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WO2016136984A1 - Variante d'endo-m, et procédé de préparation d'un composé ou d'une protéine contenant une chaîne glucidique n-liée - Google Patents

Variante d'endo-m, et procédé de préparation d'un composé ou d'une protéine contenant une chaîne glucidique n-liée Download PDF

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WO2016136984A1
WO2016136984A1 PCT/JP2016/055926 JP2016055926W WO2016136984A1 WO 2016136984 A1 WO2016136984 A1 WO 2016136984A1 JP 2016055926 W JP2016055926 W JP 2016055926W WO 2016136984 A1 WO2016136984 A1 WO 2016136984A1
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sugar chain
endo
mutant
amino acid
glcnac
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憲二 山本
加藤　紀彦
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Tokyo Chemical Industries Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to an endo M mutant and a method for producing an N-linked sugar chain-containing compound or an N-linked sugar chain-containing protein.
  • N-linked sugar chains and O-linked sugar chains are known as sugar chains possessed by glycoproteins.
  • N-linked sugar chains have an important effect on the maintenance of protein function and structure.
  • N-linked sugar chains are classified into high mannose type, hybrid type and complex type sugar chains based on the difference in sugar chain structure, and it is known that the difference in sugar chain structure affects the physiological function of glycoprotein itself. It has been.
  • fucose core fucose
  • a method for preparing a glycoprotein with a controlled sugar chain structure a method of separating and purifying it from nature and a method of preparing it using cells modified by a genetic engineering technique can be considered.
  • a combination of the latter method or a method of converting the glycoprotein sugar chain to the target sugar chain by the enzyme-chemical method in which the glycoprotein obtained above is further performed in vitro is combined.
  • the method is considered advantageous.
  • the enzyme-chemical method is an attempt to introduce a chemically prepared sugar chain derivative (sugar chain donor) into the target protein by an enzymatic reaction. It requires a sugar chain donor and a sugar chain acceptor, which are substrates for the above.
  • hydrolase As an enzyme-chemical method, a method using a hydrolase is well known, but this is not only that some hydrolase hydrolyzes the substrate, but also sugar chain transfer to other substances. It is also known that it is possible to prioritize only the sugar chain transfer reaction by controlling the reaction conditions.
  • a sugar chain donor N-type is recently used by endo- ⁇ -N-acetylglucosaminidase M (endo M) derived from hair mold.
  • endo M endo- ⁇ -N-acetylglucosaminidase M
  • a method has been reported in which a large oligosaccharide chain site on the non-reducing end side of a conjugated sugar chain is transferred to a sugar chain receptor at once (see, for example, JP-A-7-59587).
  • a mutant (glycosynthase) in which the amino acid of endo M is modified can efficiently transfer a sugar chain of a sugar chain donor containing a structure derived from a target N-linked sugar chain to a sugar chain acceptor.
  • End M is a unique enzyme that hydrolyzes all of the high mannose type sugar chain, the hybrid type sugar chain and the complex type sugar chain in the N-linked sugar chain having no core fucose as a substrate of the enzyme. It is also heavily used in the preparation of sugar chain donors and sugar chain acceptors. Thus, Endo M has established a position as an important tool in enzyme-chemical glycoprotein preparation (for example, Kenji Yamamoto et al. [Journal of Applied Glycoscience, Vol. 3 (2), 31- 38, (2013)]).
  • endoenzymes that hydrolyze immunoglobulin G having an N-linked sugar chain containing core fucose endo S (for example, Collin et al., [EMBO J., Vol. 20 (12), 3046-3055, (2001)] It has also been reported that a mutant of endo S can transfer a sugar chain donor to a sugar chain and convert the sugar chain of immunoglobulin G into a target sugar chain (for example, International Publication No. 1). 2013/120066).
  • endoenzymes other than endo-M are limited in the types of N-linked sugar chains that can be used during hydrolysis and sugar chain transfer.
  • Muramatsu et al., [J. Biochem. , Vol. 129 (6), 923-928, (2001)] and Fan et al. [J. Biol. Chem. , Vol. 287 (15), 11272-111281, (2012)] has a problem that it acts only on some high-mannose sugar chains.
  • JP-A-7-59587 and Umekawa et al. [J. Biol. Chem. , Vol. 285 (1), 511-521, (2010)] can act on a wide range of N-linked sugar chains, but hydrolyzes N-linked sugar chains containing core fucose, or core fucose.
  • the sugar chain cannot be transferred to the sugar chain receptor.
  • Collin et al., [EMBO J. , Vol. 20 (12), 3046-3055 (2001)] and WO 2013/120066 can be used as a hybrid sugar chain, although complex sugar chains containing core fucose can be used as substrates. High mannose sugar chains and derivatives derived from them cannot be used as substrates. Furthermore, it has a drawback that it hardly acts other than N-linked sugar chains bound to immunoglobulin G (IgG).
  • IgG immunoglobulin G
  • an endo-M mutant having high hydrolysis activity for an N-linked sugar chain to which core fucose is bound, and an N-linked sugar chain-containing compound or N It is an object to provide a method for producing a conjugated sugar chain-containing protein.
  • Specific means for solving the problems include the following aspects. ⁇ 1> End having an amino acid sequence in which the 251st amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is asparagine or alanine, and having an activity of catalyzing a hydrolysis reaction represented by the following reaction formula (1) M variant, or having an amino acid sequence in which the 251st amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is asparagine or alanine, and other than the 251st amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 It has an amino acid sequence modified within a range of homology of 80% or more with respect to the amino acid sequence by deletion, addition or substitution of one or a plurality of amino acid residues, and is represented by the following reaction formula (1) It is an endo M variant having an activity of catalyzing the hydrolysis reaction represented.
  • X represents a saccharide-derived group
  • Y represents a monovalent substituent
  • GlcNAc represents an N-acetylglucosaminyl group
  • ⁇ 1-4 represents position 1 of GlcNAc and 4 of GlcNAc
  • Fuc represents a fucosyl group
  • ⁇ 1-6 represents an ⁇ glycoside bond between position 1 of Fuc and position 6 of GlcNAc
  • GlcNAc-OH is a hydroxyl group on the reducing end carbon of GlcNAc.
  • H-GlcNAc indicates that a hydrogen atom is bonded to the 4-position oxygen atom of GlcNAc.
  • GlcNAc that binds to Y does not include an oxygen atom or hydroxyl group of a glycosidic bond that binds to the reducing terminal carbon at position 1 of GlcNAc.
  • Y in the reaction formula (1) is an acylamino group including a structure derived from a peptide or protein.
  • ⁇ 3> N-bonding by reacting a sugar chain donor with a sugar chain acceptor represented by the following general formula (1) in the presence of the endo M mutant according to ⁇ 1> or ⁇ 2>
  • This is a method for producing an N-linked sugar chain-containing compound or an N-linked sugar chain-containing protein for producing an N-linked sugar chain-containing protein.
  • Y represents a monovalent substituent.
  • GlcNAc represents an N-acetylglucosaminyl group
  • Fuc represents a fucosyl group
  • ⁇ 1-6 represents an ⁇ -glycoside bond between position 1 of Fuc and position 6 of GlcNAc.
  • ⁇ 3> wherein the molar ratio of the sugar chain donor to the sugar chain acceptor (number of moles of sugar chain donor / number of moles of sugar chain acceptor) is 0.2 to 20.0. It is a manufacturing method of the N-linked sugar chain-containing compound or N-linked sugar chain-containing protein described.
  • an endo-M mutant having high hydrolysis activity for an N-linked sugar chain to which core fucose is bound and a method for producing an N-linked sugar chain-containing compound or an N-linked sugar chain-containing protein are provided. it can.
  • FIG. 1A is a diagram showing a three-dimensional model of a catalyst region in a co-crystal of endo A and an M3N1-thiazoline derivative.
  • FIG. 1B is a diagram showing a three-dimensional model in the vicinity of the catalyst region in a co-crystal of endo D and an M3N1-thiazoline derivative.
  • FIG. 2 is a diagram showing the alignment results for the primary amino acid sequences of End A, End D, and End M.
  • FIG. 3 is a diagram showing the results of SDS-PAGE of endo M and endo M mutants used in the examples. The arrow indicates the position of the band of End M (WT) or End M mutant.
  • FIG. 1A is a diagram showing a three-dimensional model of a catalyst region in a co-crystal of endo A and an M3N1-thiazoline derivative.
  • FIG. 1B is a diagram showing a three-dimensional model in the vicinity of the catalyst region in a co-cry
  • FIG. 4 is a diagram showing an outline of a synthesis route of M3F-biotin and M3-biotin.
  • FIG. 5 is a diagram showing the results of measuring the solution after hydrolysis of M3N1-biotin by Endo M (WT) by HPLC. Each arrow indicates the position of the retention time corresponding to the indicated compound.
  • FIG. 6 is a diagram showing the results of TLC measurement of samples after adding and incubating endo M (WT) and endo M mutants to a solution containing M3-biotin and M3F-biotin, respectively.
  • FIG. 5 is a diagram showing the results of measuring the solution after hydrolysis of M3N1-biotin by Endo M (WT) by HPLC. Each arrow indicates the position of the retention time corresponding to the indicated compound.
  • FIG. 6 is a diagram showing the results of TLC measurement of samples after adding and incubating endo M (WT) and endo M mutants to a solution containing M3-biot
  • FIG. 7 is a diagram showing the results of incubation after adding or not adding the W251N mutant to a solution containing M3F-biotin, and measuring by MALDI-TOF MS.
  • FIG. 8A is a diagram showing the result of MALDI-TOF MS measuring the sugar chains released after a hydrolysis reaction with endo M (WT) on a glycopeptide prepared from Rituxan.
  • FIG. 8B is a diagram showing the results of MALDI-TOF MS measuring the sugar chains released after a hydrolysis reaction with a W251N mutant on a glycopeptide prepared from Rituxan.
  • FIG. 8C is a diagram showing the result of MALDI-TOF MS measuring the sugar chains released after the hydrolysis reaction with endo S for the glycopeptide prepared from Rituxan.
