JP2017055675A - Modified complex-type sugar chain hydrolyzing enzymes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide modified Endo-Oms with high specific activities compared with parent Endo-Om being an Endo-β-N-acetylglucosaminidase already excellent in specific activity etc.SOLUTION: Using a structure modelling, the inventors predicted that the amino acid residue constituting the active center of Endo-Om is the 295th tryptophan (W295) and studied the amino acid substitution of W295. As a result, the invention provides a variant W295F exhibiting a higher specific activity in comparison with Endo-Om as well as a variant W295Y that shows stronger activity to biantennary complex sugar chains than Endo-Om does.SELECTED DRAWING: None

Description

  The present invention relates to a modified endo-β-N-acetylglucosaminidase having high specific activity and a gene thereof.

In recent years, in the field of antibody drugs, the market size of which is increasing year by year as one of biopharmaceuticals, anti-body drug conjugate technology has been developed, and several drug-conjugated antibody drugs have been launched. Has been. However, ADC has not established a technique for binding a drug in a site-specific manner, and the ratio of drug to antibody cannot be controlled. As a solution, introduction of drugs into two glycosylation sites in one antibody molecule has begun to be studied (Non-patent Document 1).
Endo-β-N-acetylglucosaminidase (ENGase) is an enzyme that catalyzes the reaction of transducing sugar chains to complex carbohydrates in addition to the reaction of cleaving sugar chains by acting on N-linked sugar chains of glycoproteins. It has been widely used for the analysis of sugar chain structure of glycoprotein, modification of glycoconjugate sugar chain, preparation of Neoglycoprotein, homogenization of sugar chain part of glycoprotein, etc. There is an increasing expectation for the introduction of sugar chains modified by drugs in the production of biopharmaceuticals.
The present inventors previously isolated a novel ENGase (Endo-Om) from the methanol-assimilating yeast Ogataea minuta (Patent Document 1). Endo-Om, like ENGase (Endo-M) derived from Mucor hiemalis (Non-patent Document 2), has hydrolytic activity and transfer to any high-mannose, hybrid, or complex N-type sugar chain. It can exhibit activity, has a different substrate specificity, and has a higher specific activity than Endo-M. Therefore, in the introduction of drug-binding sugar chains of biopharmaceuticals, there is a great expectation for Endo-Om with high specific activity, including the manufacturing cost.
For industrial use of ENGase, it is desired to provide enzymes with different substrate specificities and enzymes with higher specific activity.
For this reason, attempts have been made to produce a variant in which a mutation has been introduced into ENGase. As an example of increasing the substrate specificity, glycosylation can be achieved by introducing a mutation into Endo-M or Endo-A. Reports that the degradation activity was suppressed and the transglycosylation activity was improved (Non-patent Documents 3 to 5), and that the ability to cleave a sugar chain in which core fucose was added to Endo-M could be added (Non-patent Document 6). )
However, none of them improved the specific activity of ENGase. In general, in order to improve the specific activity of an enzyme, it is necessary to analyze the catalytic mechanism of the enzyme in detail, identify the amino acid residues involved, and create and evaluate many substitutions. There are no reports of studies to improve specific activity systematically.

International Publication 2013-051608 (WO2013 / 051608)

Li X, et al., Angew Chem Int Ed. 2014 53: 7179-82. Fujita et al., (2004) Arch Biochem Biophy. 432: p41-49 Umekawa M, et al., J Biol Chem. 2008 Feb 22; 283 (8): 4469-79. Umekawa M, et al., J Biol Chem. 2010 Jan 1; 285 (1): 511-21. Huang W, et al., Chembiochem. 2010 Jul 5; 11 (10): 1350-5. Norihiko Kato et al., Abstracts of Annual Meeting 2015 Agricultural Chemistry Society of Japan p.713

  An object of the present invention is to provide a modified ENGase that further enhances the specific activity of Endo-Om, which is an ENGase excellent in terms of specific activity.

