JP7343890B2 - Enzyme agents with sialidase activity and their use - Google Patents

Description

The present specification relates to an enzyme agent having sialidase activity and its use.

Sialidase is an enzyme that hydrolyzes the O-glycosidic bond of sialic acid to liberate sialic acid. Sialic acid is a general term for substances in which the basic skeleton is neuraminic acid and the hydrogen atoms of its amino groups and hydroxyl groups have been substituted, and is a nine-carbon sugar having an amino group and a carboxy group in the molecule. In vivo, sialic acid generally exists at the non-reducing end of sugar chains in glycoproteins and glycolipids. Sialic acids include, for example, N-glycolylneuraminic acid (Neu5Gc) in which the hydrogen atom of the amino group at the 5-position of neuraminic acid is substituted with glycolic acid, and N-acetyl neuraminic acid in which the hydrogen atom is similarly substituted with an acetyl group. Naturally occurring neuraminic acid (Neu5Ac) and deaminoneuraminic acid (2-keto 3-deoxy-D-glycero-D-galactonononic acid, KDN) in which the nitrogen at the 5th position is replaced with a hydroxyl group .

Sialidase exists widely from bacteria to humans, and is thought to be involved in assimilation by bacteria, involvement in host infection, in vivo metabolism in animals, and cell regulation by exposing galactose residues. Sialidases are also used to remove sialic acids in in vitro and in vivo research and for medical purposes. Various sialidases have been provided (Patent Document 1, Non-Patent Documents 1 to 6).

Japanese Patent Application Publication No. 8-9972

Uchida Y, et al., Enzymatic properties of neuraminidases from Arthrobacter ureafaciens. J Biochem. Nov;86(5):1573-85(1979). Bouwstra JB, et al., Purification and kinetic properties of sialidase from Clostridium perfringens. Biol Chem Hoppe Seyler. Mar;368(3):269-75(1987). Ada, G. L. et al., Purification and properties of neuraminidase from Vibrio cholerae. J. Gen. Microbiol. 24, 409-421 (1961). Scanlon KL, et al.,. Purification and properties of Streptococcus pneumoniae neuraminidase. Enzyme. 41(3):143-50(1989). Rogerieux F, et al., Determination of the sialic acid linkage specificity of sialidases using lectins in a solid phase assay. Anal Biochem. Jun;211(2):200-4(1993). Corfield AP et al., The release of N-acetyl- and N-glycolloyl-neuraminic acid from soluble complex carbohydrates and erythrocytes by bacterial, viral and mammalian sialidases. Biochem J. Aug 1;197(2):293-9 (1981 ).

However, many sialidases have an optimum pH on the acidic side of pH 6.2 or lower, and some sialidases are known to use Neu5Ac as a substrate, but some sialidases do not use other sialic acids. is unknown. In addition, the bonding modes of glycosidic bonds that are subject to hydrolysis include α(2→3) bonds, α(2→6) bonds, and α(2→8) bonds, but the extent of these bonds is not necessarily clear. do not have.

Thus, at present, no sialidase has been provided that eliminates multiple sialic acids as suitable substrates. When desorbing sialic acid from sugar chains, it may be advantageous in research and the like to target multiple sialic acid substrates.

The present specification provides an enzyme agent having sialidase activity that uses a plurality of sialic acids as suitable substrates, and uses thereof.

The present inventors speculated that one of the proteins possessed by bacteria belonging to the genus Sphingobacterium is sialidase, obtained the protein and confirmed its activity, and found that it is a sialidase with novel substrate specificity. Obtained. Based on this knowledge, the disclosure of this specification provides the following means.

[1] An enzyme agent having sialidase activity containing at least one protein selected from the group consisting of (a) to (c) below.
(a) A protein having the amino acid sequence represented by SEQ ID NO: 1 or 2 (b) Having an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, and having sialidase activity Protein (c) A protein having sialidase activity, having one or more amino acids substituted, inserted, deleted and/or added in the amino acid sequence represented by SEQ ID NO: 1 or 2 [2] Neu5Ac The enzyme agent according to [1], which acts on one or more substrates selected from the group consisting of , Neu5Gc, and KDN.
[3] Has release activity of α-glycoside-bonded sialic acid residues of α(2→3) bonds, α(2→6) bonds, α(2→8) bonds, and α(2→9) bonds; The enzyme agent according to [1] or [2].
[4] The enzyme preparation according to any one of [1] to [3], further having an optimum pH of 6.5 or more and 7.5 or less.
[5] A method for treating sugar chains, comprising a treatment step of treating a sugar chain-containing compound with the enzyme agent according to any one of [1] to [4].
[6] The processing method according to [5], wherein the sugar chain-containing compound is selected from glycoproteins and glycolipids.
[7] The treatment method according to [5] or [6], wherein the treatment step is a step of treating the sugar chain-containing compound presented on the cell surface with the enzyme agent.
[8] A treatment step of treating the sugar chain-containing compound with the enzyme agent according to any one of [1] to [4];
a detection step of detecting sialic acid residues released by treatment with the enzyme agent;
A method for determining glycan properties, comprising:
[9] A method for producing a sugar chain degradation product, comprising:
a treatment step of treating a sugar chain-containing compound with the enzyme agent according to any one of [1] to [4];
a step of recovering the sialic acid residues and/or the sialic acid residues removed from the treatment step;
A production method comprising:
[10] A sialidase expression vector comprising a DNA encoding a protein having sialidase activity, including at least one protein selected from the group consisting of (a) to (c) below.
(a) A protein having the amino acid sequence represented by SEQ ID NO: 1 or 2 (b) Having an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, and having sialidase activity Protein (c) A protein having sialidase activity and having one or more amino acids substituted, inserted, deleted and/or added in the amino acid sequence represented by SEQ ID NO: 1 or 2 [11] Sialidase An expression kit comprising:
A kit comprising the sialidase expression vector described in [10].