  • FIG. 9 is a diagram showing the results of SDS-PAGE of the solutions after hydrolysis of endo S, endo M (WT) and W251N mutants against human lactoferrin.
  • FIG. 10A is a graph showing the results of MALDI-TOF MS measuring the sugar chains released after a hydrolysis reaction with endo M (WT) on a glycopeptide prepared from human lactoferrin.
  • FIG. 10B is a diagram showing the result of MALDI-TOF MS measuring the sugar chain released after hydrolyzing the glycopeptide prepared from human lactoferrin with the W251N mutant.
  • FIG. 10A is a graph showing the results of MALDI-TOF MS measuring the sugar chains released after a hydrolysis reaction with endo M (WT) on a glycopeptide prepared from human lactoferrin.
  • FIG. 10B is a diagram showing the result of MALDI-TOF MS measuring the sugar chain released after hydrolyzing the glycopeptide prepared from human lactoferrin with the W
  • FIG. 11 shows the reaction product obtained after hydrolyzing the glycoprotein (Rituxan) containing core fucose with the W251N mutant for each time (0 to 72 hours) using MALDI-TOF MS. It is a figure which shows the measurement result.
  • FIG. 12 is a diagram showing an outline of a sugar chain transfer reaction by an endo M mutant.
  • FIG. 13 is a diagram showing the results of measuring the solution after the transglycosylation reaction by MALDI-TOF MS.
  • FIG. 14 is a diagram showing the results of further MS / MS analysis of m / z 2719.8 and m / z 2573.9 signals obtained by MALDI-TOF MS of the solution after the transglycosylation reaction.
  • End M represents a kind of “End Enzyme”
  • End Enzyme has the same meaning as “End- ⁇ -N-acetylglucosaminidase”, and is a position where the tip of the arrow in the following general formula (2) is extended. That is, an enzyme that hydrolyzes the glycosidic bond between GlcNAc and GlcNAc.
  • Endoenzyme variant refers to a deletion, addition and / or substitution of one or more amino acid residues of an endoenzyme amino acid.
  • the amount of each component of the N-linked sugar chain-containing compound, N-linked sugar chain-containing glycoprotein, sugar chain donor, sugar chain acceptor, endo M and endo M mutant is particularly Unless otherwise indicated, it means the total amount of a plurality of substances present in the reaction solution.
  • “Substrate” refers to a substance that is subject to hydrolysis or sugar chain transfer of an endoenzyme or endoenzyme mutant.
  • the substance to be subjected to sugar chain transfer refers to a sugar chain donor and a sugar chain acceptor in a sugar chain transfer reaction.
  • X, Y, GlcNAc and ⁇ 1-4 have the same meanings as reaction formula (1) described later.
  • F represents a hydrogen atom or a fucosyl group that is glycosidically bonded to the 6-position of GlcNAc at ⁇ 1-6.
  • the arrow represents the position where the endoenzyme or endoenzyme variant is hydrolyzed.
  • Catalyzing the hydrolysis reaction means that the ⁇ -glycosidic bond between GlcNAc 1-position and GlcNAc 4-position of (A) is represented by End M or End M mutant, as shown in Reaction Formula (1) described later.
  • generates (B) and (C) is said, and it may hereinafter be called a hydrolysis activity.
  • the hydrolysis reaction may be simply referred to as “reaction”.
  • the hydrolysis activity is represented by the amount of product ( ⁇ mol / min ⁇ mg) produced by hydrolysis in a certain time.
  • “Glycosyl transfer” refers to a part of the sugar chain structure of a sugar chain donor, for example, in the case of the general formula (2), the part on the left side from the position where the tip of the arrow is extended ( ⁇ 1-4 glycoside bond part). , Refers to binding (transfer) to a sugar chain receptor.
  • the sugar chain receptor in the present disclosure refers to a sugar-containing substance having (Fuc ⁇ 1-6) GlcNAc or GlcNAc at the non-reducing end as represented by the general formula (1).
  • “Transglycosylation activity” refers to the ability of Endo M or Endo M mutant to transfer a sugar chain donor to a sugar chain acceptor to generate a new product (transfer to a sugar chain).
  • Transglycosylation yield refers to the amount of glycoprotein to which a complex type glycan generated after the reaction is transferred, unless otherwise specified. “Transglycosylation yield” refers to the molarity of the product of glycan transfer. The ratio of the number to the number of moles of sugar chain receptor used in the reaction.
  • endo M mutant refers to an endo enzyme mutant in which the amino acid of the amino acid sequence of endo M (see SEQ ID NO: 1) has been deleted, added, or substituted.
  • End M is an endoenzyme derived from the hair mold Mucor Himaris (GenBank Accession No. BAB43869)
  • End A is an endoenzyme derived from Arthrobacter protoformier (GenBank Accession No. AAD10851)
  • End “D” means Streptococcus pneumoniae-derived endoenzyme (GenBank Accession No. BAB62042.1)
  • End S means Streptococcus pyogenes-derived endoenzyme (GenBank Accession No. AAK00850).
  • WT wild type End M
  • “Homology” refers to residues in a protein amino acid sequence variant that are identical after aligning the sequence by introducing gaps, if necessary, to achieve maximum homology (percent). Defined as a percentage. Methods and computer programs for alignment are well known in the art and use ClustralW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) herein. . In addition, as a method of describing amino acid residues, it is expressed by either one of three letters or one letter.
  • the 251st tryptophan residue is indicated as W251.
  • an amino acid residue substituted that is, an amino acid residue substituted with another amino acid
  • a residue obtained by substituting the 251st tryptophan residue of endo M with an asparagine residue (or alanine) is W251N ( Alternatively, End M, which is indicated as W251A) and includes this, may be referred to as the W251N mutant of End M, or simply the W251N mutant.
  • endo M mutant of the present disclosure a mutant having an amino acid sequence in which the 251st amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is asparagine or alanine is referred to as “endo M mutant of the present disclosure”. There is.
  • the amino acid sequence at position 251 of the amino acid sequence represented by SEQ ID NO: 1 has an amino acid sequence that is asparagine or alanine, and 251 of the amino acid sequence represented by SEQ ID NO: 1 Having an amino acid sequence modified within a range of homology of 80% or more to the amino acid sequence by deletion, addition or substitution of one or more amino acid residues other than the amino acid residue;
  • the “end of the present disclosure” is particularly preferred.
  • M mutant homologue Sometimes referred to as “M mutant homologue”.
  • the hydrolysis activity by the endo M mutant is, for example, by adding the endo M mutant to a solution in which the substrate represented by (A) in the reaction formula (1) is dissolved, and after a predetermined temperature and a predetermined time, The oligosaccharide produced in the solution can be confirmed, for example, by measuring with TLC, HPLC and MALDI-TOF MS.
  • TLC As TLC, after the reaction solution is spotted on thin layer chromatography, it is developed with, for example, a developing solvent (1-butanol / acetic acid / water: 3/2/2), colored by the orcinol method, and image capturing device (for example, the product can be quantitatively confirmed by GT-X970 (manufactured by Epson Corporation), and the reaction rate, that is, the hydrolysis activity of the endo M mutant can be calculated.
  • a developing solvent (1-butanol / acetic acid / water: 3/2/2
  • the product can be quantitatively confirmed by GT-X970 (manufactured by Epson Corporation), and the reaction rate, that is, the hydrolysis activity of the endo M mutant can be calculated.
  • the HPLC analytical column may be either a normal phase system or a reverse phase system, such as an amino column in the normal phase system and an ODS (octadecylsilyl) column in the reverse phase system.
  • Examples of the MALDI-TOF MS include the following method. That is, a certain amount of acetone was added to the solution after the reaction, and the dissolved portion was dried, and then a certain amount of DHBA solution (20 mg / mL 2,5-dihydroxybenzoic acid dissolved in 50% aqueous methanol solution) was added. Dissolve. Thereafter, a part of the dissolved solution was spotted on a plate for MALDI-TOF MS analysis, dried, and then subjected to UltraXtrem JA-1 (manufactured by Bruker Daltonics) or an autoflex speed-tko1 reflector system (manufactured by Bruker Daltonics).
  • Measurement mode positive ion mode and reflector mode
  • Measurement voltage 25 kv and 1.5 kv to 2.5 kv
  • Measurement molecular weight range 0 to 4000 (m / z) and 45000 to 60000 (m / z)
  • Number of integration 20000-40000 and 1000-8000
  • the transglycosylation activity of the endo-M mutant since the endo-M mutant has hydrolytic activity, an accurate value of the transglycosylation activity cannot be known.
  • the sugar chain transfer yield and the sugar chain transfer yield the solution obtained by adding the End M mutant to a solution in which the sugar chain donor and the sugar chain acceptor are dissolved, and incubating at a predetermined temperature for a predetermined time.
  • the product produced therein can be identified, for example, by measuring with the above-mentioned TLC, HPLC and MALDI-TOF MS, and the yield and yield can be confirmed.
  • the carbohydrate portion is described with reference to the nomenclature usually used for describing oligosaccharides. These nomenclatures are described, for example, in Hubbard et al. [Ann. Rev. Biochem. , Vol. 50, 555 (1981)].
  • Mannose is represented by Man
  • 2-N-acetylglucosamine is represented by GlcNAc
  • galactose is Gal
  • fucose is Fuc
  • glucose Glc
  • Sialic acid is represented by the abbreviated notation NeuAc for 5-N-acetylneuraminic acid and NeuGc for 5-glycolylneuraminic acid.
  • the N-acetylglucosaminyl group may be referred to as a GlcNAc residue
  • the mannosyl group may be referred to as a Man residue.
  • the monosaccharide refers to only the above-mentioned sugars such as Gal and GlcNAc itself.
  • the position of the carbon that forms the sugar is represented by the 1st position of the reducing end and the 2nd position of the adjacent carbon atom.
  • a combination of these two or more monosaccharides is called an oligosaccharide, and its derivative is called an oligosaccharide derivative. That is, the N-linked sugar chain is an oligosaccharide.