The present inventors considered that amino acid residues constituting the active center of Endo-Om (Patent Document 1) previously developed by the present inventors were estimated by structural modeling, and amino acid substitution of the amino acid residues was performed. It was. Many examples of changing the substrate specificity from structural modeling are known, but in general, it is not easy to improve the activity, but rather the activity is often reduced by mutation. Moreover, structural modeling of Endo-Om (Fig. 1) revealed that the tryptophan (W) residue at position 295, which is supposed to interact with the substrate sugar chain, is conserved as a catalytic residue in the GH85 family. In addition, the GH85 ENGase family has low conservative properties and is far away from the 196th glutamic acid (E), 194th asparagine (N), and 231th tyrosine (Y) residues. There are no examples that have been scientifically substituted, and from the study of related enzymes, the tryptophan (W) residue at position 295 cannot be inferred as a residue involved in specific activity.
Therefore, the present inventors have never predicted that specific activity will be improved by amino acid substitution at W (W295) at position 295 of Endo-Om, and the substrate specificity is slightly increased (glycan structure that is not cleaved). When various amino acid substitutions were tried in anticipation of the possibility, a mutant enzyme with an unexpectedly high specific activity could be obtained.

Specifically, it is as follows.
Amino acid residues constituting the active center of the enzyme (Endo-Om) disclosed in Patent Document 1 are estimated by structural modeling, and amino acid substitution is performed to estimate amino acids that affect substrate specificity and specific activity. Went. As a result, it was predicted that the 295th tryptophan (W295) was important for retaining the sugar chain as a substrate. In addition, in relation to substrate specificity, it was suggested that Endo-Om could cause steric hindrance with the sugar chain structure that cannot be cleaved. Therefore, if steric hindrance could be suppressed by amino acid substitution, substrate specificity would be expected to expand. Therefore, when W295 amino acid substitution was performed, contrary to expectation, the substrate specificity did not change, but compared with the enzyme before mutation, the W295F variant exhibiting strong activity against various sugar chains and glycoproteins and It was possible to isolate a W295Y variant exhibiting strong activity against a specific sugar chain. In particular, the improvement in activity in the W295F variant was extremely high.
Moreover, when the reactivity with respect to ribonuclease (RNase) B having a high mannose sugar chain and transferrin having a complex biantennary sugar chain was confirmed, the same tendency was confirmed.
Obtaining the above knowledge, the present invention has been completed.

That is, the present invention is as follows.
[1] In the amino acid sequence of endo-β-N-acetylglucosaminidase (Endo-Om), the amino acid residue corresponding to the tryptophan (W) residue at position 295 of SEQ ID NO: 2 is a phenylalanine (F) residue or tyrosine (Y) Endo-Om variants (W295F, W295Y) substituted with residues, have the same substrate specificity as the pre-mutation Endo-Om, and at least have a specific activity to complex sugar chains Endo-Om variants that are better than the previous Endo-Om.
[2] The Endo-Om variant according to [1], wherein the amino acid sequence of Endo-Om before mutation of the Endo-Om variant has the amino acid sequence represented by SEQ ID NO: 2.
[3] A nucleic acid encoding the Endo-Om variant according to [1] or [2].
[4] A recombinant vector comprising the nucleic acid according to [3] above and capable of expressing the nucleic acid in a host cell.
[5] A transformant into which the nucleic acid according to [3] is introduced and expresses an Endo-Om variant.
[6] A method for producing an Endo-Om variant having the same substrate specificity as that of the pre-mutation Endo-Om and having at least a specific activity for complex sugar chains that is higher than that of the pre-mutation Endo-Om. In the amino acid sequence of endo-β-N-acetylglucosaminidase (Endo-Om), the amino acid residue corresponding to the tryptophan (W) residue at position 295 of SEQ ID NO: 2 is substituted with phenylalanine (F) residue or tyrosine ( Y) A method for producing a modified Endo-Om, comprising performing substitution to mutate a residue.
[7] A method for cleaving an asparagine-linked sugar chain from a target glycoprotein, comprising the step of causing the Endo-Om variant according to [1] or [2] to act on the target glycoprotein.
[8] A method for transferring an asparagine-linked sugar chain to a target acceptor molecule, comprising a step of causing the Endo-Om variant according to [1] or [2] to act on a target acceptor molecule.

