2F-peptide was prepared by reacting B4GalT1 here. In addition, SGP was subjected to an acid treatment to partially remove sialic acid to obtain A1aG2-peptide and A1bG2-peptide, and then sialidase and galactosidase were allowed to act thereon to prepare G1a-peptide and G1b-peptide.

  In order to examine the substrate specificity of each enzyme, 0.25 μg of the enzyme and 0.25 mM of each glycopeptide were mixed in a 50 mM citrate buffer (pH 5.0) (reaction volume: 10 μL), and the mixture was mixed at 45 ° C. for 30 minutes. After the reaction, the cleavage status of the glycopeptide sugar chain was analyzed by mass spectrometry, and the result of determining the specific activity (mU / mg) is shown in FIG. As a result, Endo-Tsp1006 and Endo-Bac1008 were considered to efficiently cleave complex sugar chains having α2,6 sialic acid or galactose at the non-reducing end of the sugar chains. In addition, both enzymes also had the activity of cleaving G2B type sugar chains, which are bisecting GlcNAc sugar chains having galactose at the non-reducing end. On the other hand, Endo-Tsp1263 was considered to have a cleavage activity for G0B-type sugar chains and G2B-type sugar chains, which are bisecting GlcNAc sugar chains, but N-acetylglucosamine is present at the non-reducing terminal of the sugar chains. It was found that the complex type sugar chain was efficiently cleaved. Therefore, this enzyme had a very low activity of cleaving G2B-type sugar chains, which are bisecting GlcNAc sugar chains having galactose at the non-reducing end.

<Example 3> Method for comparing substrate specificities of various endoglycosidases using various glycoproteins as substrates Based on the above substrate specificities of Endo-Tsp1263, Endo-Tsp1006 and Endo-Bac1008, FIG. 7 shows RNase B as a substrate. The results of sugar chain cleavage reactions with various ENGases using PSA, PSA, ovomucoid and α1-AGP are shown. For the reaction, 0.2 to 0.5 μg of the enzyme and 1 to 3 μg of various unmodified glycoproteins were mixed in a recommended buffer for the reaction of each enzyme (reaction volume: 10 μL). For 18 hours. That is, Endo-Tsp1457, Endo-Tsp1263, Endo-Tsp1006, and Endo-Bac1008 were reacted at 45 ° C. in a 50 mM citrate buffer (pH 5.0). Endo-M, Endo-CC, EndoH, and EndoF1 were reacted at 37 ° C. in 50 mM citrate buffer (pH 6.0). EndoF2 and EndoF3 were reacted at 37 ° C. in 50 mM citrate buffer (pH 4.5). Regarding PNGase F (Glycopeptidase F, TAKARA BIO), the reaction was carried out under non-denaturing conditions and denaturing conditions according to the instructions attached to the product. After completion of the reaction, the resultant was subjected to SDS-PAGE to analyze the state of cleavage of the glycoprotein sugar chain.

  The sugar chain of RNase B (high mannose type sugar chain) was completely cleaved in the reaction using Endo-M, Endo-CC, EndoH, EndoF1, EndoF2, EndoF3, and Endo-Tsp1457. With regard to the sugar chain of PSA (mainly a complex type sugar chain having 2 to 3 chains), only a part of the sugar chain was cleaved in the reaction using Endo-M, Endo-CC, and Endo-Tsp1263. , EndoF2, EndoF3, Endo-Tsp1006, and Endo-Bac1008 resulted in almost complete cleavage. In addition, in the reaction using Endo-M, Endo-CC, EndoF2, and EndoF3, only a part of the sugar chain of α1-AGP (mainly a complex two- to four-chain sugar chain) is cleaved. However, the reaction using Endo-Tsp1006 and Endo-Bac1008 resulted in almost complete cleavage. According to Non-Patent Documents 8 and 9, it is suggested that sialic acid or galactose is present in most of the non-reducing ends of the sugar chains of PSA and α1-AGP, and Endo-Tsp1006 and Endo-Bac1008 have high efficiency. It is thought that it has a sugar chain structure that can be cleaved well.