  • a monosaccharide moiety in an oligosaccharide is sometimes referred to as a “sugar unit”.
  • “Glycosidic bond” refers to a bond in which the hydroxyl group at the 1-position of a sugar unit in a sugar chain and the hydroxyl group of another sugar are dehydrated and condensing with each other via an oxygen atom, such as an ⁇ 1-6 glycoside bond.
  • the term “sugar” refers to a glycosidic bond in which the 1-position (carbon) of a sugar and the 6-position (oxygen atom at the 6-position) of another sugar are bonded in an ⁇ -type.
  • the hydroxyl group at the 1-position of the sugar has ⁇ type and ⁇ type.
  • the glucosaminyl group (GlcNAc) on the non-reducing end side of the chitobiosyl site (-GlcNAc ⁇ 1-4GlcNAc-) is the oxygen atom at the 1-position of the GlcNAc. Is included, and an oxygen atom at the 4-position is included.
  • GlcNAc that binds to Y on the reducing end side does not include the oxygen atom at the 1-position and includes oxygen atoms at the 4-position and the 6-position.
  • the fucosyl group (Fuc) does not include the oxygen atom at the 1-position and includes a hydroxyl group from the 2-position to the 4-position.
  • the 2nd, 3rd, 4th and 6th carbons that are not glycosidic bonded indicate that a hydroxyl group is bonded.
  • a sugar chain including three monosaccharides may be referred to as three sugars, and a five sugar chain including five sugars.
  • Core sugar chain refers to the sugar chain moiety represented by C-1 below in the N-linked sugar chain
  • trimannosyl refers to the three mannose moieties in the core sugar chain.
  • Man binding at ⁇ 1-6 on the non-reducing end side is Man2
  • Man binding at ⁇ 1-3 on the non-reducing end side is Man3.
  • Man on the reducing end side is referred to as Man1.
  • the fucosyl group (Fuc) bonded to the 6-position of GlcNAc is referred to as core fucose.
  • an oligosaccharide as shown on the left side of the position where the tip of the arrow in the general formula (2) is extended may be referred to as a trimannosyl GlcNAc-containing sugar chain.
  • Protein and “protein” are generally referred to as peptides having a small number of amino acid residues, and proteins having a large number of amino acid residues, and there is no clear difference in the number of amino acid residues. Those having 50 residues or more are called proteins.
  • a protein may also be referred to as a polypeptide.
  • the “compound” includes a peptide and does not include a protein.
  • “Glycopeptide” or “glycoprotein” means that at least one N-linked sugar chain is present in the peptide or polypeptide portion, and unless otherwise specified, a plurality of N-linked types The types of sugar chains include one type and two or more types.
  • the endo M mutant of the present disclosure has a high hydrolysis activity and transglycosylation activity with respect to an N-linked sugar chain having a core fucose, so that a wide variety of N-linked types having a core fucose that could not be achieved by the prior art.
  • Sugar chains can be hydrolyzed, and furthermore, a wide variety of N-linked sugar chain-containing compounds or N-linked sugar chain-containing proteins having core fucose can be produced by sugar chain transfer.
  • the reason for having such an effect in the present disclosure is not clear, but the inventor thinks as follows. In a carbohydrate hydrolase, a reaction proceeds by recognizing a sugar chain serving as a substrate in the catalytic region of the enzyme and an active residue acting on the substrate.
  • Endo M does not act on the N-linked sugar chain having core fucose is that any amino acid in the catalytic region of End M prevents the core fucose moiety from being accommodated. As a result of searching for residues, it was found to be the 251st tryptophan. Furthermore, generally, when an amino acid residue in the vicinity of a catalyst residue is substituted, the enzyme activity is often significantly reduced. However, in the present disclosure, endo A, endo D and endo M having similar catalytic domain structures are described in detail.
  • the N-linked sugar chain containing core fucose is strongly accommodated in the catalytic region. It can be recognized that high hydrolytic activity and transglycosylation activity could be achieved.
  • endo-M mutant of the present disclosure maintains the properties of endo-M having a wide variety of N-linked sugar chains as substrates, hydrolysis of N-linked sugar chains having core fucose It can be said that sugar chain transfer has the same advantage. Further, it has been known from the previous reports that endo M has a substrate having a wide variety of substituents for the reducing terminal GlcNAc of the N-linked sugar chain. In addition, the reducing terminal GlcNAc is also known to be a substrate for endo M in other structures as long as it has a 4,6 cis diol site, as disclosed in JP-A-2008-22779. Yes. Therefore, the endo M mutant can also use the same wide range of compounds as substrates.
  • the endo M mutant of the present disclosure has an amino acid sequence in which the 251st amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is asparagine or alanine, and has a hydrolysis reaction represented by the following reaction formula (1).
  • Endo M mutant having catalytic activity or amino acid residue 251 of the amino acid sequence represented by SEQ ID NO: 1 has an amino acid sequence of asparagine or alanine, and 251st amino acid sequence represented by SEQ ID NO: 1 Having an amino acid sequence modified within a range of homology of 80% or more to the amino acid sequence by deletion, addition or substitution of one or more amino acid residues other than It is an endo M mutant (endo M mutant homolog) having an activity of catalyzing the hydrolysis reaction represented by the reaction formula (1).
  • X represents a saccharide-derived group
  • Y represents a monovalent substituent.
  • GlcNAc represents an N-acetylglucosaminyl group
  • ⁇ 1-4 represents a ⁇ -glycoside bond between position 1 of GlcNAc and position 4 of GlcNAc.
  • Fuc represents a fucosyl group
  • ⁇ 1-6 represents an ⁇ -glycoside bond between position 1 of Fuc and position 6 of GlcNAc.
  • GlcNAc-OH indicates that a hydroxyl group is bonded to the carbon at the reducing end of GlcNAc.
  • H-GlcNAc in (C) indicates that a hydrogen atom is bonded to the 4-position oxygen atom of GlcNAc.
  • the endo M mutant of the present disclosure is a mutant in which a mutation is introduced into the amino acid of endo M.
  • the endoenzyme represented by SEQ ID NO: 1 is an endo ⁇ -N acetylglucosaminidase (GenBank Accession No. BAB43869) derived from Mucor Himalis.
  • the endo M mutant is a W251N mutant or W251A mutant, or the mutant homolog of the present disclosure described above, so that the N-linked sugar chain having core fucose can be sufficiently hydrolyzed. Furthermore, a W251N mutant or a W251A mutant is more preferable, and a W251N mutant is particularly preferable.
  • the endo M mutant of the present disclosure can be prepared by a normal genetic engineering technique, and can be prepared using various types of hosts and corresponding appropriate protein expression vectors.
  • the host include Escherichia coli, Brevibacillus, cyanobacteria, lactic acid bacteria, yeast, insect cells and animal cells.
  • E. coli Escherichia coli
  • Brevibacillus cyanobacteria
  • lactic acid bacteria yeast
  • insect cells insect cells and animal cells.
  • As a specific production method in the case of Escherichia coli, Umekawa et al. [J. Biol. Chem. Vol. 285 (1), 511-521, (2010)], and yeast is described in detail in JP-A-11-332568.
  • the endo-M mutant and endo-M mutant homologue of the present disclosure are a fusion-type endo-M variant (or a fusion-type endo) that is fused with other peptides or proteins on their C-terminal side or N-terminal side in a hydrolysis reaction. M mutant homologues).
  • the peptide or protein that can be fused is not particularly limited as long as it does not inhibit the hydrolysis reaction.
  • a hexahistidine peptide (amino acid sequence from the N-terminus to HHHHHH), a flag peptide (amino acid sequence from the N-terminus) DYKDDDDK), influenza HA polypeptide (amino acid sequence is YPYDVPDYA from the N-terminus), glutathione-S-transferase, luciferase, avidin, chitin-binding protein, c-myc, thioredoxin, disulfide isomerase (DsbA), maltose-binding protein (MBP) ), And green fluorescent protein (GFP).
  • hexahistidine peptides and flag peptides are particularly preferable from the viewpoint of ease of preparation of endo M mutant or endo M mutant homolog.
  • fusion-type endo M mutant or fusion-type endo M mutant homolog there is a gap between the polypeptide part of the peptide or protein to be fused and the polypeptide part of the endo-M mutant or endo-M mutant homolog.
  • the linker region may include an amino acid sequence portion (protease site) that is hydrolyzed by a protease.
  • protease site For example, a factor Xa site, a thrombin site, an enterokinase site, a precision protease site is mentioned.
  • saccharide hydrolases found from nature have a hundreds of glycosyl hydrolase families (from the homology of amino acid sequences, etc.).
  • GH family glycosyl hydrolase family 85
  • other proteins belonging to the GH family 85 are known to be widely distributed in biological species from humans to bacteria.
  • End M mutant homolog in the present disclosure has an amino acid sequence length of about 500 residues including the endo M catalytic region, it is expected to have hydrolytic activity as an endo M mutant homolog. it can.
  • the hydrolysis activity of the endo M mutant homologue of the present disclosure is 150 of the hydrolysis activity of endo M (WT) when the hydrolysis reaction represented by reaction formula (1) is performed under the same conditions. It means that it is more than%.
  • the endo M mutant homolog in the present disclosure is the 251st amino acid as long as it has a homology of 80% or more with the W251N mutant or W251A mutant and hydrolyzes the N-linked sugar chain having core fucose. Any amino acid residue other than the residue may be prepared by substitution, deletion and addition with another amino acid.
  • an endo M mutant homolog resulting from deletion, addition and / or substitution of one or more amino acid residues other than the 251st amino acid residue of endo M (SEQ ID NO: 1).
  • the homology to the endo M (SEQ ID NO: 1) is 80% or more, the hydrolysis or transglycosylation activity can be sufficiently maintained. Further, it is preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.