The modified Endo-Om W295F of the present invention exhibits a high specific activity of 2 to 3 times with respect to all of the main sugar chains to which the original Endo-Om reacts, especially in the M9A sugar chain of the high mannose type sugar chain. The specific activity was improved by more than 3 times. The specific activity against complex type biantennary sugar chain (agalacto biantennary) was also close to 3 times. In the case of the modified Endo-Om W295Y, the specific activity of Oligomannose-type sugar chains such as Endo-Om decreased to about 0.6 to 0.7 times compared to the original Endo-Om, but the complex type bifurcated sugar chain The activity against (agalacto biantennary) was 2.3 times higher.
By using these Endo-Om modifying enzymes, it is possible to hydrolyze sugar chains and glycoproteins in a smaller amount than before, and it is possible to provide enzymes that are superior in terms of production costs.
Since it was possible to provide an Endo-Om variant with increased specific activity, particularly specific activity for complex sugar chains, without changing the substrate specificity of Endo-Om having a wide range of substrate specificity and high specific activity, In addition to using the modified enzyme itself as a reagent for sugar chain analysis, a reagent for introducing a sugar chain, or the like, a standard glycopeptide composition for sugar chain analysis or sugar chain introduction, or a sugar chain structure such as a biopharmaceutical is uniform. Applications such as the production of a modified composition are expected.

A: Structural modeling of Endo-Om from Endo-A and Endo-D structures (using SWISS-MODEL and DeepView). B: Prediction of binding between N-type sugar chain (Man3GlcNAc2-Asn) and active center by pivot modeling method based on the result of co-crystallization of Endo-A and substrate. Amino acid sequence conserved region within Endo-Om's GH85 ENGase family. It can be seen that Endo-Om W295 is not storable. The figure which isolate | separated various Endo-Om protein used for activity measurement by SDS-polyacrylamide electrophoresis, and was stained with Coomassie. It can be seen that Endo-Om with an expected molecular weight of 92 kDa has been purified almost uniformly. RNase B and transferrin treated with Endo-Om, separated by SDS-polyacrylamide electrophoresis and stained with Coomassie. Since the mobility of RNase B and transferrin changes depending on the presence or absence of sugar chains, the strengths of various Endo-Om activities against these glycoproteins can be compared. The asterisk in the figure is a visualization of Endo-H.

1. Endo-β-N-acetylglucosaminidase (ENGase)
(1-1) Action of endo-β-N-acetylglucosaminidase Endo-β-N-acetylglucosaminidase is an enzyme that cleaves chitobiose present at the reducing end of N-type sugar chains by hydrolysis. These enzymes simultaneously have transglycosidase activity that efficiently catalyzes the reaction of transferring the excised sugar chain to the acceptor carbohydrate or complex carbohydrate.
So far, Endo-A from Arthrobacter, Endo-D from Streptococcus pneumoniae, Endo-F from Flavobacterium, Endo-H from Streptomyces plicatus, Endo-Os from rice, Endo-M from Mucor hiemalis (non-patented) Document 6) is known.
Therefore, endo-β-N-acetylglucosaminidase can be used to analyze glycoprotein sugar chain structure, modify glycoconjugate sugar chains, prepare neoglycoprotein, and homogenize the sugar chain part of glycoproteins. This is a useful group of enzymes.