  In existing ENGases, no enzyme capable of efficiently releasing bisecting GlcNAc sugar chains from various glycoproteins has not been found so far. In FIG. 7 as well, in the enzymatic reaction using the existing ENGase, Endo-Tsp1006, or Endo-Bac1008, the ovomucoid sugar chain to which the complexing sugar chain containing bisecting GlcNAc was mainly hardly cleaved. However, when Endo-Tsp1263 was used, most of the sugar chain could be efficiently released without denaturing ovomucoid, which is a substrate glycoprotein. According to Non-Patent Document 10, N-acetylglucosamine exposed at the non-reducing end is present in most of the multi-branched complex-type sugar chains of ovomucoid, including the bisecting GlcNAc structure, and Endo-Tsp1263 is efficiently used. It has a sugar chain structure that can be easily cleaved.

<Example 4> Method of investigating the substrate specificity of endoglycosidase of Endo-Tsp1263, Endo-Tsp1006 and Endo-Bac1008 using galactosylated ovomucoid as a substrate. According to FIG. Cleavage activity for the G0B-type bisecting GlcNAc sugar chain in which galactose is not bound to the non-reducing end is low, but cleavage activity for the G2B-type bisecting GlcNAc sugar chain in which two galactose molecules are bound to the non-reducing end of this sugar chain. Is expensive. On the other hand, Endo-Tsp1263 has a high cleavage activity on the G0B-type bisecting GlcNAc sugar chain, but has a low cleavage activity on the G2B-type bisecting GlcNAc sugar chain. Then, B4GalT1 of human galactose transferase was allowed to act on ovomucoid to prepare galactosylated ovomucoid in which galactose was added to the non-reducing end of the sugar chain of ovomucoid. I checked the activity.

  The enzymatic reaction was carried out by mixing 2.4 μg of ovomucoid or galactosylated ovomucoid and 0.25 μg of the enzyme in a 50 mM citrate buffer (pH 5.0) (reaction volume: 10 μL), and after reacting at 45 ° C. for 23 hours, It was subjected to SDS-PAGE to analyze the state of cleavage of the glycoprotein sugar chain. The reaction of PNGase F (Glycopeptidase F, TAKARA BIO) was performed under denaturing conditions in accordance with the instructions attached to the product. As a result, most of the ovomucoid sugar chain was cleaved by Endo-Tsp1263 as in FIG. 7, but hardly cleaved by Endo-Tsp1006 and Endo-Bac1008 (FIG. 8). On the other hand, the sugar chain of galactosylated ovomucoid was hardly cleaved by Endo-Tsp1263, but was mostly cleaved by Endo-Tsp1006 and Endo-Bac1008. That is, with respect to the bisecting GlcNAc-containing multibranched complex-type sugar chain in which galactose is present at the non-reducing end of the sugar chain, if Endo-Tsp1006 and Endo-Bac1008 are used, the sugar chain is not subjected to denaturation treatment, Can be efficiently liberated.

Example 5 Method of Releasing Sugar Chain of Specific Structure Including Bisting GlcNAc Sugar Chain Under Non-denaturing Conditions Using a plurality of Endoglycosidases Generally, sugar chains of various structures can be bound to a glycoprotein. When releasing sugar chains from various glycoproteins using ENGase, depending on the type of sugar chain bound to the glycoprotein and the type of ENGase used in the cleavage reaction, the effect of enzyme substrate specificity may be affected. As a result, a sugar chain that cannot be cleaved is generated, and a glycoprotein or glycopeptide in which a sugar chain having a specific structure remains bound is generated. Therefore, when it is desired to completely release sugar chains from glycoproteins to which sugar chains having various structures are bound using ENGase, or to release only sugar chains having a specific structure, the substrate specificity of ENGase used for the cleavage reaction In consideration of the nature, it is necessary to carry out the cleavage reaction using a single enzyme or a combination of a plurality of enzymes. In this example, assuming such a situation, a method of releasing a sugar chain having a specific structure including a bisecting GlcNAc sugar chain under non-denaturing conditions using a plurality of endoglycosidases was examined.