  • the concentration of endo M mutant (or endo M mutant homolog) in the reaction solution is 0.01 ⁇ g / ⁇ L to 2 ⁇ g / ⁇ L is preferable. If it is 0.01 ⁇ g / ⁇ L or more, the hydrolysis reaction can proceed more rapidly, and if it is 2 ⁇ g / ⁇ L or less, the solubility of the endo-M mutant in the buffer solution can be improved. The decomposition reaction can proceed more rapidly.
  • the concentration of endo M mutant (or endo M mutant homolog) in the reaction solution Is preferably 0.01 ⁇ g / ⁇ L to 40 ⁇ g / ⁇ L.
  • the hydrolysis reaction can proceed more rapidly, and by being 40 ⁇ g / ⁇ L or less, the solubility of the endo M mutant in the buffer solution is improved, and more Economical.
  • 0.01 ⁇ g / ⁇ L to 30 ⁇ g / ⁇ L is preferable, and 0.02 ⁇ g / ⁇ L to 20 ⁇ g / ⁇ L is particularly preferable.
  • the degree of purification of the endo M mutant or endo M mutant homolog used in the reaction is preferably 50% or more from the viewpoint of further increasing the hydrolysis activity and shortening the reaction time. It is preferably 70% or more, particularly preferably 80% or more, and most preferably 90% or more. When the degree of purification is 50% or more, the hydrolysis activity of the endo M mutant or endo M mutant homolog can be further increased.
  • the degree of purification can be calculated from the image data after staining after SDS-PAGE.
  • the endo M mutant in the present disclosure hydrolyzes the substrate represented by (A) in the reaction formula (1), that is, the substrate represented by the following general formula (3).
  • X, Y, GlcNAc, ⁇ 1-4, Fuc and ⁇ 1-6 have the same meanings as the reaction formula (1), respectively.
  • the general formula (3) preferably has a structure represented by the following general formula (4).
  • X 1 , X 2 , X 3 , X 4 , X 5 and X 6 each independently represent a hydrogen atom or a saccharide-derived group.
  • GlcNAc represents an N-acetylglucosaminyl group.
  • Fuc represents a fucosyl group
  • Man represents a mannosyl group.
  • ⁇ 1-6 represents an ⁇ -glycoside bond between the 1-position of Man and the 6-position of Man or an ⁇ -glycoside bond between the 1-position of Fuc and the 6-position of GlcNAc
  • ⁇ 1-3 represents the 1-position of Man and the 3-position of Man. Represents an ⁇ -glycoside bond.
  • ⁇ 1-4 represents a ⁇ glycosidic bond between position 1 of GlcNAc and position 4 of GlcNAc or ⁇ glycoside bond between position 1 of Man and position 4 of GlcNAc.
  • Z 1 represents a hydrogen atom or GlcNAc, and GlcNAc contained in Z 1 is bonded to Man linked to GlcNAc at ⁇ 1-4 at ⁇ 1-4.
  • Y is synonymous with the general formula (3).
  • X 1 and X 2 Specific examples of X 1 and X 2, GlcNAc ⁇ 1-2, Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2 , NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuGc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2 and NeuGc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1 -2, heterologous antigen: Gal ⁇ 1-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, mannose 6-phosphate: Man ⁇ 1- (6PO4), polylactosamine: [Gal ⁇ 1-4GlcNAc ⁇ 1-3] nGal ⁇ 1-4GlcNAc ⁇ 1-2 (n is an arbitrary number), keratan Sulfuric acid [Gal ⁇ 1-4GlcNAc (6SO3) ⁇ 1-3] nGal ⁇ 1-4GlcNAc ⁇ 1-2 (n is an arbitrary number), [Gal (6SO3) ⁇ 1-4GlcNAc 6SO3) ⁇ 1-3]
  • Examples of X 3 and X 4 include groups in which the reducing terminal GlcNAc of each of the aforementioned saccharide-derived groups is ⁇ 1-4.
  • Examples of X 5 and X 6 include groups in which the reducing terminal GlcNAc of each of the aforementioned saccharide-derived groups is ⁇ 1-6.
  • Z 1 may be a hydrogen atom or GlcNAc ⁇ 1-4.
  • X 1 to X 6 may have any structure derived from a high mannose sugar chain (M4 to M9), and X 1 to X 6 in each case of M4 to M9 are M4 : X 1 to X 4 and X 6 are hydrogen atoms and X 5 is Man ⁇ 1-6, M5: X 1 to X 2 , X4 and X6 are hydrogen atoms, X 5 is Man ⁇ 1-6 and X 3 is Man ⁇ 1 -3, M6: X 1 to X 2 , X4 and X6 are hydrogen atoms, X 5 is Man ⁇ 1-6 and X 3 is Man ⁇ 1-2Man ⁇ 1-3, M7: X 1 to X 2 , X4 and X6 are hydrogen atoms , X 5 is Man ⁇ 1-2Man ⁇ 1-6, X 3 is Man ⁇ 1-2Man ⁇ 1-3, M8: X 1 to X 2 and X 6 are hydrogen atoms, X 5 is Man ⁇ 1-2Man ⁇ 1-6, and X 3 is Man ⁇ 1 -2Man ⁇ 1-3
  • either or both of X 1 and X 2 are GlcNAc ⁇ 1-2, Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-3Gal ⁇ 1 -4GlcNAc ⁇ 1-2, NeuGc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2 and NeuGc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, and X 3 to X 6 are hydrogen atoms, or X 1 to X 6 are all hydrogen atoms Preferably there is.
  • both X 1 and X 2 Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, any NeuGc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2 and NeuGc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2 More preferably, X 3 to X 6 are hydrogen atoms, or X 1 to X 6 are all hydrogen atoms.
  • Y is a substituent containing a structure in which an oxygen atom, a nitrogen atom, a carbon atom and a sulfur atom are directly bonded to the 1-position carbon of GlcNAc. . Y is not particularly limited as long as it does not reduce the hydrolysis activity of the endo M mutant.
  • alkoxy group alkoxy group, acylamino group, aryl Oxy, alkenyloxy, acyloxy, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy and arylsulfonyloxy Group, and the like.
  • substituents may further have a substituent.
  • Y is a hydroxyl group, an optionally substituted alkoxy group having 1 to 30 carbon atoms, and an optionally substituted aryl group having 6 to 30 carbon atoms for ease of preparation.
  • An oxy group, an optionally substituted alkenyloxy group having 1 to 30 carbon atoms and an optionally substituted acylamino group are preferred.
  • Y is an alkoxy group which may have a substituent having 1 to 12 carbon atoms, an aryloxy group which may have a substituent having 6 to 12 carbon atoms, and a substituent.
  • An acylamino group which may have a hydrogen atom is preferred.
  • the compound represented by the general formula (3) other than the acylamino group which Y may have a substituent can be obtained by purchasing from a commercially available reagent maker, and can be obtained by a chemical synthesis method, It can also be obtained by enzymatic preparation. Specifically, as a chemical synthesis method, for example, Nakano et al. [Carbohydr. Res. , Vol. 342 (5), 675-695 (2007)].
  • Y in General Formula (3) is an acylamino group which may have a substituent
  • Y is preferably an acylamino group containing a structure derived from a peptide or protein.
  • the molecular weight of the protein in this case is not particularly limited, but is preferably 5,000 to 500,000, more preferably 10,000 to 150,000, from the viewpoint of facilitating the hydrolysis reaction. is there.
  • Examples of the peptide or protein include, for animal cells, glycoproteins and glycopeptides that are secreted extracellularly or on the cell surface and have an N-linked sugar chain. Examples include various hormones, cells Examples include adhesion factors, various receptors, extracellular matrix, various enzymes, various cytokines, antibacterial or antiviral peptides, and various antibodies.
  • glycoproteins can be purchased as reagents and foods, and general methods for extracting glycoproteins from purchased foods and natural products, such as [The Biochemical Society of Japan “Basic Biochemical Experimental Methods”, Vol. 5, Tokyo Chemical Doujin, 2000].
  • the degree of purification (purity) of the glycoprotein used in the reaction is not particularly limited, but from the viewpoint of more promptly carrying out the hydrolysis reaction of the endo M mutant, the purity (the ratio of the glycoprotein to the total mass excluding water) ) Is preferably 50% by mass or more. Furthermore, it is preferable that it is 70 mass%, and it is especially preferable that it is 80 mass% or more.
  • the polypeptide part of the glycoprotein may be one type or two or more types. The purity of the glycoprotein can be similarly confirmed by, for example, a method using SDS-PAGE.
  • an alkoxy group which may have a substituent having 1 to 12 carbon atoms is particularly preferable from the viewpoint of solubility of the substrate.
  • These compounds having an N-linked sugar chain to which an optionally substituted alkoxy group is bonded are substrates for simply investigating the hydrolysis activity of endo M mutant and endo M mutant homologues. Useful as.
  • the monovalent substituent may further have an alkyl group having 1 to 12 carbon atoms.
  • the number of substituents that may be present is 1 to 20, preferably 1 to 10.
  • oligosaccharide derivatives obtained by reductive amination of GlcNAc at the reducing end of the N-linked sugar chain as shown in the following general formula (5) should also be used. Can do.
  • X, GlcNAc, and Fuc have the same meanings as in general formula (3), respectively.
  • ⁇ 1-4 represents a ⁇ glycoside bond between position 1 of GlcNAc and position 4 of HexNAcol
  • ⁇ 1-6 represents an ⁇ glycoside bond between position 1 of Fuc and position 6 of HexNAcol.
  • R 1 represents a monovalent substituent.
  • HexNAcol bonded to R 1 is represented by the following general formula (6).
  • R 2 represents a monovalent substituent. * Represents a binding position with GlcNAc on the non-reducing terminal side of the general formula (6), and ** represents a binding position with Fuc of the general formula (6).
  • R 2 is not particularly limited, and examples thereof include an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 1 to 12 carbon atoms, an aralkyl group having 7 to 20 carbon atoms, an aryl group having 6 to 16 carbon atoms, and a pyridyl group. Can be mentioned.
  • the hydrogen atom on the aromatic ring and the heterocyclic ring further includes a halogen atom, an alkylamino group having 1 to 6 carbon atoms, an amino group, a cyano group, an alkylcyano group having 1 to 6 carbon atoms, and a carbamoyl group.