FIG. 2 is a diagram showing an overview of the sialidase activity of this drug. FIG. 2 is a diagram showing the purification results of recombinant sialidase (with tag). It is a figure showing the purification result of recombinant sialidase (no tag). FIG. 2 is a diagram showing the pH dependence of sialidase activity of recombinant sialidase. FIG. 2 is a diagram showing the temperature dependence of sialidase activity of recombinant sialidase. FIG. 3 is a diagram showing the substrate specificity (sialic acid molecular species) of sialidase activity of recombinant sialidase. FIG. 2 is a diagram showing the substrate specificity (binding mode specificity) of sialidase activity of recombinant sialidase. FIG. 2 is a diagram showing the results of evaluating changes in polysialic acid on the cell surface due to treatment of CHO cells with recombinant sialidase by flow cytometry using an anti-polysialic acid antibody.

The present specification relates to a novel enzyme agent having sialidase activity, a method for treating a sugar chain-containing compound using the enzyme agent, a method for determining sugar chain characteristics, a method for producing a degradation product of a sugar chain-containing compound, and the like.

The protein having sialidase activity disclosed herein (hereinafter also referred to as the present protein) was obtained by the present inventors from a bacterium belonging to the genus Sphingobacterium (at the time the present inventors were dealing with the bacterium, Sphingobacterium spp. It is related to the amino acid sequence represented by SEQ ID NO: 1, which was obtained from the genome information of M. multivorum. This amino acid sequence contains sequences conserved in the sialidase family, but its activity was unknown.

This protein can have the amino acid sequence represented by SEQ ID NO:1. The protein specified by the amino acid sequence represented by SEQ ID NO: 1 is classified as GH33 based on amino acid sequence similarity on a database of carbohydrate-related enzymes called CAZy (Carbohydrate active enzymes). A characteristic of GH33 is that it has a 6-fold-β-propeller domain. This amino acid sequence has four aspartate box motifs (S-X-D-X-G-X-T-W, where X is any amino acid) that bacterial sialidases have, and near the N-terminus of the amino acid sequence, RIP, which is often found in sialidases. It also has a motif called array.

Therefore, it cannot be said that the sialidase activity of this protein cannot be inferred from this amino acid sequence. However, it has been reported that sialidase from fungi has sialidase activity even with just one aspartate box, and like this protein, other sialidase-like proteins found on the genomes of bacteria belonging to the genus Sphingobacterium are Although both are classified as GH33 in CAZy and have one or two aspartate boxes, no sialidase activity has been found. Considering the above, it cannot be said that it is easy to infer that a protein has sialidase activity based only on the amino acid sequence.

Furthermore, even if it is possible to infer the sialidase activity of a protein from the amino acid sequence, it is currently extremely difficult to infer the substrate specificity of sialidase from the amino acid sequence or three-dimensional structure. In addition, the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 has a novel substrate specificity that uses all the sialic acids of Neu5Ac, Neu5Gc, and KDN as substrates, which has not yet been reported. Even those skilled in the art were unable to infer the gender.

According to the present inventors, the enzyme agent having sialidase activity disclosed herein (hereinafter also simply referred to as this agent) provides a novel sialidase having substrate specificity different from that of conventional sialidases. be able to. The agent can have broader substrate activity than, for example, conventional sialidases. Furthermore, for example, the optimal pH of this agent is more neutral, which is advantageous for sialidase treatment of gangliosides, which are glycoproteins and glycolipids on the cell surface. Furthermore, for example, the present agent can be used to more reliably carry out processing of chain-containing compounds, determination of sugar chain properties, production of decomposition products of sugar chain-containing compounds, etc. based on a wide range of substrate properties.

Hereinafter, representative and non-limiting specific examples of the present disclosure will be described in detail with reference to the drawings as appropriate. This detailed description is merely intended to provide those skilled in the art with details for implementing a preferred example of the disclosure, and is not intended to limit the scope of the disclosure. Additionally, the additional features and inventions disclosed below can be used separately or together with other features and inventions in order to provide a further improved "enzyme agent having sialidase activity and its use." can.

Additionally, the features and combinations of steps disclosed in the following detailed description are not essential to practicing the present disclosure in its broadest sense, and are intended solely for the purpose of specifically illustrating representative embodiments of the present disclosure. shall be described. Moreover, various features of the exemplary embodiments above and below, as well as those recited in the independent and dependent claims, are hereby described in providing additional and useful embodiments of the present disclosure. They do not have to be combined exactly as shown in the specific examples given or in the order listed.

All features recited in this specification and/or in the claims may be found individually, as limitations on the original disclosure and specificity claimed, apart from the features recited in the examples and/or in the claims. and are intended to be disclosed independently of each other. Furthermore, all references to numerical ranges and groups or populations are intended to disclose intermediate configurations thereof as limitations on the original disclosure and the specific claimed subject matter.