(1-2) Types of main sugar chains on which ENGase acts The main N-linked sugar chains of main glycoproteins that control the main biological activities of the body are the standard 3-mannose oligosaccharides From the sugar chain structure bound to the non-reducing end of the chain (M3B), it is classified into high mannose type (oligomannose type, mannan type sugar chain), hybrid type and complex type (Essentials of Glycobiology Chapter 8 http: // (See www.ncbi.nlm.nih.gov/books/NBK1908/).
A high mannose type (oligomannose type, mannan type) sugar chain is a type in which many mannoses having a mannose number of 3 to 9, mainly 5 to 9, are bonded. Specific examples include M3B, M4A, M4B, M5A, M6A, M6B, M6C, M7A, M7B, M7C, M7D, M8A, M8B, M8C, and M9A sugar chains. Typical high mannose-type sugar chains are referred to as “MαA” [M5A], “Manα1-3 [Manα1-3 (Manα1-6) Manα1-6] Manβ1-4GlcNAcβ1-4GlcNAc-”, “M6B”. `` Manα1-2Manα1-3 [Manα1-3 (Manα1-6) Manα1-6] Manβ1-4GlcNAcβ1-4GlcNAc- '', referred to as `` M8A '', `` Manα1-2Manα1-2Manα1-3 [Manα1-3 (Manα1-2Manα1 -6) Manα1-6] Manβ1-4GlcNAcβ1-4GlcNAc- ”, referred to as“ M9A ”,“ Manα1-2Manα1-2Manα1-3 [Manα1-2Manα1-3 (Manα1-2Manα1-6) Manα1-6] Manβ1-4GlcNAcβ1 -4GlcNAc- ".
A hybrid (hybrid type) sugar chain is a sugar chain having an oligomannose chain and an oligosaccharide composed of N-acetylglucosamine, galactose, and sialic acid. Typical hybrid sugar chains include “Galβ1-4GlcNAcβ1-2Manα1-3 [Manα1-3 (Manα1-6) Manα1-6] Manβ1-4GlcNAcβ1-4GlcNAc-” and “hybrid-type”, There is “GlcNAcβ1-2Manα1-3 (GlcNAcβ1-4) [Manα1-3 (Manα1-6) Manα1-6] Manβ1-4GlcNAcβ1-4GlcNAc-”, which is called “hybrid-type (bisecting GlcNAc)”.
Complex sugar chains refer to N-linked sugar chains including N-acetylglucosamine, galactose, sialic acid, and fucose, and are further branched, branched, and branched according to the number of branches on the non-reducing end. , Sometimes divided into bisecting types. Examples of biantennary complex type sugar chains include sialyl biantennary, asialo biantennary, agaracto biantennary, agaracto biantennary (bisecting GlcNAc) type sugar chains, or structures in which some of these sugar chains are missing. The three-branched complex sugar chains include `` sialyl (2,6) -branched triantennary '', `` asialo (2,6) -branched triantennary '', `` agalacto (2,6) -branched triantennary '', `` sialyl Examples include (2,4) -branched triantennary ”,“ asialo (2,4) -branched triantennary ”,“ agalacto (2,4) -branched triantennary ”type glycans, or structures lacking some of these glycans. It is done. Examples of the four-branching complex type sugar chain include “sialyl tetraantennary”, “asialo tetraantennary”, “agalacto tetraantennary” type sugar chains, or a structure in which some of these sugar chains are deleted. These bi-branched, tri-branched, and tetra-branched sugar chains in which GlcNAc is added by β1,4-bonded mannose are called bisecting types. As a typical complex type sugar chain,
`` Siaobiantennary '' or `` sialyl biantennary '' called `` Siaα2-6Galβ1-4GlcNAcβ1-2Manα1-3 (Siaα2-6Galβ1-4GlcNAcβ1-2Manα1-6) Manβ1-4GlcNAcβ1-4GlcNAc- '', `` asialo biantennary '' Galβ1-4GlcNAcβ1-2Manα1-3 (Galβ1-4GlcNAcβ1-2Manα1-6) Manβ1-4GlcNAcβ1-4GlcNAc- There is "4GlcNAc-".

  In general, when evaluating the specific activity of various ENGases, M3B-PA, in which PA (pyridylamino group) is bonded to M3B sugar chain, is usually used. When evaluating the length, PA is bound to a typical sugar chain of each sugar chain group. In the case of a high mannose type sugar chain, it is M9A-PA, further M7B-PA, M6B-PA, M5A-PA, and the hybrid sugar chain is hybrid-type-PA, further hybrid-type (bisecting GlcNAc) As the complex type sugar chain, agalacto biantennary-PA and further asialo (2,4) -branched triantennary-PA are used.

  There is a bias in the types of sugar chains that can be released by various ENGases, and there are few types of ENGases that have been confirmed to be reactive with all three types of sugar chains. In particular, there are only a few types such as Endo-M other than Endo-Om that are reported to have the activity of cleaving complex sugar chains. Regarding the modification of complex-type sugar chains, the substrate specificity of transglycosidase activity is the same as the degradation activity based on conventional knowledge, so only these enzymes can transfer complex-type sugar chains to acceptors. Absent.