  RNase B to which a high-mannose type sugar chain is bound, α1-AGP to which mainly a two- to four-chain complex type sugar chain is bound, and bisecting wherein N-acetylglucosamine is exposed at the non-reducing end of the sugar chain. For a mixed solution (50 mM citrate buffer, pH 5.0) of ovomucoid (1 μg each) to which GlcNAc-containing complex-type sugar chains are mainly bound, Endo-Tsp1407, Endo-Tsp1263, Endo-Tsp1006, and Endo-Bac1008 Using each enzyme (each 0.6 μg) alone or in combination, a sugar chain cleavage reaction was performed (45 ° C., 20 hours). The result of subjecting the reaction product to SDS-PAGE is shown in FIG.

  From the results shown in FIG. 9, for example, in order to release all sugar chains from these glycoproteins, including complex GlcNAc-containing complex-type sugar chains in which N-acetylglucosamine is exposed at the non-reducing end of the sugar chains, Endo- A combination of Tsp1407, Endo-Tsp1263, Endo-Tsp1006, a combination of Endo-Tsp1407, Endo-Tsp1263, Endo-Bac1008, or a combination of Endo-Tsp1407, Endo-Tsp1263, Endo-Ed-E100 using Endo-Tsp1263, Endo-Tsp8 A sugar chain cleavage reaction may be performed. In addition, in order to release a sugar chain other than the bisecting GlcNAc-containing complex type sugar chain in which N-acetylglucosamine is exposed at the non-reducing end of the sugar chain from these glycoproteins, a combination of Endo-Tsp1407 and Endo-Tsp1006 Alternatively, the sugar chain cleavage reaction may be carried out using an enzyme having a combination of Endo-Tsp1407 and Endo-Bac1008.

  By using ENGases having different substrate specificities singly or in combination as described above, it is possible to release a sugar chain having a specific structure including a glycosylated GlcNAc from a glycoprotein, or to convert only a sugar chain having a specific structure into a glycoprotein. It is possible to leave on.

Example 6 Method for Investigating Glycosyltransferring Activity of Endo-Tsp1006 N220Q Mutant For ENGase belonging to the GH85 family according to the classification of the CAZy database, a mutant having an amino acid substitution introduced into the NxE motif or the like in the active site thereof Is known to exhibit glycosyltransferase activity (Non-Patent Document 11). This is considered to be due to the fact that the introduction of a mutation into the active site of ENGase and the like suppressed the sugar chain cleavage activity and also revealed the glycosyl transfer activity inherent in ENGase. Therefore, it was determined whether a mutant exhibiting such a glycosyltransfer activity could be prepared for Endo-Tsp1006.

  The asparagine in the NxE motif of Endo-Tsp1006 is N220. Therefore, it was decided to produce an Endo-Tsp1006 N220Q mutant in which this asparagine was substituted with glutamine. Using the primers for mutation introduction 5′-AACTACCAGTGGGGAAGATACGCGTTAT-3 ′ and 5′-TTCCCACTGGTAGTTTAATGCCATCACT-3 ′, mutation was introduced into the Tsp1006 gene by polymerase chain reaction. This gene was inserted into a pGEX6P-1 vector (GE Healthcare) to construct an Endo-Tsp1006 N220Q mutant expression vector. Then, an Endo-Tsp1006 N220Q mutant was obtained in the same manner as in the method for preparing the wild-type Endo-Tsp1006 described above.