In addition, in this specification, the word "more than" has the meaning not only as a lower limit but also as more than the above. In addition, the term "less than or equal to" has the meaning of less than the following as an upper limit value. Therefore, the ranges described as "more than" and "less than" have the meaning of "more than" and "less than", as well as more than and less than, more than and less than, and more than and less than.

Hereinafter, various embodiments regarding this agent and its use will be described in detail.
(Sialidase and enzyme agents)
As used herein, sialidase refers to an enzyme that releases sialic acid residues bound by O-glycosidic bonds by hydrolysis of the O-glycosidic bonds. Furthermore, sialidase activity refers to the activity of releasing sialic acid residues by hydrolysis of O-glycosidic bonds.

The sialidase contained in this agent (hereinafter also simply referred to as sialidase) is any protein selected from the group consisting of (a) to (c) below.
(a) A protein having the amino acid sequence represented by SEQ ID NO: 1 or 2 (b) Having an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, and having sialidase activity Protein (c) A protein that has one or more amino acids substituted, inserted, deleted, and/or added in the amino acid sequence represented by SEQ ID NO: 1 or 2, and has sialidase activity.

As mentioned above, the amino acid sequence represented by SEQ ID NO: 1 of this sialidase is derived from a bacterium belonging to the genus Sphingobacterium (at the time the present inventors were working on this bacterium, it was estimated to be Sphingobacterium multivorum). It was obtained from genome information. Furthermore, the amino acid sequence represented by SEQ ID NO: 2 is a sequence obtained by removing positions 1 to 19 of the amino acid sequence represented by SEQ ID NO: 1. Although this sequence is considered to be a secretory signal sequence, it is not necessarily necessary except when producing the present sialidase as a secreted protein.

Further, the present sialidase may have an amino acid sequence having a certain degree or more of identity with the amino acid sequence represented by SEQ ID NO: 1 or 2.

As used herein, identity or similarity is a relationship between two or more proteins or two or more polynucleotides as determined by comparing their sequences, as is known in the art. In the art, "identity" refers to the identity between protein or polynucleotide sequences, as determined by alignment between the protein or polynucleotide sequences, or, as the case may be, by alignment between a stretch of such sequences. It means the degree of array invariance of . Similarity also refers to the correlation between protein or polynucleotide sequences, as determined by alignment between the protein or polynucleotide sequences or, in some cases, by alignment between a stretch of partial sequences. means the degree of More specifically, it is determined by sequence identity and conservation (specific amino acids in a sequence or substitutions that maintain physicochemical properties in the sequence). Note that the similarity is referred to as "Similarity" in the BLAST sequence homology search results described below. Preferably, the method for determining identity and similarity is one designed to provide the longest alignment between contrasting sequences. Methods for determining identity and similarity are provided as publicly available programs. For example, the BLAST (Basic Local Alignment Search Tool) program by Altschul et al. It can be determined using the alignment function of SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., Nucleic Acids Res. 25: p3389-3402 (1997)) or UniProtKB. Conditions when using software such as BLAST or UniProt KB are not particularly limited, but default values can be used.

The present sialidase can have, for example, 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1, for example, 85% or more, or 86% or more, or, for example, 87% or more. For example, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 95% or more, and For example, it is 96% or more, for example, 97% or more, for example, 98% or more, for example, 99% or more, and for example, 99.5% or more.

The present sialidase can have, for example, 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2, for example, 85% or more, or 86% or more, or, for example, 87% or more. For example, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 95% or more, and For example, it is 96% or more, for example, 97% or more, for example, 98% or more, for example, 99% or more, and for example, 99.5% or more.

When the present sialidase is aligned with the amino acid sequence represented by SEQ ID NO: 1, for example, 1 or Two or more, for example three or more, or for example all, aspartic acid boxes (S-X-D-X-G-X-T-W, where X is any amino acid) can be provided.

The present sialidase may also have one or more amino acid mutations in the amino acid sequence represented by SEQ ID NO: 1 or 2. The amino acid mutation is one or more types selected from substitution, insertion, deletion, and addition. Further, the number of mutations is one or more, but for example, 50 or less, for example, 40 or less, for example, 30 or less, for example, 25 or less, and for example, , 20 or less, for example 15 or less, for example 10 or less, for example 8 or less, for example 6 or less, and for example 4 or less be.

Note that the mutation sites are not particularly limited, but for example, when aligned with the amino acid sequence represented by SEQ ID NO: 1, for example, positions 72 to 79, positions 140 to 147, positions 272 to 279, and When an aspartic acid box (S-X-D-X-G-X-T-W) is present at one or more positions corresponding to positions 327 to 334, for example at three or more positions, or for example at all these positions, amino acid substitution at a position other than the aspartic acid box It can have displacement, etc. Also, for example, one or more of the positions corresponding to 72nd to 79th, 140th to 147th, 272nd to 279th, and 327th to 334th, for example, 3 or more, or for example in all of these positions. When it has an aspartic acid box, it can have an amino acid substitution mutation at the position corresponding to X (any amino acid residue) of the aspartic acid box (S-X-D-X-G-X-T-W).