(1-2) Ogataea minuta-derived endo-β-N-acetylglucosaminidase (Endo-Om)
Endo-Om is an endo-β-N-acetylglucosaminidase (SEQ ID NO: 1, 2) previously obtained from the methanol-utilizing yeast Ogataea minuta strain IFO10746 by the present inventors. The physicochemical properties are as follows. (Patent Document 1)
(1) Action: It acts on an N-linked glycoprotein in an endo-type to release a sugar chain and has transglycosidase activity to transfer the released sugar chain to an acceptor compound.
(2) Substrate specificity; with high mannose type, mixed type, cleaves between N, N'-diacetylchitobiose existing in the core structure of biantennary complex type sugar chain to produce oligosaccharide, and acceptor molecule Transfer. Here, the acceptor molecule that can be transferred is a monosaccharide such as glucose or GlcNAc or a derivative thereof, but it can also be transferred to glycopeptides and glycoproteins having them.
(3) Specific activity: Endo-Om has a specific activity as high as 13 times that of Endo-M and a Vmax as high as 55 times that of Endo-M.
(4) Reactivity to each sugar chain; high hydrolysis activity for high mannose type sugar chains, and further hydrolyzes hybrid sugar chains and bifurcated complex sugar chains, but complex sugar chains with 4 or more branches Sugar chains with a core fucose structure cannot be decomposed. For tri-branched sugar chains, agalacto (2,6) -branched triantennary can be degraded, but agalacto (2,4) -branched triantennary cannot be degraded. In particular, agaracto biantennary, M3B, M6B, and M9A sugar chains are more reactive than Endo-M.

3. Endo-Om variants Endo-Om W295F and Endo-Om W295Y, which are amino acid variants of the Endo-Om of the present invention, are obtained by replacing the tryptophan (W) residue at the amino acid sequence position 295 of Endo-Om with a phenylalanine (F) residue. Group or a mutated enzyme mutated to a tyrosine (Y) residue and can be mass-produced by an E. coli host.
From the three-dimensional structure modeling of Endo-Om and the predicted binding to the substrate, it was assumed that W located at position 295 is not related to the specific activity of the enzyme, but exclusively determines the substrate specificity. In both cases, the substrate specificity of Endo-Om before mutation was not changed at all except that the specificity of W295Y for agalacto biantennary was improved (Tables 1 to 3).
On the other hand, the activity for each substrate was greatly different, and Endo-Om W295F showed an improvement of 2 to 3 times or more for both high mannose type sugar chains and biantennary complex type sugar chains. In contrast, in W295Y, the activity against high mannose-type sugar chains does not reach the enzyme activity before mutation, but it shows 2.34 times the specific activity for biantennary complex-type sugar chains as compared to the original Endo-Om. It was.
In addition, none of the Endo-Om 295 W amino acid mutants W295F and W295Y other than the amino acid mutants (W295A, W295E, W295Q) showed any enzyme activity.
As described above, the Endo-Om W295F and Endo-Om W295Y mutants provided in the present invention change the substrate specificity of Endo-Om having a wide substrate specificity and high specific activity. In particular, the specific activity for complex-type glycans is improved, so that, as with Endo-Om, a wide range of applications such as introduction of drug-bound glycans into biopharmaceuticals can be expected, and a reagent with high specific activity is inexpensive. The excellent effect that can be provided is expected.

4). Method for Producing Endo-Om Variant The variant of the present invention uses a cDNA encoding Endo-Om (for example, SEQ ID NO: 1) as a template and a codon encoding tryptophan (W) at position 295 as phenylalanine (F) or tyrosine. Although it is generally performed using a PCR primer designed to substitute for the codon (Y), a known point mutation method can also be applied. In the examples of the present invention, PCR was performed using pOMEA1-6H3F-Endo-Om, which is an expression vector for Escherichia coli described in Patent Document 1, as a template, the amplified DNA fragment was cleaved with NdeI and NotI, and pET30a (Novagen) Inserted into the NdeI-NotI site.
The obtained cDNA mutant can be obtained in large quantities by introducing and expressing it in Escherichia coli, yeast, insect cells, or animal cells using expression vectors that can be amplified in the respective hosts.
For mass production, it is preferable to use Escherichia coli or yeast as a host. In the examples of the present invention, Escherichia coli BL21 (DE3) pLysS (Biodynamics Research Laboratories) induced by the heat shock method was used. It is not limited. As the recombinant vector, a known vector suitable for the host is used. For Escherichia coli, Escherichia coli plasmids such as pDONR (trademark) 201, pBluescript, pUC18, pUC19, pBR322, pTAPlus, pDrive, and pETBlue-1 are widely used, but are not limited thereto.
In addition, methanol-utilizing yeast (Ogataea minuta IFO10746 strain), which is an Endo-Om producing microorganism before mutation introduction, is also preferable as a host for mass production. By appropriately introducing a known yeast vector and applying a culture method using methanol induction, a large amount of the modified product of the present invention can be obtained. For example, an overexpression strain of the variant of the present invention using the O. minuta TK10-1-2 strain as a host can be produced according to the description in (Patent Document 1).
Methods for introducing the recombinant vector into the host cell include calcium chloride method or calcium chloride / rubidium chloride method, electroporation method, electroinjection method, method using chemical treatment such as PEG, method using gene gun, etc. Is mentioned.
The variant of the present invention can be produced by culturing transformed cells containing the expression vector prepared as described above in a known nutrient medium such as LB medium (Difco). The culture conditions such as the culture temperature, the pH of the medium and the culture time are appropriately selected so that the modified product of the present invention is produced in large quantities.
The modified product of the present invention is separated from the culture obtained by the above culture using operations such as ultrasonic disruption, centrifugation, and filtration, and is subjected to an isolation / purification process.
As a method of isolation / purification, a normal protein isolation / purification method can be applied. Specifically, affinity chromatography, ion exchange chromatography, gel filtration, hydrophobic chromatography, isoelectric point, etc. Examples thereof include chromatography, and a method of combining them.

EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and the techniques used to implement the present invention are not specifically limited to the techniques that clearly indicate the source. Can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in the technical literature and patent gazette cited in this specification is regarded as a part of the disclosure content of this specification.

(Example 1) Estimation of amino acids constituting the active center of Endo-Om by in silico analysis
The conformational modeling of the Endo-Om N-terminal region (Pro74-Ile377) is based on the structure of Endo-A (PDB ID: 3FHQ) and Endo-D (PDB ID: 2W92). SWISS-MODEL (http: //swissmodel.expasy.org) and DeepView (http://spdbv.vital-it.ch) (FIG. 1A). Subsequently, based on the result of co-crystallization of Endo-A and the substrate, prediction of the binding (fitting) between the N-type sugar chain (Man3GlcNAc2-Asn) and the active center was performed by the pivot modeling method (FIG. 1B). As a result, it was predicted that a total of 14 amino acids constitute the active center of Endo-Om and are amino acid residues that bind to the substrate. Of these, Asn-194, Glu-196, and Tyr-231 located in the lower right direction in FIG. 1B are amino acids that are well conserved in the GH85 ENGase family. Analysis of Endo-A, Endo-D, and Endo-M Thus, it is reported that the residue recognizes chitobiose present at the cleavage site at the reducing end of the N-type sugar chain. Therefore, amino acid residues involved in Endo-Om sugar chain scission activity are thought to exist near the site of action.
On the other hand, W295 present on the opposite side and far from the point of cleaving action may be an amino acid residue that is not related to the cleaving activity and that exclusively determines the substrate specificity.
Endo-Om is active on biantennary complex-type sugar chains in which GlcNAc is β1,2 linked to α1,3-linked mannose, but GlcNAc is linked to α1,3-linked mannose by β1,2 and β1,4 bonds. It has been clarified that the tribranched glycan linked with two molecules shows no reactivity. From the results of sugar chain fitting, Endo-Om's 295th tryptophan (W295) is the α1,3-linked mannose of the N-type sugar chain that is the substrate, and N-acetylglucosamine that binds β1,2 to the mannose It was predicted to be located near the (GlcNAc) residue. Therefore, it was estimated that the substrate specificity of Endo-Om was defined by W295.
Therefore, in order to further expand the substrate specificity of the Endo-Om complex sugar chain, the present inventors have made steric hindrance so that even a bulky three-branched complex sugar chain that Endo-Om cannot cleave is brought closer to the cleavage site. Attempts were made to replace the tryptophan (W) residue with an amino acid residue having a functional group with less steric hindrance.