  • the above reductive aminated oligosaccharide derivative can be prepared by a general method.
  • R 2 is a 2-aminopyridyl group
  • a known method [Agric. Biol. Chem. , Vol. 55 (1), 283-284 (1991)]
  • a sample obtained by releasing an N-linked sugar chain from a glycoprotein is obtained by 2-aminopyridylation.
  • the 2-aminopyridylation can be prepared, for example, by using a 2-aminopyridylation kit manufactured by Takara Bio.
  • many types of 2-aminopyridylated N-linked sugar chains are already commercially available (for example, manufactured by Cosmo Bio).
  • the hydrolysis reaction is carried out in a solution in which the endo M mutant and the substrate represented by the general formula (1) (A) are dissolved.
  • the solution used for the reaction is not particularly limited as long as it does not inhibit the hydrolysis activity of the endo M mutant.
  • phosphate buffer, citrate buffer, carbonate buffer, Tris-HCl buffer, MOPS buffer Liquid, HEPES buffer, borate buffer, and tartrate buffer may be used alone or in combination.
  • a phosphate buffer is preferable, specifically, sodium phosphate, potassium phosphate, and magnesium phosphate are preferable, and sodium phosphate and potassium phosphate are more preferable.
  • the concentration of the phosphate buffer is preferably 10 mM to 250 mM, more preferably 10 mM to 150 mM, and more preferably 20 mM to 100 mM.
  • concentration 10 mM or more
  • the buffering capacity is increased to increase the hydrolysis activity of the endo M mutant, and by setting the concentration to 250 mM or less, the hydrolysis activity of the endo M mutant can also be increased.
  • the pH of the solution in the reaction is preferably 5.0 to 8.5, more preferably 5.5 to 7.0, and particularly preferably 6.0 to 7.0.
  • the pH is 5.0 to 8.5, which is effective because the hydrolysis activity of the endo M mutant or endo M mutant homolog is further improved.
  • the stability of the substance having the substrate represented by (A) in the general formula (1) is further improved, which is effective.
  • the temperature in the reaction is preferably 4 ° C. to 45 ° C., more preferably 20 ° C. to 40 ° C., more preferably 25 ° C. from the viewpoint of increasing the hydrolysis activity of the endo M mutant or endo M mutant homolog.
  • a temperature of ⁇ 35 ° C. is particularly preferred.
  • the concentration of the substance having the substrate represented by (A) in the general formula (1) is not particularly limited, but is preferably 100 mM or less from the viewpoint of completing the hydrolysis reaction more quickly. Furthermore, it is preferable that it is 20 mM or less.
  • the reaction time in the reaction represented by the general formula (1) is preferably adjusted as appropriate depending on the reaction temperature, the concentration of the substrate represented by (A), and the concentration of the endo M mutant.
  • the reaction temperature is 25 ° C. to 40 ° C.
  • the substrate represented by (A) is 0.5 mM to 20 mM
  • the endo M mutant is 0.1 ⁇ g / ⁇ L to 10 ⁇ g / ⁇ L
  • the reaction time is 3
  • the time is preferably from 100 to 100 hours.
  • a hydrolysis reaction can be further advanced by being 3 hours or more, and a hydrolysis product with higher purity can be obtained by being within 100 hours.
  • N-linked sugar chain-containing compound or N-linked sugar chain-containing protein ⁇ Method for producing N-linked sugar chain-containing compound or N-linked sugar chain-containing protein ⁇
  • an N-linked sugar chain-containing compound or a sugar chain donor is reacted with a sugar chain acceptor represented by the following general formula (1) in the presence of the above-mentioned endo M mutant.
  • An N-linked sugar chain-containing compound or a method for producing an N-linked sugar chain-containing protein for producing an N-linked sugar chain-containing protein can be provided.
  • Y represents a monovalent substituent.
  • Fuc represents a fucosyl group
  • GlcNAc represents an N-acetylglucosaminyl group.
  • ⁇ 1-6 represents an ⁇ -glycoside bond between position 1 of Fuc and position 6 of GlcNAc.
  • sugar chain receptor is not particularly limited as long as it does not inhibit the sugar chain transfer activity of the endo M mutant as long as it is represented by the general formula (1). Moreover, the preferable range of Y in General formula (1) is the same as the said General formula (4).
  • Y does not contain a protein-derived structure
  • Y is an acylamino group that does not contain a protein-derived structure
  • that contains a peptide-derived structure is a chemical compound that includes a peptide extension step as shown in a patent document (Japanese Patent Laid-Open No. 10-45788).
  • a glycopeptide or N-linked sugar chain derivative obtained by a reagent manufacturer can be easily prepared by hydrolysis with the endo M mutant of the present disclosure or a commercially available endo enzyme.
  • Y containing a protein is a glycoprotein obtained by purchasing a commercially available reagent or food, or a glycoprotein obtained by a general extraction / separation method of a food or natural product. It can be easily prepared by hydrolysis with the endo M mutant of the present disclosure or a commercially available endo enzyme.
  • Endoenzymes include Endo H (manufactured by New England Biolabs), Endo S (manufactured by Sigma-Aldrich and New England Biolabs), End D (manufactured by Cosmo Bio), and End F1 to F3 (Sigma-Aldrich). Etc.).
  • Non-commercial enzymes include glycosyl hydrolases belonging to GH family 85, or enzymes that belong to GH family 18 and have been shown to hydrolyze N-linked sugar chains. Can do.
  • the endo M mutant of the present disclosure is preferable due to the wide range of substrate specificity.
  • a non-reducing end side site of an unnecessary N-linked sugar chain of a glycoprotein that is a target of sugar chain conversion is released, and a non-reducing end side site of a desired N-linked sugar chain is This is advantageous because it can be newly introduced.
  • the sugar chain donor is not particularly limited as long as the endo M mutant can transfer a sugar chain to a sugar chain acceptor.
  • the reducing terminal GlcNAc of an N-linked sugar chain-containing compound, peptide and protein as shown in the general formula (3), or non-reducing trimannosyl GlcNAc-containing sugar chain as shown in the following general formula (7) Oxazoline derivatives are mentioned.
  • the non-reducing terminal side sugar chain is efficiently transferred to the sugar chain by the endo M mutant.
  • the reducing terminal GlcNAc of a trimannosyl GlcNAc-containing sugar chain represented by the following general formula (7) is: Oxazolineated derivatives are preferred.
  • X 1 to X 6 , Z 1 , Man, ⁇ 1-6, ⁇ 1-3, and ⁇ 1-4 are synonymous with the general formula (4).
  • GlcNAc-oxa represents a structure as shown below. * Indicates the position where Man and ⁇ 1-4 are bonded.
  • the saccharide-derived groups in X 1 to X 6 and Z 1 have the same meanings as those in the general formula (4), respectively, and the preferred ranges are also the same.
  • Derivatives derived from such oxazolineated N-linked sugar chains include, for example, Noguchi et al. [J. Org. Chem. , Vol. 74 (5), 2210-2212 (2009)], and the oxazoline derivative having a structure derived from a complex type sugar chain is, for example, a commercially available oligosaccharide having a structure derived from an N-linked sugar chain.
  • GlcNAc trimannosyl GlcNAc-containing sugar chain.
  • a commercially available reagent for oxazolinization CDMBI, manufactured by Fushimi Pharmaceutical Co., Ltd.
  • CDMBI commercially available reagent for oxazolinization
  • reaction that is, the sugar chain transfer reaction, is performed in a solution in which the endo M mutant, sugar chain donor, and sugar chain acceptor are dissolved.
  • the solution used for the reaction is not particularly limited as long as it does not inhibit the transglycosylation activity of the endo M mutant.
  • phosphate buffer, citrate buffer, carbonate buffer, Tris-HCl buffer, MOPS Examples include a buffer solution, a HEPES buffer solution, a borate buffer solution, and a tartrate buffer solution. These buffers may be used alone or in combination.
  • a phosphate buffer is preferable, and specifically, sodium phosphate, potassium phosphate, and magnesium phosphate are more preferable. Sodium phosphate and potassium phosphate are preferred.
  • the concentration of the phosphate buffer is preferably 10 mM to 250 mM, more preferably 20 mM to 150 mM, and more preferably 50 mM to 100 mM, from the viewpoint of the transglycosylation activity of the endo M mutant or endo M mutant homolog. is there.
  • the transglycosylation activity of the endo M mutant or endo M mutant homolog can be enhanced, and the transglycosylation yield and transglycosylation yield can be increased. it can.
  • the pH of the solution in the reaction is preferably 5.5 to 8.5, more preferably 6.0 to 8.0, and more preferably 6.5 to 7 from the viewpoint of increasing the sugar chain transfer yield and the sugar chain transfer yield. .5 is particularly preferred.
  • the temperature in the reaction includes the viewpoint of the transglycosylation activity of the endo M mutant or endo M mutant homolog, the transglycosylation yield, the viewpoint of increasing the transglycosylation yield, and the stability of the sugar chain receptor, endo M mutant, etc. From the viewpoint of properties, it is preferably 4 ° C. to 40 ° C., more preferably 20 ° C. to 40 ° C., and particularly preferably 25 ° C. to 35 ° C. By setting it to 4 degreeC or more, glycan transfer activities, such as an endo M variant, can be improved, and when it is set to 40 degrees C or less, glycan transfer yield and glycan transfer yield can be increased.
  • the concentration of endo M mutant (or endo M mutant homolog) in the reaction solution is preferably 0.005 ⁇ g / ⁇ L to 0.5 ⁇ g / ⁇ L.
  • the sugar chain transfer yield is improved by being 0.005 ⁇ g / ⁇ L or more, and the product after the sugar chain transfer of endo M mutant (or endo M mutant homolog) is 0.5 ⁇ g / ⁇ L or less. It is possible to suppress the rehydrolysis of glycan and improve the sugar chain transfer yield and the sugar chain transfer yield.