For example, examples of such amino acid substitutions as mutations are preferably conservative substitutions, and specifically include substitutions within the parenthesized groups below. For example, (glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

(Sialidase activity)
The present sialidase has, for example, any one or more of the following sialidase activities (1) to (3).
(1) Using one or more sialic acid molecular species selected from the group consisting of Neu5Ac, Neu5Gc and KDN as a substrate (2) α(2→3) bond, α(2→6) bond, α (2→8) bond and α(2→9) bond have α-glycosidic bonding activity of Neu5Ac (3) The optimum pH is 6.5 or more and 7.5 or less (4) The optimum temperature is Temperature is 25℃ or more and 40℃ or less

The present sialidase can use one or more types selected from Neu5Ac, Neu5Gc, and KDN as a substrate as a molecular species of sialic acid. Target molecular species can each be used as a substrate, for example, Neu5Ac and Neu5Gc, for example Neu5Ac and KDN, for example Neu5Gc and KDN, and for example Neu5Ac, Neu5Gc and KDN. All of these can be used as substrates.

The sialic acid molecular species targeted by this sialidase can be obtained by preparing and reacting a synthetic substrate corresponding to the molecular species, such as 4MU-Neu5C, and emitting a fluorescent signal based on the resulting decomposition product 4MU. This can be done by, for example, detecting.

Regarding sialidase, there are few reports that explicitly specify Neu5Gc as a molecular species to be degraded, and an existing sialidase that specifically degrades KDN has been reported (Patent Document 1), but it has not been reported that Neu5Gc is a molecular species to be degraded. There are no known sialidases that do this.

For example, if it has molecular species characteristics that target all of Neu5Ac, Neu5Gc, and KDN, it may have higher activity against Neu5Ac than the other two species. For example, when the activity against Neu5Ac is assumed to be 100, and the reaction is carried out under the same conditions with only the substrate changed, the free activity against Neu5Gs is, for example, 15 to 20, and the activity against KDN is over 0 and 20.

This sialidase can hydrolyze o-glycosides with various bonding modes to release sialic acid molecular species. For example, the free activity of the previously described sialic acid molecules with α-glycosidic bonds of α(2→3) bonds, α(2→6) bonds, α(2→8) bonds, and α(2→9) bonds is can have Regarding existing sialidases, it has been described that α(2→3), α(2→6), and α(2→8) are the target binding modes, but α(2→9) is the target binding mode. No sialidase has been reported.

For example, with respect to Neu5Ac, the o-glycosidic bond of all the above-mentioned bonding modes can be hydrolyzed to liberate the molecular species.

The binding mode targeted by this sialidase is, for example, by preparing disaccharides with various binding modes (one is sialic acid), reacting them, and separating and detecting the decomposition products by thin layer chromatography. This can be done by

The optimum pH of the present sialidase is, for example, 6.5 or more and 7.0 or less. The optimum pH can be defined by the conditions described in the Examples below. For example, a synthetic substrate can be prepared using a molecular species targeted by the present sialidase, and an optimum pH can be obtained for the synthetic substrate. Typically, the optimum pH for 4MU-Neu5Ac and/or 4MU-KDN can be referred to. Also, for example, only the optimum pH for 4MU-Neu5Ac can be referred to.

Existing sialidase has an optimum pH on the more acidic side (6.2 at the most neutral side). This sialidase allows sialidase treatment under conditions closer to neutrality, and allows sialidase treatment under conditions with reduced cytotoxicity.

The optimum temperature of the present sialidase is, for example, 30°C or higher and 37°C or lower. The optimum temperature can be defined by the conditions described in the Examples described later. For example, a synthetic substrate can be prepared using a molecular species targeted by the present sialidase, and the optimum temperature can be obtained for the synthetic substrate. Typically, reference can be made to the optimal temperature for the synthetic substrates 4MU-Neu5Ac and/or 4MU-KDN. Also, for example, only the optimum temperature for 4MU-Neu5Ac can be referred to.

This drug contains at least the present sialidase. In addition to the present sialidase, the present agent may contain other sialidases having sialic acid molecular species specificity that can be used simultaneously. Furthermore, this agent may also contain enzymes other than sialidase.

(Manufacture of sialidase)
The method for producing the present sialidase is not particularly limited, and it can be obtained by any known protein production method. For example, the present sialidase can be isolated and purified by culturing bacteria of the genus Sphingobacterium, or it can also be obtained by chemical synthesis.

Furthermore, for example, the present sialidase can be obtained as a recombinant protein by genetic engineering techniques. That is, the amino acid sequence represented by SEQ ID NO: 1 or 2, an amino acid sequence having a certain identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, or, for example, the amino acid sequence represented by SEQ ID NO: 1 or 2. A DNA encoding an amino acid sequence having an amino acid mutation in the sequence is synthesized or obtained by a known method such as PCR using DNA from a bacterium belonging to the genus Sphingobacterium as a template DNA, and the DNA is transformed into a protein-encoding gene in an appropriate host cell. The present sialidase can be obtained as a culture product by transforming a host cell with a DNA construct that can be expressed as a DNA construct and culturing the host cell.

In addition, for a protein having a certain identity or one or more amino acid mutations with the amino acid sequence represented by SEQ ID NO: 1 or 2, for example, at least a portion of the DNA encoding the amino acid sequence or its complementary strand may be used as a probe. It can be obtained by hybridization, error prone PCR, or known mutagenesis methods. Such a protein can be confirmed to be the present sialidase by evaluating its amino acid sequence and sialidase activity, and can be produced as the present sialidase in the same manner as described above.