(Example 2) Expression of recombinant Endo-Om in E. coli In order to express Endo-Om (SEQ ID NOs: 1 and 2) in E. coli, the following plasmids were constructed. To make 10 histidine residues bind to the N-terminal side of Endo-Om,
Primer A:
5'-AAGGAGATATACATATGCATCACCATCACCATCACCATCACCATCACGACTACAAAGACCATGACGG-3 '(SEQ ID NO: 3) and
Primer B:
5'-TGCTCGAGTGCGGCCGCTCACACCCAAACCTCACTC-3 '(SEQ ID NO: 4)
Then, PCR was performed using pOMEA1-6H3F-Endo-Om in Patent Document 1 as a template. The amplified DNA fragment was cleaved with NdeI and NotI and inserted into the NdeI-NotI site of pET30a (Novagen). The obtained plasmid was designated as pET30-10H3F-EOm.
Next, gene mutations were introduced into pET30-10H3F-EOm using a QuickChange site-directed mutagenesis kit (Agilent) to prepare each mutant expression vector.
For mutagenesis, W295F
Primer C:
5'-GACGTCTTCGGCCGTGGAACGCTGGTT-3 '(SEQ ID NO: 5) and primer D:
5'-ACGGCCGAAGACGTCGTAGCCGACGTA-3 '(SEQ ID NO: 6)
W295Y
Primer E:
5'-GACGTCTACGGCCGTGGAACGCTGGTT-3 '(SEQ ID NO: 7) and primer F:
5'-ACGGCCGTAGACGTCGTAGCCGACGTA-3 '(SEQ ID NO: 8)
W295A
Primer G: 5'-GACGTCGCCGGCCGTGGAACGCTGGTT-3 '(SEQ ID NO: 9) and Primer H: 5'-ACGGCCGGCGACGTCGTAGCCGACGTA-3') (SEQ ID NO: 10)
W295Q
Primer I: 5′-GACGTCCAAGGCCGTGGAACGCTGGTT-3 ′ (SEQ ID NO: 11) and Primer J: 5′-ACGGCCTTGGACGTCGTAGCCGACGTA-3 ′ (SEQ ID NO: 12)
W295E
Primer K: 5'-GACGTCGAGGGCCGTGGAACGCTGGTT-3 '(SEQ ID NO: 13) and Primer L: 5'-ACGGCCCTCGACGTCGTAGCCGACGTA-3' (SEQ ID NO: 14)
It was used. The obtained expression vector was amplified in E. coli and then subjected to DNA sequencing to confirm the introduction of mutation.
Using each expression vector, E. coli BL21 (DE3) pLysS (Biodynamics Laboratory) was transformed using the heat shock method. The resulting transformant was cultured overnight at 37 ° C in LB medium (Difco) containing 50 μg / ml kanamycin and 34 μg / ml chloramphenicol, and then 100 ml Overnight Express Instant LB medium (Merck Millipore ) And cultured at 16 ° C. for 2 days. After culturing, the cells were collected by centrifugation and suspended in a storage buffer (20 mM sodium phosphate buffer (pH 7.4) containing 500 mM sodium chloride, 50 mM imidazole, 1 mM phenylmethylsulfonyl fluoride). For ultrasonic crushing, a Q125 ultrasonic homogenizer (Qsonica) was used, and crushing conditions were determined to repeat Amplitude 40%, 1 sec pulse, 4 sec interval 6 times. The crushed liquid was centrifuged at 15,000 × g for 20 minutes at 4 ° C. to remove unnecessary materials. The soluble fraction was subjected to HisTrap HP (1 mL, GE Healthcare) equilibrated with a storage buffer containing 50 mM imidazole, washed with a storage buffer containing 200 mM imidazole, and then washed with a storage buffer containing 500 mM imidazole. The fraction containing Endo-Om was eluted. The obtained amino acid variant fractions of W295 were each dialyzed with a storage buffer, and it was confirmed by SDS-PAGE that each variant was purified uniformly (FIG. 3), and then stored at −80 ° C.

(Example 3) Analysis of a substitution product of the 295th tryptophan of Endo-Om
Substitution of W295 for other amino acid residues was performed, and changes in enzyme activity and substrate specificity were analyzed. The activity was measured by the method described in Patent Document 1 using a PA (pyridylamino) sugar chain (Takara Bio Inc.). The results are shown in (Table 1). W295A, W295E, and W295Q did not show activity against any of the substrates, whereas W295Y and W295F had the same activity against the same substrate as that of WY before mutagenesis. Except for the specificity of W295Y for agalacto biantennary, substrate specificity It was confirmed that the sex was not changed.
(Table 2) shows the relative activities of WY, W295F and W295Y in Table 1 with respect to each sugar chain with respect to Man3GlcNAc2-PA (M3B).
Moreover, as (Table 3), the activity with respect to each sugar chain of W295F and W295Y was shown by the relative activity with WY.