  • the hydrolysis activity, sugar chain transfer yield, and sugar chain transfer yield of the endo M mutant should be confirmed by, for example, a measuring method using TLC, SDS-PAGE, HPLC, or MALDI-TOF MS, as described above. Can do.
  • the concentration of the sugar chain receptor in the reaction solution is not particularly limited as long as it does not inhibit the sugar chain transfer reaction of the endo M mutant, and the desired sugar chain transfer yield. Or the solubility of the sugar chain receptor in the reaction solution and the like.
  • the sugar chain receptor represented by the general formula (1) does not contain a protein
  • 0.1 mM to 200 mM is preferable.
  • it is 0.1 mM or more, a higher sugar chain transfer yield can be obtained, and when it is 200 mM or less, sugar chain transfer can be achieved by a sugar chain transfer reaction by an endo M mutant having higher sugar chain transfer activity.
  • the yield can be increased.
  • 1 mM to 50 mM is more preferable.
  • the molar ratio of the sugar chain donor to the sugar chain acceptor is the type of sugar chain donor or sugar chain acceptor used. In addition, it can be appropriately set in consideration of recovery of the reaction after the reaction. However, when preparing an N-linked sugar chain-containing compound by a sugar chain transfer reaction, the molar ratio is preferably 0.2 to 20.0, more preferably 0.5 to 15.0. 1.0 to 10.0 is particularly preferable, and 1.0 to 5.0 is most preferable. When the molar ratio is 0.2 or more, a more sufficient transglycosylation yield can be obtained, and when the molar ratio is 20.0 or less, a higher transglycosylation yield can be obtained.
  • the reaction time in the transglycosylation reaction is appropriately set depending on the reaction temperature, the mass of the endo M mutant, and the like, but is preferably 10 minutes to 600 minutes. By being 10 minutes or more, the sugar chain transfer yield can be made higher, and by being 600 minutes or less, the influence of hydrolysis of the product after the sugar chain transfer can be more effectively prevented, The sugar chain transfer yield and the sugar chain transfer yield can be increased. Among the above reaction times, it is more preferably 30 minutes to 120 minutes.
  • a sugar chain donor and an endo M mutant may be further added after a predetermined time from the start of the reaction, and this operation can further increase the sugar chain transfer yield. In this case, from the viewpoint of increasing the sugar chain transfer yield, it is preferable to carry out the reaction from 3 hours to 48 hours after the initial sugar chain donor is charged.
  • the pharmaceutical composition includes a pharmaceutically acceptable stabilizer, buffer, excipient. , Binders, disintegrating agents, flavoring agents, coloring agents, fragrances, and the like can be added as appropriate to form dosage forms such as injections, tablets, capsules, granules, fine granules, and powders.
  • N-linked sugar chain-containing compound or N-linked sugar chain-containing protein of the present disclosure many types of sugar chain donors are used, and many corresponding types of N-linked sugar chains are increased. Since a compound containing a ratio or a protein containing an N-linked sugar chain is obtained, the obtained product is also useful as a sugar chain preparation.
  • Endo A and End D are both known to belong to the GH family 85, and both are enzymes that hydrolyze N-linked sugar chains, based on a comparison of the primary structures of amino acid sequences. As described above, End A does not hydrolyze N-linked sugar chains containing core fucose, but End D does. On the other hand, End A is described in Yin et al. 4, 4658 (2009)] and Ling et al. [J. Mol. Biol. , Vol. 389 (1), 1 (2009)], End D is a document of Abbott et al. [J. Biol. Chem. , Vol.
  • FIG. 1A shows a stereo model of the catalyst region in the co-crystal of endo A and M3N1-thiazoline derivative
  • FIG. 1B shows a stereo model near the catalyst region in the co-crystal of endo D and M3N1-thiazoline derivative.
  • a co-crystal three-dimensional structure with the same compound having the same amino acid is superposed by PyMol (manufactured by DeLano Scientific LLC), and the difference in the distribution of amino acid residues in the vicinity of the active residue is examined, and an amino acid that may allow core fucose Amino acid residues (endo A) that might inhibit residue or core fucose tolerance were estimated.
  • PyMol is a general molecular graphic tool capable of 3D imaging of proteins, small molecules, electron density, molecular surfaces, and orbitals.
  • the estimated amino acid residues were compared (aligned) with the primary sequences of Ends A, M and D as shown in FIG.
  • the amino acid to be mutated, the amino acid number, and the amino acid after the mutation are represented, for example, as W251N for the amino acid of endo M.
  • the source of the arrow indicates the amino acid and amino acid number of the corresponding endo A.
  • the 175th and 177th encircled lines of endo M and the solid triangle symbol are active amino acids in the hydrolysis reaction of endo M, endo A and endo D and acidic amino acids directly involved in the active residues Indicates residue.
  • the other box shows the candidate amino acid residues to be mutated for End M and the corresponding End A and End D amino acid residues.
  • endo M mutant was prepared as follows.
  • endo M mutants As endo M mutants, G125W mutant, Q128A mutant, Q128S mutant, W228H mutant, W251A mutant and W251N mutant were prepared as follows.
  • the endo-M mutant used in this example is disclosed in Umekawa et al. [J. Biol. Chem. Vol. 285 (1), 511-521, (2010)].
  • Plasmid DNA (pET23b-EndoM-His6) introduced into BamHI and XhoI sites at the multiple cloning site of a commercially available protein expression vector (pET23b, manufactured by Novagen) as a template using DNA encoding the full length of the amino acid sequence of SEQ ID NO: 1 as a template
  • protein expression for preparation of each endo M mutant was performed using oligo DNA for forward primer and oligo DNA for reverse primer (SEQ ID NOs: 2 to 13) encoding amino acids to be mutated.
  • a vector was constructed. By this preparation method, a protein in which six histidines (6 ⁇ His, histag) are fused to the C-terminal region of each endo M mutant can be obtained.
  • DNA polymerase KOD-plus, manufactured by Toyobo Co., Ltd.
  • plasmid DNA pET23b-EndoM-His6 serving as a template
  • dNTP nucleic acid donor
  • MgSO4 magnesium sulfate
  • a protein expression vector (pET23b- ⁇ EndoM-His6) encoding each mutated endo M gene was introduced (transformed) into Escherichia coli BL21 (DE3) strain for protein expression to obtain a transformant. .
  • End M (WT) was transformed with a vector (pET-EndoM-His6) into which DNA encoding endo M was introduced by the method described in the above document without performing the above-described mutation introduction procedure.
  • BL21 (DE3) was prepared, and endo-M was expressed and purified as described below.
  • BL21 (DE3) having pET23b- ⁇ EndoM-His6 was precultured (5 ° C., 12 hours) with 5 mL of LB culture solution containing 100 ⁇ g / mL of ampicillin (Amp), and then 1 mL (OD value, 0.5-2) 0.0) was added to 100 mL of an LB culture solution containing 100 ⁇ g / mL of ampicillin (Amp), followed by shaking culture (19 ° C., 38 hours).
  • the cultured cells are collected by centrifugation (3300 ⁇ g, 10 minutes, 4 ° C.), then added with 5 mL of BugBusterMasterMix (manufactured by Novagen) containing 1 mM PMSF solution and incubated at room temperature for 10 minutes with infiltration. Thus, a lysate of bacterial cells was obtained. The resulting lysate is centrifuged at room temperature at 21500 ⁇ g for 15 minutes to remove the precipitate, and a 500 mM imidazole solution is added to the resulting supernatant to a final concentration of 20 mM, and purification of the Histag for 1 mL is performed.
  • the sample was applied to a column for use (Ni 2+ -charged Hi-trap chelating column, manufactured by GE Healthcare (GE)) as it was. Thereafter, 10 ml of 20 mM phosphate buffer (pH 7.5) containing 20 mM imidazole, 0.5 M NaCl, and 1 mM dithiotriitol (DTT) is added to the column, and the protein having no Histag bound to the column, etc. 4 ml of 20 mM phosphate buffer (pH 7.5) containing 100 mM imidazole, 0.5 M NaCl and 1 mM DTT was added to the protein bound to the column, and 1 ml of the solution flowing out of the column was added.
  • 20 mM phosphate buffer pH 7.5
  • DTT dithiotriitol
  • Fractionate each fraction then add 4 mL of 20 mM phosphate buffer (pH 7.5) containing 150 mM imidazole, 0.5 M NaCl and 1 mM DTT, and add 1 ml of the solution flowing out of the column. (Fraction). SDS-PAGE was performed on each fraction (1 mL, 8 total) to confirm the contained protein. After that, among these fractions, the fraction containing the purified protein was collected, and a centrifugal filter unit having an ultrafiltration membrane (fractionated molecular weight 30,000, Amicon Ultra, manufactured by Millipore) was used to add 0.15M NaCl.
  • a centrifugal filter unit having an ultrafiltration membrane fractionated molecular weight 30,000, Amicon Ultra, manufactured by Millipore
  • FPLC system AKTA explorer, manufactured by GE
  • N175Q mutant the thing made from Tokyo Chemical Industry Co., Ltd. was used as it was.
  • the N175Q mutant is disclosed in Umekawa et al. [J. Biol. Chem. Vol. 285 (1), 511-521, (2010)], a derivative in which the reducing terminal GlcNAc of the non-reducing trimannosyl GlcNAc-containing sugar chain represented by the general formula (7) is oxazolineated It is known that the sugar chain is efficiently transferred to a sugar chain receptor having no core fucose.
  • the specific preparation method is shown below.
  • the synthesized product was confirmed by thin layer chromatography and NMR. Thin layer chromatography was confirmed by developing color with 10% ethanol sulfate using 60F254 manufactured by Merck.
  • NMR ECA-400 manufactured by JEOL Ltd. was used.
  • the following “glucosaminyl group” and “fucosyl group” include those in which those hydroxyl groups are substituted with other groups.
  • a commercially available rituximab preparation (Rituxan, manufactured by Zenyaku Kogyo Co., Ltd.) having the following properties was used as Example 3.