The host cell is not particularly limited, and known host cells can be used. For example, in addition to bacteria such as Escherichia coli and Bacillus subtilis, and yeasts such as Candida utilis, Saccaromyces cerevisiae, and Pichia pastoris, Rhizopus niveus and Rhizopus delmar (Rhizopus delemar) and higher eukaryotes (for example, CHO cells) can be used. For example, the present sialidase has sialidase activity and can be obtained in high yield by using a host cell derived from Escherichia coli (K-12 strain) such as Rosetta gami2.

Further, a vector as a DNA construct can be appropriately selected in consideration of the type of host cell to be used. For example, the DNA construct may be a plasmid, cosmid, or viral expression vector. The DNA construct may also be capable of replicating within the host cell, or may be configured to be integrated into the host chromosome so as to be capable of replication. DNA constructs can be constructed using various commercially available plasmids as appropriate.

Note that the DNA construct can have various DNA sequences necessary for gene expression, such as a promoter, a terminator, a ribosome binding site, a transcriptional regulatory signal such as a transcription termination signal, a translational regulatory signal, and the like. In addition, it may contain appropriate selection markers such as drug resistance genes and auxotrophic genes. Those skilled in the art can carry out the genetic engineering techniques described above, for example, as appropriate according to the methods described in Molecular Cloning: A Laboratory Manual.

The transformed host cells can be cultured using general methods depending on the host cells used. Usually, enzymes are produced and accumulated in the intracellular or extracellular culture by culturing for about 1 to 4 days. Regarding culture conditions (medium, pH, temperature, etc.), for example, the temperature is generally 25 to 37°C for bacteria, 25 to 30°C for yeast, and about 37°C for eukaryotic cells. For culture conditions, reference can be made to, for example, the Gene Expression Experiment Manual (Kodansha).

Isolation and purification of the recombinant enzyme produced by the transformant can be carried out according to conventional methods by appropriately combining known separation and purification methods. These separation and purification methods are not particularly limited, but include, for example, methods that utilize differences in solubility such as salt precipitation and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylic electrophoresis. Methods that utilize differences in molecular weight such as ion exchange chromatography, methods that utilize differences in charge such as ion exchange chromatography, methods that utilize differences in hydrophobicity such as hydrophobic chromatography and reversed phase chromatography, and methods that utilize differences in hydrophobicity such as isoelectric point Other methods include methods that utilize differences in isoelectric points, such as electrophoresis, and affinity chromatography. In addition to the production methods described in Examples, for general separation and purification methods, reference may be made to, for example, Basic Experimental Methods for Proteins and Enzymes (Nankodo).

(Sialidase expression vector and kit)
According to the present specification, in addition to the sialidase expression vector, a sialidase expression kit comprising the sialidase expression vector is also provided. Additionally, such kits can also include host cells, such as E. coli, suitable for expression of sialidase and the like. Furthermore, other known reagents for use in this type of expression vector can also be provided as appropriate.

(Use of enzyme agent)
Utilizing its sialidase activity, this agent can be used in the treatment of sugar chain-containing compounds, determination of sugar chain properties, and methods for producing sugar chain degradation products. According to this agent, due to its sialic acid molecular species specificity, binding mode specificity, and optimal pH, it acts on various sugar chains, liberates sialic acid molecular species, and causes sialic acid residues to detach. It is possible to prepare sugar chains that have been removed, determine the characteristics of sugar chains by analyzing the released sialic acid residues, and obtain the molecular species of released sialic acids. In addition, new knowledge regarding the significance of sialic acid modification was obtained from the differences in the physiological functions of sugar chain-containing compounds or cells presenting the compounds before sialic acid release (sialic acid modification state) and after sialic acid release. Obtainable.

(Method for processing sugar chains)
The method for treating sugar chains disclosed herein can include a treatment step of treating a sugar chain-containing compound with the present agent. According to this treatment method, sialic acid residues can be efficiently released from the sugar chains of the sugar chain-containing compound, and a sugar chain-containing compound from which the sialic acid residues have been removed can be obtained.

As used herein, the term "sugar chain" refers to a compound in which sugars are linked via glycosidic bonds. A sugar chain-containing compound refers to a compound consisting of such a sugar chain, as well as a compound in which such a sugar chain is bonded to another compound, such as a glycoprotein in which a protein and a sugar chain are bonded, or a compound in which a lipid and a sugar chain are bonded. Examples include complex carbohydrates such as glycolipids and proteoglycans. Note that the sugar chain in the sugar chain-containing compound may be one in which one or more monosaccharides are bonded.

Here, a glycoprotein is one in which one or more sugar chains are bound to a part of amino acids constituting the protein. There are generally several to several dozen sugar chains, and they are mainly cell membrane proteins and secreted proteins. Amino acids to which sugar chains are attached typically include asparagine (N-type sugar chain), serine, and threonine (O-type sugar chain). In addition, monosaccharides constituting sugar chains constituting glycoproteins include galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, Neu5Ac, and xylose.

Proteoglycans refer to complexes in which glycosaminoglycans are bound to proteins, and have sugar chains consisting of about 100 to 200 monosaccharides such as hyaluronic acid, chondroitin sulfate, heparan sulfate, and keratan sulfate. Carbohydrate chains generally comprise repeating structures of disaccharides. Proteoglycans typically exist to fill spaces between fibrous proteins.