  Next, a donor substrate required for the transglycosylation reaction was prepared. The α1-AGP sugar chain was cleaved with Endo-Tsp1006 and treated with sialidase to prepare a mixture of 2 to 4 complex sugar chains having a non-reducing terminal of galactose. This sugar chain mixture contains any one of G3F-type sugar chains in which fucose is bound to N-acetylglucosamine of any one of G2-type sugar chains, G3GN3b-type sugar chains, G4GN4-type sugar chains, and G3GN3b-type sugar chains. G4F-type sugar chains in which fucose is bound to such N-acetylglucosamine are included. An oxazoline form of this sugar chain mixture was used as a donor substrate. In addition, Fmoc-Asn (GnF) -OH in which N-acetylglucosamine with core fucose was bound to asparagine into which an Fmoc group had been introduced was used as an acceptor substrate for the transglycosylation reaction.

  The glycosyltransfer reaction was performed using 0.5 mM Fmoc-Asn (GnF) -OH, 3.0 mM oxazoline-linked sugar chains (45% of 4-chain sugar chains, 45% of 3-chain sugar chains, 10% of 2-chain sugar chains), 2 μg of Endo-Tsp1006 or Endo-Tsp1006 N220Q mutant was mixed in 50 mM Tris-HCl (pH 7.4) (reaction volume: 8 μL) and reacted at 25 ° C. for up to 60 hours. Thereafter, the reaction products were subjected to high performance liquid chromatography for analysis, and the ratio of each glycosyl transfer product was determined.

  FIG. 10 shows the result. In the case of wild-type Endo-Tsp1006, only trace amounts of glycosyltransfer products were detected, and most of unreacted Fmoc-Asn (GnF) -OH (abbreviated as Fmoc-GNF) remained. This is probably because the glycosyltransferase activity is hardly exhibited by the wild-type enzyme, or even if a glycosyltransferase product is generated, the sugar chain of the glycosyltransferase product may be cleaved by the sugar chain cleavage activity. On the other hand, in the case of the Endo-Tsp1006 N220Q mutant, the ratio of Fmoc-Asn (GnF) -G3 (abbreviated as G3), which is a glycosyltransfer product of G3GN3b-type sugar chain, is 22.6%, and that of G4GN4 type sugar chain. The ratio of the product Fmoc-Asn (GnF) -G4 (abbreviated as G4) is 19.3%, and the ratio of Fmoc-Asn (GnF) -G2 (abbreviated as G2), which is a glycosyltransfer product of G2-type sugar chain, is reduced. 12.1%, the ratio of Fmoc-Asn (GnF) -G3F (abbreviated as G3F), which is a glycosyltransfer product of G3F type sugar chain, is 7.2%, and Fmoc-Asn, a glycosyltransfer product of G4F type sugar chain, The ratio of (GnF) -G4F (abbreviated as G4F) was 5.2%, and the glycosyl transfer activity of this mutant was confirmed. Therefore, it can be said that Endo-Tsp1006 can be used as a glycosyltransferase (glycosynthase) by mutagenesis.

  INDUSTRIAL APPLICABILITY The present invention can release complex sugar chains containing various hyperbranched sugar chains from glycoproteins, glycopeptides, or sugar chains under non-denaturing conditions using endoglycosidase. Further, glycoproteins and glycopeptides in which all sugar chains or only sugar chains having a specific structure are released from glycoproteins and glycopeptides can be prepared. Therefore, the present invention can be applied to the preparation of sugar chain samples such as hyperbranched sugar chains and bisecting GlcNAc sugar chains, the preparation of glycoproteins as acceptor molecules used for sugar chain remodeling, and the functional analysis of various sugar chains. It can also be used in the pharmaceutical industry and the like.

Claims (12)