  • the obtained Rituxan peptide was dried and dissolved in 5% acetic acid, and the resulting solution was applied to a C18 cartridge column. Thereafter, the C18 column was washed with 5% acetic acid, and then the peptide / glycopeptide bound to the column was sequentially washed with 5% acetic acid containing 20% 2-propanol and 5% acetic acid containing 40% 2-propanol. The solution that was eluted and collected after elution was dried by centrifugal concentration to obtain a Rituxan peptide.
  • the obtained Rituxan peptide is a mixture which has about 10 mass% of glycopeptides having N-linked sugar chains with respect to the mass of all Rituxan peptides.
  • TLC ⁇ Various measurement methods> (TLC) 2 ⁇ L of the solution after the following reaction was spotted on a thin layer chromatography (TLC, Silica gel 60, manufactured by Merck), developed with a developing solvent (1-butanol / acetic acid / water: 3/2/2), the orcinol method, That is, after being immersed in a 0.2% by volume orcinol solution dissolved in 2M sulfuric acid, it was colored by heating at 110 ° C. for about 10 minutes. The colored plate was imaged with a commercially available scanner device.
  • HPLC HPLC 10 ⁇ L of 0.1 vol% trifluoroacetic acid (TFA) was added to 10 ⁇ L of the solution after the reaction, and used as a sample for HPLC measurement.
  • HPLC measurement was performed using an HPLC system equipped with an analytical column [Cosmosil 5C18 AR-II (pore size 4.6 mm ⁇ length 150 mm, manufactured by Nacalai Tesque)] [Separation module: e2569 (Waters), photodiode array. Detector: 2999 (manufactured by Waters)] was performed under the following conditions.
  • MALDI-TOF MS 80 ⁇ L of cold acetone was added to the solution after the reaction, and the mixture was allowed to stand at ⁇ 80 ° C. for 30 minutes. Thereafter, the mixture was centrifuged at 17400 ⁇ g for 10 minutes, the supernatant was transferred to another tube, dried by concentration centrifugation, and dissolved in 10 ⁇ L of pure water. 2 ⁇ L of the obtained solution was confirmed by TLC, and 1 ⁇ L was dissolved in 3 ⁇ L of DHBA solution (20 mg / mL 2,5-dihydroxybenzoic acid dissolved in 50% aqueous methanol). 2 ⁇ L each of the obtained solution was spotted on a plate for MALDI-TOF MS analysis and dried.
  • Measurement mode positive ion mode and reflector mode
  • Measurement voltage 25 kv and 1.5 kv to 2.5 kv
  • Measurement molecular weight ranges m / z 0 to 4000 and 45000 to 60000 ⁇ Number of integration: 20000-40000 and 1000-8000
  • the MS / MS measurement was performed using argon gas using a signal having a predetermined m / z value obtained by the MALDI-TOF MS measurement as a precursor ion.
  • SDS-PAGE Measurement by SDS-PAGE was performed using a commercially available slab gel apparatus (a constant temperature double mini slab gel electrophoresis apparatus manufactured by Nihon Aido Co., Ltd.), and after electrophoresis of 10% polyacrylamide gel at 40 mA for 60 minutes, was stained with Coomassie brilliant blue and the resulting gel was evaluated.
  • As the marker Protein Marker Broad Range (2-212 kDa) (manufactured by NEB) was used.
  • a gel imaging device Scanner GT-X970, manufactured by Epson Corporation was used.
  • Example 1 In a 1.5 mL microtube, 1 ⁇ g of the W251A mutant as an endo M mutant and 10 ⁇ g of M3-biotin or M3F-biotin as a substrate (Scheme (1) (A)) were added to 30 mM phosphoric acid. After dissolving in 10 ⁇ L of an aqueous sodium solution (pH 6.0), it was incubated at 30 ° C. in a thermostatic bath. After incubation for a predetermined time, the reaction was stopped by heat treatment at 95 ° C. for 5 minutes to obtain a solution after the reaction. The obtained solution was measured by TLC, HPLC and MALDI-TOF MS, and the hydrolysis activity was calculated.
  • Example 2 Comparative Examples 1 to 5
  • Table 2 in the same manner as in Example 2 or Example 2, Solutions after the reactions in Comparative Examples 1 to 5 were obtained. Each solution obtained was measured by the same method as in Example 1, and the hydrolysis activity was calculated.
  • FIG. 7 shows the measurement results by MALDI-TOF MS of the solution obtained after adding the W251N mutant to the solution containing M3F-biotin and incubating for 20 minutes under the above conditions. From the results, when the W251N mutant was added, the signal (m / z 1363.6) of M3F (Na + addition) detected when the W251N mutant was not added disappeared, and instead F1N1-biotin (Na + addition) signal (m / z 673.6) and M3N1 (Na + addition) signal (m / z 730.6) appeared. This revealed that M3F was hydrolyzed by the W251N mutant to produce F1N1-biotin and M3N1.
  • the amount of the product after hydrolysis with respect to M3F-biotin is calculated from the analysis by HPLC, and the specific hydrolysis activity is determined from the reaction time (incubation time) and the mass of the added endo M mutant or endo M. ( ⁇ mol / (min ⁇ mg)) and relative activity were calculated.
  • the relative activity is shown as the relative activity of endo M mutant to each substrate when the activity of endo M against M3F-biotin is 1. The results are shown in Table 2.
  • FIG. 5 shows the result of HPLC measurement when End M is added to M3-biotin.
  • FIG. 6 the three charts show the results when the end M is not added to the reaction solution from the top, when 10 ng is added, and when 100 ng is added.
  • the horizontal axis represents column retention time, and the vertical axis represents detection sensitivity by UV absorption.
  • the arrow indicates the retention time of the resulting product.
  • the endo M mutants of the present disclosure have a hydrolytic activity for M3F-biotin, which is an N-linked sugar chain having a core fucose, and endo M or other endo M mutants. It was shown to be significantly higher than Thus, it was shown that substitution of the 251st tryptophan residue of endo M with an appropriate amino acid is extremely important for hydrolysis of the N-linked sugar chain having core fucose.
  • Example 3 As Example 3, a hydrolysis reaction was performed on a Rituxan peptide containing a glycopeptide of the W251N mutant. 200 ⁇ g of the Rituxan peptide prepared above was dissolved in 400 ⁇ l of 100 mM sodium phosphate buffer (pH 6.5), 4 ⁇ g of W251N mutant was added, and reacted at 30 ° C. for 16 hours. After completion of the reaction, the post-reaction solution was dried by centrifugal concentration, redissolved in 5% acetic acid, and the obtained solution was applied to a C18 column. After the application, the fraction not bound to the column was collected and lyophilized to obtain a dried product of the N-linked sugar chain fraction released by hydrolysis with the endo M mutant. The dried product was subjected to sugar chain analysis by the following mass spectrometry.
  • Example 6 In Example 3, except that 4 ⁇ g of Endo M (WT) was used in place of 4 ⁇ g of the W251N mutant, the hydrolysis reaction for the Rituxan peptide was performed and the sugar chain analysis by mass spectrometry was performed in the same manner as in Example 3. .
  • Example 3 In Example 3, except that 4Ug of W251N mutant and 100 mM sodium phosphate buffer (pH 6.5) were used, End S (manufactured by New England Biolabs) 100U and NEB G6 buffer were used. In the same manner, a hydrolysis reaction for the Rituxan peptide was performed, and a sugar chain analysis by mass spectrometry was performed.
  • FIGS. 8A to 8C show the results of MALDI-TOF MS measurement of the released sugar chain after the hydrolysis reaction with endo M (WT) on the glycopeptide prepared from Rituxan
  • FIG. 8B shows the result prepared from Rituxan
  • FIG. 8C shows the result of MALDI-TOF MS measurement of the released glycan after hydrolyzing the glycopeptide with the W251N mutant.
  • FIG. 8C shows the result of endo S against the glycopeptide prepared from Rituxan. The result of measuring the sugar chain released after the hydrolysis reaction by MALDI-TOF MS is shown.
  • 8B m / z 1171.7, 1416.8, 1620.9, 1825.0, 2187.1, 25488.3 and HexNAc2Nex2, HexNAc3Hex3, HexNAc3Hex4, HexNAc3Hex5, NeuAc1HexNAc3Hex5 , A peak corresponding to NeuAc2HexNAc3Hex5 was detected.
  • 8A to 8C show sugar chain structures estimated from the fragment patterns obtained from the results of MALDI-TOF / TOF analysis of each peak. In the estimated sugar chain structure, it was shown that Example 3 and Reference Example 1 shown in FIG. Thus, the W251N mutant was shown to have hydrolytic activity similar to Endo S against glycopeptides to which various types of complex-type sugar chains containing core fucose were bound.
  • Example 4 As Example 4, hydrolysis of the W251N mutant to human lactoferrin (hLF) was performed. 2 ⁇ g of W251N mutant was added to 20 ⁇ l of 100 mM sodium phosphate buffer (pH 6.5) in which 10 ⁇ g of hLF (L3770, manufactured by Sigma-Aldrich) was dissolved, and reacted at 30 ° C. for 16 hours. An equal amount of SDS-PAGE sample buffer was added to the solution after the reaction, and the sample was heated in boiling water for 5 minutes to obtain a sample for SDS-PAGE.
  • hLF human lactoferrin
  • each lane was applied to a well of a 7.5% polyacrylamide gel for SDS-PAGE so that 0.5 ⁇ g of hLF was contained, and SDA-PAGE was performed.
  • the gel after SDA-PAGE was stained by Coomassie staining.
  • Example 7 a hydrolysis reaction for hLF was performed in the same manner as in Example 4 except that 2 ⁇ g of endo M (WT) was used instead of the W251N mutant, and SDS-PAGE was performed on the solution after the reaction.
  • WT endo M
  • Example 4 was the same as Example 4 except that End U (manufactured by New England Biolabs) 100U and NEB G6 buffer were used instead of 20 ⁇ l of the W251N mutant and 100 mM sodium phosphate buffer (pH 6.5). Then, hydrolysis reaction for hLF was performed, and SDS-PAGE was performed on the solution after the reaction.