Glycolipids refer to lipids with linked sugar chains, and are broadly classified into glyceroglycolipids, which contain ether bonds between sugars and glycerin, and glycosphingolipids, which contain glycosidic bonds between sugars and sphingosine. Glycosphingolipids include simple cerebrosides that have one molecule of hexose as a sugar, hexose sugars such as galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, and fucose, gangliosides that contain sialic acid, and sulfatides that contain sulfated sugars. and so on.

Since glycoproteins and glycolipids often contain sialic acid residues as sugar chain-containing compounds to be subjected to the treatment process, this agent is useful for treating such complex carbohydrates. Furthermore, the treatment step of the present treatment method includes treating the sugar chain-containing compound present in the cell surface layer with the present agent in addition to treating the sugar chain-containing compound in a free state with the present agent. This agent is useful for cell treatment because it can exhibit high sialidase activity under conditions with low cytotoxicity.

In the treatment step, the sugar chain-containing compound and this drug are brought into contact in a liquid medium under conditions that allow the sialidase to exert its sialidase activity, thereby allowing the sialidase activity to liberate sialic acid residues. . The reaction concentration in the treatment step is not particularly limited as long as it is within a temperature range in which the present sialidase acts stably, but may be, for example, 30°C or higher and 37°C or lower, or around these. Further, the pH in the treatment step is not particularly limited as long as it is a pH range in which the present sialidase acts stably, but it can be, for example, pH 6.5 to 7.0 or around these.

In addition, the reaction temperature in the treatment step does not need to be constant throughout the reaction time; if it is desirable to increase the activity of other enzymes used together at the beginning of the reaction time, the temperature at which the activity of that enzyme increases The temperature can be adjusted as appropriate, such as by setting the temperature at the initial stage of the reaction time, and setting the temperature at a temperature range in which the activity of the enzyme of the present invention increases during the middle and late stages of the reaction time.

The reaction time in the treatment step can be appropriately determined in consideration of the reaction temperature, the concentration of the substrate, and the activity of the enzyme used when other enzymes are used together. When using the present sialidase alone, the time period can be set as appropriate, for example, within the range of 10 minutes to 16 hours, although there is no particular limitation.

According to this treatment method, the sialidase activity of the present sialidase can liberate sialic acid residues in a wider variety of sialic acid molecular species and binding modes than ever before. Therefore, it is possible to obtain a sugar chain-containing compound from which sialic acid residues are removed more than ever before. Therefore, for example, it becomes easier to evaluate the influence of sialic acid modification.

In this treatment method, one or more types selected from sialidases other than the present sialidase and other glycosidases, such as galactosidase, hexosaminidase, fucosidase, mannosidase, and other exoglycosidases, may be treated simultaneously or sequentially, as appropriate. You can also do that.

(Method for determining glycan characteristics)
The method for determining sugar chain properties disclosed herein (hereinafter also simply referred to as the present determination method) consists of a treatment step of treating a sugar chain-containing compound with this drug, and a step of treating a sugar chain-containing compound with this drug. and a detection step of detecting acid residues. Here, the sugar chain characteristics refer to the composition and order of the constituent sugars of the sugar chain of the sugar chain-containing compound. The processing steps in this determination method can be carried out in the same manner as the processing steps in the present processing method described above. In addition, the detection step in this determination method is not particularly limited, but for example, sialic acid residues in the reaction solution after the treatment step are detected by various mass spectrometry, various chromatography, various NMR, various electromagnetic This can be a step of separating and identifying by electrophoresis. Note that, prior to or simultaneously with the separation and identification, after the separation, the free sialic acid residue may be bound to a lectin or specific antibody to which a signal element such as a fluorescent label has been added or formed to be capable of being added. . For such sugar chain fingerprints, known techniques can be applied as appropriate.

Furthermore, this determination method includes a step of allowing a sialidase-treated sugar chain compound to interact with a glycosyltransferase, and detecting the sugar chain end exposed by the sialidase treatment based on its reactivity and a specific antibody. Good too. As the glycosyltransferase, any known glycosyltransferase can be used as appropriate.

In addition, in this determination method, similarly to the present treatment method, sialidases other than the present sialidase and various glycosidases can be treated simultaneously or sequentially. The sugar chain properties can also be determined by detecting the residues and remaining ends released by these glycosidases.

(Method for producing sugar chain degradation products)
The method for producing sugar chain degradation products disclosed herein (hereinafter also referred to as the present production method) includes a treatment step of treating a sugar chain-containing compound with the present sialidase, and sialic acid residues produced by the treatment step. and/or a recovery step of recovering the sialic acid residue removed product. The processing steps in this production method can take the same form as the processing steps in the present processing method described above.

The recovery step in this production method is a step of separating and recovering at least a portion of the decomposition products in the treatment step, that is, the liberated sialic acid residues and the sialic acid residues removed. This production method only needs to be a step of recovering at least a portion of the expected free sialic acid residues and sialic acid residues removed, and these separation and recovery methods are used, for example, in the determination in this determination method. The separation and identification step can be performed as a recovery step.

In addition, in this production method, similarly to the present treatment method, sialidases other than the present sialidase and various glycosidases can be treated simultaneously or sequentially. The residues and remaining ends released by these glycosidases can also be recovered.

According to the present specification, after treating a sugar chain-containing compound using this agent, a step of newly binding a sugar chain to the remaining sugar chain terminal using one or more glycosyltransferases is provided. Also provided are methods for producing sugar chains.