  A method for releasing a complex-type sugar chain from a glycoprotein, a glycopeptide, or a sugar chain, wherein the complex-type sugar chain is undenatured using a microorganism-derived endoglycosidase that recognizes a difference in sugar at the non-reducing terminal side of the sugar chain. A method of releasing sugar chains.   The complex-type sugar chain is a double-chain complex-type sugar chain, a multi-branched complex sugar chain having three or more chains, or a complex-type sugar chain containing a bisecting GlcNAc structure, and α2,6 is added to the non-reducing end of the sugar chain. The method according to claim 1, wherein the complex type sugar chain contains sialic acid.   The complex type sugar chain is a double-chain complex type sugar chain, a multi-chain complex sugar chain having three or more chains, or a complex type sugar chain containing a bisecting GlcNAc structure, and galactose is present at the non-reducing end of the sugar chain. 2. The method according to claim 1, wherein the sugar chain is a complex type sugar chain.   The complex-type sugar chain is a double-chain complex-type sugar chain, a multi-chain complex sugar chain having three or more chains, or a complex-type sugar chain containing a bisecting GlcNAc structure, and N-acetyl is added to the non-reducing end of the sugar chain. The method according to claim 1, wherein glucosamine is a complex type sugar chain in which glucosamine is present. The endoglycosidase is Endo-Tsp1006, which is endo-β-N-acetylglucosaminidase derived from a bacterium of the genus Tannerella, and is a protein of the following (a), (b) or (c): A method according to claim 2 or claim 3.
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 3 in the Sequence Listing; (b) an amino acid sequence of SEQ ID NO: 3 in which one or several amino acids are deleted, substituted or added, and Endo-Tsp1006 Protein having enzymatic activity (c) A protein consisting of an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 3 in the sequence listing, and having Endo-Tsp1006 enzyme activity The said endoglycosidase is Endo-Bac1008 which is endo- (beta) -N-acetylglucosaminidase derived from bacteria of the genus Muribaculum, and is a protein of the following (a), (b) or (c): A method according to claim 2 or claim 3.
(A) a protein comprising the amino acid sequence of SEQ ID NO: 7 in the Sequence Listing; (b) 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: 7, and Endo-Tsp1008 Protein having enzymatic activity (c) A protein comprising 90% or more amino acid sequence homology with the amino acid sequence of SEQ ID NO: 7 and having Endo-Tsp1008 enzyme activity The endoglycosidase is Endo-Tsp1263, which is endo-β-N-acetylglucosaminidase derived from a bacterium belonging to the genus Tannerella, and is a protein of the following (a), (b) or (c): 4. The method according to 4.
(A) a protein comprising the amino acid sequence of SEQ ID NO: 1 in the Sequence Listing; (b) 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: 1, and Endo-Tsp1263 Protein having enzymatic activity (c) A protein comprising an amino acid sequence having 90% or more homology to the amino acid sequence of SEQ ID NO: 1 and having an Endo-Tsp1263 enzyme activity The endoglycosidase is Endo-Bno1263 which is an endo-β-N-acetylglucosaminidase derived from a bacterium belonging to the genus Bacteroides, and is a protein of the following (a), (b) or (c): A method according to claim 2 or claim 3.
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 9 in the Sequence Listing; (b) an amino acid sequence of SEQ ID NO: 9 in which one or several amino acids are deleted, substituted or added, and Endo-Bno1263 Protein having enzymatic activity (c) Protein comprising an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 9 in the sequence listing, and having Endo-bno1263 enzymatic activity   A method for releasing a complex-type sugar chain from a glycoprotein, a glycopeptide or a sugar chain, wherein the complex-type sugar chain having a specific structure under non-denaturing conditions using a plurality of endoglycosidases that recognize differences in the sugar at the non-reducing end How to release   The plurality of endoglycosidases are endo-β-N-acetylglucosaminidases derived from bacteria belonging to the genus Tannerella Endo-Tsp1457, Endo-Tsp1603, Endo-Tsp1263, Endo-Tsp1006, endo-β-N-acetylglucosa derived from bacteria belonging to the genus Muribaculum. 10. The method according to claim 9, wherein Endo-Bac1008 is a combination or all of Endo-Bno1263 which is an endo-β-N-acetylglucosaminidase derived from a bacterium belonging to the genus Bacteroides.   A method for producing a glycoprotein, glycopeptide or sugar chain in which a complex type sugar chain has been cleaved using the method according to any one of claims 1 to 8. A method for producing a glycoprotein, glycopeptide or sugar chain in which a complex type sugar chain having a specific structure is cleaved using the method according to claim 9 or 10.

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