  • End U manufactured by New England Biolabs
  • NEB G6 buffer 100U and NEB G6 buffer
  • FIG. 9 shows the results of SDS-PAGE for the solution after each reaction.
  • the upper part of each lane shows the type of endoenzyme used for the hydrolysis reaction.
  • the start point of the arrow represents the protein name estimated in each band indicated by the end point.
  • DG-hLF means hLF in which one or two N-linked sugar chains are hydrolyzed by an endoenzyme.
  • Native hLF means hLF whose N-linked sugar chain is not hydrolyzed. From the results, in hLF (Control) that has not been hydrolyzed, a band corresponding to native hLF having two sugar chains slightly above 80 kDa is observed. A small part is further separated because it is presumed to have three sugar chains.
  • Comparative Example 8 that is, in the case of the hydrolysis reaction with endo S, a band appears on the lower molecular weight side than Control. Therefore, at least one sugar chain from hLF (native hLF) is obtained by the hydrolysis reaction with endo S. Is considered liberated.
  • Comparative Example 7 that is, in the hydrolysis reaction with endo M (WT), a band appears on the lower molecular weight side than Control, so it is considered that at least one sugar chain was detached from the hLF.
  • End M (WT) does not hydrolyze N-linked sugar chains that do not have core fucose, but it is considered that N-linked sugar chains that do not have core fucose are also included in the hLF.
  • Example 5 As Example 5, a hydrolysis reaction for hLF of the W251N mutant was performed. 100 ⁇ g of hLF prepared above was dissolved in 100 ⁇ l of 100 mM sodium phosphate buffer (pH 6.5), 20 ⁇ g of W251N mutant was added, and reacted at 30 ° C. for 16 hours. After completion of the reaction, the post-reaction solution was dried by centrifugal concentration, redissolved in 5% acetic acid, and the obtained solution was applied to a C18 column. After the application, the fraction not bound to the column was collected and lyophilized to obtain a dried product of the N-linked sugar chain fraction released by hydrolysis with the endo M mutant. The dried product was subjected to sugar chain analysis by the following mass spectrometry.
  • Example 9 a hydrolysis reaction for hLF was performed in the same manner as in Example 5 except that 20 ⁇ g of endo M (WT) was used instead of 20 ⁇ g of the W251N mutant, and sugar chain analysis by mass spectrometry was performed.
  • WT endo M
  • FIGS. 10A and 10B show the results of MALDI-TOF MS measurement of the sugar chain released after the hydrolysis reaction of hLF with endo M (WT), and FIG. 10B shows the result of W251N mutant against hLF.
  • WT endo M
  • FIGS. 10A and 10B black squares indicate N-acetylglucosamine, gray circles indicate mannose, white circles indicate galactose, and black diamonds indicate N-acetylneuraminic acid.
  • Example 6 As Example 6, a hydrolysis reaction of W251N mutant against Rituxan (antibody, IgG1) was performed. 200 ⁇ g of the above-mentioned commercially available Rituxan was dissolved in 100 ⁇ l of 50 mM sodium phosphate buffer (pH 6.25), and at 4 ° C. with a membrane concentrator [fraction molecular weight 10,000, Amicon Ultra 15 (manufactured by Merck Millipore)] The buffer was exchanged. To 100 ⁇ L of 50 mM sodium phosphate buffer solution (pH 6.25) in which 200 ⁇ g of Rituxan (IgG1) obtained by buffer exchange was dissolved, 1 mg of W251N mutant was added after 0 hours, 24 hours, and 48 hours, respectively.
  • reaction time 0 hour (A), 24 hours (B), 48 hours (C), and 72 hours (D)
  • reaction time 0 hour (A)
  • a part of the post-reaction solution is used as a sample.
  • Each sample was further reduced and subjected to MS analysis with MALDI-TOF.
  • FIG. 11 As shown in FIG. 11, as the reaction time elapses, the peak at the position corresponding to the molecular weight of the heavy chain to which the N-linked sugar chain is bound decreases, and instead of the mass of the heavy chain having no N-linked sugar chain.
  • the W251N mutant has hydrolytic activity also on the fucose-containing N-linked sugar chain of IgG.
  • Example 7 The sugar chain transfer reaction was performed by preparing 20 ⁇ L of a solution having the following composition conditions in a 1.5 mL microtube and then incubating at 30 ° C. for 60 minutes in a thermostatic bath. ⁇ conditions ⁇ Sugar chain donor: 5 mM SG-oxazole Sugar chain receptor: 5 mM F1N1-biotin Endo M mutant: 0.66 ⁇ g W251N mutant Buffer concentration and pH and total volume of the solution: 25 mM sodium phosphate (pH 6.5) 20 ⁇ L After incubation, the reaction was stopped by heat treatment at 95 ° C. for 5 minutes to obtain a solution after the reaction. The resulting solution was dried, dissolved in 20 mg / mL DHBA solution and subjected to MALDI-TOF MS analysis.
  • Comparative Examples 10 to 12 As shown in Table 3 below, in Comparative Example 10, the sugar chain receptor in Example 7 was the same concentration of N1-biotin, and in Comparative Example 11, the endo M mutant in Example 7 was the same mass of the N175Q mutant. In Comparative Example 12, the reaction was performed under the same conditions except that the sugar chain receptor in Comparative Example 11 was changed to the same concentration of F1N1-biotin, and the solution after the reaction was similarly subjected to MALDI-TOF MS analysis.
  • FIG. 12 the outline
  • N1-biotin or F1N1-biotin and SG-oxazoline were condensed by the transglycosylation reaction of W251N mutant or N175Q mutant, respectively.
  • Product, SG-biotin or SGF-biotin was detected. Details are shown below.
  • Example 7 The analysis result in MALDI-TOF MS is shown in FIG.
  • B a product obtained by condensing F1N1-biotin and SG-oxazoline, that is, a sodium ion adduct of SGF-biotin (NeuAc2Gal2GlcNAc2Man3GlcNAc2Fuc-biotin [corresponding to M + 3Na-2H +)
  • a possible signal (m / z 2719.8) was detected.
  • Comparative Example 10 using N1-biotin as the sugar chain receptor no peak corresponding to the condensation product could be detected.
  • Comparative Example 11 C in FIG.
  • Example 7 MS / MS analysis was further performed on the signals obtained in Example 7 and Comparative Example 11 (B and C in FIG. 13). The results are shown in the upper part of FIG. 14 for Example 7 and the lower part of FIG. 14 for Comparative Example 11.
  • ⁇ white rhombus
  • ⁇ white circle
  • ⁇ black circle
  • Man Man
  • ⁇ white square
  • GlcNAc GlcNAc
  • Fuc Fuc
  • the endo M mutant of the present disclosure efficiently transfers a sugar chain donor to a sugar chain acceptor to which core fucose is bound.

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Abstract

La présente invention concerne, selon un mode de réalisation : une variante d'endo-M possédant une séquence d'acides aminés dans laquelle le résidu d'acide aminé en position 251 de la séquence d'acides aminés représentée par SEQ ID NO : 1 est l'asparagine ou l'alanine, et ayant une activité de catalyse d'une réaction d'hydrolyse représentée par la formule réactionnelle suivante (1) (X représente un groupe dérivé des glucides et Y est un substituant monovalent); ou une variante d'endo-M possédant une séquence d'acides aminés dans laquelle le résidu d'acide aminé en position 251 de la séquence d'acides aminés représentée par SEQ ID NO : 1 est l'asparagine ou l'alanine, possédant une séquence d'acides aminés modifiée présentant au moins 80 % d'homologie avec la séquence d'acides aminés suite à une délétion, addition ou substitution d'un ou de plusieurs résidus d'acides aminés autres que le résidu d'acide aminé en position 251 de la séquence d'acides aminés représentée par SEQ ID NO : 1, et ayant une activité de catalyse de la réaction d'hydrolyse représentée par la formule réactionnelle suivante (1).
PCT/JP2016/055926 2015-02-26 2016-02-26 Variante d'endo-m, et procédé de préparation d'un composé ou d'une protéine contenant une chaîne glucidique n-liée Ceased WO2016136984A1 (fr)

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CN110036109A (zh) * 2016-12-02 2019-07-19 第一三共株式会社 新颖的内-β-N-乙酰基氨基葡糖苷酶

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WO2013120066A1 (fr) * 2012-02-10 2013-08-15 University Of Maryland, Baltimore Glyco-ingénierie chimio-enzymatique d'anticorps et de leurs fragments fc

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WO2013120066A1 (fr) * 2012-02-10 2013-08-15 University Of Maryland, Baltimore Glyco-ingénierie chimio-enzymatique d'anticorps et de leurs fragments fc

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110036109A (zh) * 2016-12-02 2019-07-19 第一三共株式会社 新颖的内-β-N-乙酰基氨基葡糖苷酶
KR20190088483A (ko) * 2016-12-02 2019-07-26 다이이찌 산쿄 가부시키가이샤 신규 엔도-β-N-아세틸글루코사미니다아제
EP3550018A4 (fr) * 2016-12-02 2020-05-27 Daiichi Sankyo Company, Limited NOUVELLE ENDO-ß-N-ACÉTYLGLUCOSAMINIDASE
US11208442B2 (en) 2016-12-02 2021-12-28 Daiichi Sankyo Company, Limited Endo-beta-N-acetylglucosaminidase
KR102399639B1 (ko) 2016-12-02 2022-05-18 다이이찌 산쿄 가부시키가이샤 신규 엔도-β-N-아세틸글루코사미니다아제
TWI777992B (zh) * 2016-12-02 2022-09-21 日商第一三共股份有限公司 新穎內-β-N-乙醯基胺基葡萄糖苷酶
AU2017368597B2 (en) * 2016-12-02 2023-12-14 Daiichi Sankyo Company,Limited Novel endo-β-N-acetylglucosaminidase
AU2017368597C1 (en) * 2016-12-02 2024-03-28 Daiichi Sankyo Company,Limited Novel endo-β-N-acetylglucosaminidase

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