(kit)
The kits disclosed herein can include the present sialidase. This kit is a kit for treating a sugar chain-containing compound using the present sialidase, determining the sugar chain characteristics, or using the sugar chain degradation product for production, etc. In addition to the present sialidase, this kit may further include various glycosidases including other sialidases. In addition, depending on the purpose of this kit, this kit may also contain an optionally labeled antibody that specifically binds to the sialic acid residue that is expected to be released or the sugar chain-containing compound after sialic acid removal. It can also contain lectins and the like. This kit can also be equipped with reagents and devices applicable to glycan research and glycan analysis.

Examples that embody the present disclosure will be described below, but the present disclosure is not limited to these examples. In this specification, unless otherwise specified, "%", "part", etc. are based on mass. In addition, unless otherwise specified, the operating procedures followed the method described in Molecular Cloning: A Laboratory Manual (Sambrook, Maniatis et al., Cold Spring Harbor Laboratory Press (1989)).

(Preparation of recombinant sialidase)
A cDNA sequence (SEQ ID NO: 3) corresponding to the amino acid sequence represented by SEQ ID NO: 1 was prepared by removing the hydrophobic region sequence (1-19 aa (1-57 bp)) presumed to be a secretion signal. This cDNA sequence was cloned into pET32a vector according to a conventional method, and this vector was used to transform Rosetta-gami2 (DE3) (Merck & Co., Ltd.) to obtain a single colony on an LA plate. A single colony was inoculated into 5 ml of LA medium and cultured with shaking at 37°C for 16 hours. The entire volume of this culture solution was transferred to 200 ml of LA medium, and further shaking culture was performed at 37° C. for 2 hours, and it was confirmed that the OD600 was around 0.4.

IPTG (isopropyl β-D-1-thiogalactoside) was added to the culture solution to a final concentration of 0.4 mM, and the culture was further cultured with shaking at 15° C. for 20 hours. The culture solution was centrifuged at 6,000 g for 10 minutes at 4°C to collect bacterial cells, and the cells were washed with 40 ml of PBS. After washing, add 20ml equilibration buffer (5mM imidazole, 0.5M NaCl, 20mM Tris-HCl (pH 8.0)), and then add PMSF (phenylmethylsulfonyl fluoride) and protease inhibitor cocktail (to a final concentration of 1mM). PIC: final concentrations of 1 μg/ml leupeptin, 2 μg/ml antipain, 10 μg/ml benzamidine, 1 μg/ml pepstatin, and 1 μg/ml aprotinin) were added and suspended.

The cell suspension was disrupted by ultrasonication, and the supernatant was collected as a crude fraction by centrifugation at 6,000 g for 10 minutes at 4°C. Imidazole was added to the crudely purified fraction to a final concentration of 50 mM, applied to a 2 ml Ni-NTA agarose column equilibrated in advance with an equilibration buffer, and mixed by rotation at 4° C. for 15 minutes. After collecting the flow through fraction, 8.5 ml of wash buffer (10 mM imidazole, 1 M NaCl, 40 mM Tris-HCl (pH 8.0)) was added and washed to obtain a wash fraction. was recovered. Furthermore, 7ml elution buffer (100mM imidazole, 0.5M NaCl, 20mM Tris-HCl (pH 8.0)) was added, and the elution fraction (Elute fraction) in which the His-tagged protein was eluted was collected. Finally, 8 ml strip buffer (1M imidazole, 0.5M NaCl, 20mM Tris-HCl (pH 8.0)) was added, and a strip fraction was collected. The eluted fraction was ultrafiltered using VIVA SPIN20 (MWCO=10000) (manufactured by ThermoFischer SCIENTIFIC), and desalted and concentrated to 0.75 ml to obtain a purified fraction.

The proteins contained in each fraction were confirmed by SDS-PAGE and CBB staining. The results are shown in Figure 2. As shown in FIG. 2, the tagged sialidase was found to be purified as a 59 kDa protein in the eluted fraction (Purified fraction).

Furthermore, 1.9 units of enterokinase and 10× reaction buffer were added to 700 μl of the purified fraction, and mixed by rotation at 4° C. for 16 hours to cleave the His tag added to the N-terminus. 10 μl of benzamide Sepharose was added to the reaction solution, mixed by rotation at 4° C. for 1 hour, and centrifuged at 2,000 g for 5 minutes at 4° C. to precipitate and remove enterokinase. Add 100 μl of Ni-NTA agarose equilibrated with equilibration buffer to the supernatant, mix by rotating at 4°C for 1 hour, and precipitate the tagged sialidase by centrifuging at 2,000 g for 5 minutes at 4°C. Removed.

This supernatant was concentrated to 550 μl using VIVA SPIN20 (MWCO=10000). The concentrated fraction was dispensed into 10 μl portions and stored at -80°C. SDS-PAGE was performed to confirm that the sialidase tag had been removed, and CBB staining confirmed that sialidase was shifted before and after enterokinase digestion. At the same time, a calibration curve was created by performing SDS-PAGE on bovine serum albumin (BSA), whose protein amount was known, and measuring the brightness using CS analyzer 3.0 (ATTO, Tokyo). The concentration was determined. The results are shown in Figure 3.

As shown in FIG. 3, it was found that untagged sialidase was purified as a 40 kDa protein by enterokinase treatment. It was also found that the concentration of untagged sialidase was 3.8 μg/ml, and the yield of untagged sialidase was 530 μg per 200 ml of bacterial cells.