As a result of the above, W295F has the same tendency as WT, that is, M5A of high mannose type sugar chain and agaracto biantennary are low, and the activity to other high mannose type sugar chains is high. Although there is a tendency, it was confirmed that the relative activity to all sugar chains including the bifurcated complex type sugar chain was higher than that of WY. On the other hand, W295Y showed a tendency that the relative activity of the biantennary complex type sugar chain was higher than that of WY. None of the amino acid mutants other than W295F and W295Y (W295A, W295E, W295Q) showed any activity (Table 1).
Specifically, when the relative activity to each sugar chain of W295F and W295Y is assumed to be 100% of the enzyme activity before mutagenesis (Table 3), in the case of W295F, a high mannose-type sugar chain and two branches The specific activity was 2 to 3 times higher than that of any complex type sugar chain. In W295Y, the activity against high mannose-type sugar chains does not reach the enzyme activity before mutation, but it is not inferior to the specific activity value of conventional Endo-M (Non-patent Document 2), and is a bifurcated complex type. The specific activity of sugar chains is 2.34 times that of the original Endo-Om, which is about 8 times that of Endo-M.

(Example 4) Reactivity of Endo-Om W295F and W295Y to glycoprotein In Example 3, the specific activity of W295F and W295Y mutant enzymes with respect to PA sugar chains increased at least with respect to complex sugar chains. The reactivity to ribonuclease (RNase) B having a mannose sugar chain and transferrin having a complex sugar chain was examined. When the enzyme was reacted with native RNaseB, W295F had an increased proportion of RNaseB whose sugar chain was cleaved compared to the enzyme before mutation introduction. On the other hand, W295Y was less than or equal to the enzyme before the mutation introduction. As for denatured RNase B, RNase B with the sugar chain cleaved by any enzyme was confirmed to the same extent.
With regard to native transferrin, W295F also had an increased amount of transferrin with the sugar chain cleaved, compared to the enzyme before mutation introduction. Furthermore, when the reaction was carried out after removing the sialic acid on the non-reducing end side by sialidase treatment, transferrin with the sugar chain cleaved more markedly increased in W295F. For denatured transferrin, W295F and W295Y cleaved sugar chains more efficiently than the enzyme before mutation introduction. On the other hand, with respect to denatured transferrin after sialidase treatment, all enzymes cleaved sugar chains to the same extent. From the above, it was confirmed that Endo-Om W295F efficiently cleaves the N-type sugar chain of the native glycoprotein in comparison with Endo-Om before the introduction of mutation (FIG. 4).

Claims (8)

  In the amino acid sequence of endo-β-N-acetylglucosaminidase (Endo-Om), the amino acid residue corresponding to the tryptophan (W) residue at position 295 of SEQ ID NO: 2 is a phenylalanine (F) residue or a tyrosine (Y) Endo-Om variant (W295F, W295Y) substituted with a residue, having the same substrate specificity as that of the unmutated Endo-Om, and at least the specific activity to the complex type sugar chain Endo-Om variant that is better than -Om.   The Endo-Om variant according to claim 1, wherein the amino acid sequence of Endo-Om before mutation of the Endo-Om variant has the amino acid sequence represented by SEQ ID NO: 2.   A nucleic acid encoding the Endo-Om variant according to claim 1 or 2.   A recombinant vector comprising the nucleic acid of claim 3 and capable of expressing the nucleic acid in a host cell.   A transformant into which the nucleic acid according to claim 3 is introduced and which expresses an Endo-Om variant.   A method for producing an Endo-Om variant having the same substrate specificity as that of an unmutated Endo-Om and having at least a specific activity to a complex sugar chain that is higher than that of an unmutated Endo-Om. In the amino acid sequence of -β-N-acetylglucosaminidase (Endo-Om), the amino acid residue corresponding to the tryptophan (W) residue at position 295 of SEQ ID NO: 2 is the phenylalanine (F) residue or tyrosine (Y) residue A method for producing an Endo-Om variant, comprising performing substitution to mutate a group.   A method for cleaving an asparagine-linked sugar chain from a target glycoprotein, comprising a step of causing the Endo-Om variant according to claim 1 or 2 to act on the target glycoprotein.   A method for transferring an asparagine-linked sugar chain to a target acceptor molecule, comprising a step of causing the Endo-Om variant according to claim 1 or 2 to act on a target acceptor molecule.