Sialidase activity measurement using 2'-(4-methylumbelliferyl)-α-DN-sodium sialic acid (4MU-Sialic acid)

(1) pH dependence 10 μl of various 0.5 M buffers were added to a 96-well plate, Multiplate 96F (Sumitomo Bakelite, Tokyo). The 0.5M buffers are sodium acetate buffer in the pH range of 4.0 to 6.0, HEPES-NaOH buffer in the pH range of 6.0 to 8.0, and Tris-HCl buffer in the pH range of 8.0 to 10.0. Using. Next, BSA (final concentration 1 ng/μl), 4MU-Neu5Ac or 4MU-KDN (final concentration 20 μM), and 5.7 ng of the untagged sialidase prepared in Example 1 (the same applies hereinafter) were added as a 40 μl reaction solution. It was left standing at room temperature for 30 minutes while shielded from light. Thereafter, 50 μl of 0.25 M glycine-NaOH buffer (pH 10.4) was added to stop the reaction, and using a plate reader Enspire (PerkinElmer, Waltham, MS, USA), the excitation wavelength was 365 nm and the fluorescence wavelength was 437 nm. The fluorescence of released 4MU was measured. The measurement results of relative enzyme activity based on fluorescence intensity are shown in FIG. 4.

As shown in FIG. 4, the optimum pH of sialidase was found to be in the range of 6.5 to 7.5 or 6.5 to 7.0 for both substrates. From the above, it was found that the sialidase obtained in Example 1 had an optimum pH closer to the neutral side than conventional sialidase.

(2) Temperature dependence Add 50 μl reaction solution (0.1 M HEPES-NaOH (pH 6.5), 1 ng/μl BSA, 20 μM 4MU-Neu5Ac or 4MU-KDN, 3.8 ng sialidase) to a 1.5 ml Eppendorf tube, It was left standing at 4, 15, 25, 30, 37, and 50°C for 30 minutes under light shielding conditions. Thereafter, 50 μl of 0.25 M glycine-NaOH buffer (pH 10.4) was added, and the fluorescence of released 4MU was measured using a plate reader Enspire under conditions of an excitation wavelength of 365 nm and a fluorescence wavelength of 437 nm. The results are shown in Figure 5.

As shown in Figure 5, when 4MU-Neu5Ac is used as a substrate, at pH 6.5, sialidase exhibits good sialidase activity at temperatures above 25°C and below 40°C, more specifically at temperatures above 30°C and below 37°C. Ta. When 4MU-KDN was used as a substrate, almost the same results were obtained although the activity was lower.

(3) Substrate specificity (sialic acid molecular species)
10 μl containing 3.84 ng of sialidase, 62.5 μunits of Arthrobacter ureafaciens (Au) sialidase, 667 μunits of Clostridium perfringens (Cp) sialidase, 1 unit of Vibrio cholera (Vc) sialidase, and 8 units of Streptococcus pneumoniae (Sp) sialidase was added to a 96-well plate. Note that the definition of the unit is based on the definition of each company's data sheet. Add 40 μl reaction solution (0.1 M HEPES-NaOH (pH 7.0) or 0.1 M sodium acetate buffer (pH 5.0), 20 μM 4MU-Neu5Ac or 4MU-Neu5Gc or 4MU-KDN) to each well and store under light-protected conditions. The mixture was left standing at 37°C for 30 minutes. Thereafter, 50 μl of 0.25 M glycine-NaOH buffer (pH 10.4) was added, and the fluorescence of released 4MU was measured using a plate reader Enspire under conditions of an excitation wavelength of 365 nm and a fluorescence wavelength of 437 nm. The results are shown in FIG.

As shown in FIG. 6, this sialidase exhibited sialidase activity against Neu5Ac, Neu5Gc, and KDN. On the other hand, A.u. sialidase, C.p. sialidase, and V.c. sialidase showed sialidase activity against Neu5Ac and Neu5Gc, but did not show sialidase activity against KDN. Also, S. p. Sialidase showed sialidase activity only against Neu5Ac.

(4) Evaluation of binding mode specificity of sialidase by silica gel thin layer chromatography (TLC) As substrates, α2,3-sialyllactosamine, α2,6-sialyllactosamine, α2,8-polysialic acid, α2,9- 5 μg (Neu5Ac equivalent) of each polysialic acid was used. To each substrate, 50 μl of a reaction solution containing 0.1 M HEPES-NaOH (pH 6.5), 1 ng/ml BSA, and 1.92 μg sialidase was added and reacted at 30° C. for 12 hours. 1 μg (in terms of sialic acid) of the reaction solution was spotted on a TLC plate and developed for 5 hours (1-propanol: aqueous ammonia: water = 6:1:2.5). After development, a resorcinol reagent (2 mg/ml resorcinol, 9.6 N concentrated hydrochloric acid, 0.25 mM copper sulfate) was sprayed, heated at 100° C. or higher, and spots after development were detected. The results are shown in FIG.

As shown in Figure 7, this sialidase has various binding modes, namely α2,3-sialylgalactosamine, 2,6-sialyllactosamine, 2,8-polysialic acid, and 2,9-polysialic acid. Neu5Ac was released in both cases. From the above, it was found that the present sialidase has sialidase activity capable of releasing sialic acid residues bound by α(2→9)o-glycosidic bonds.

(Evaluation of in vivo sialidase utilization by flow cytometry)
CHO cells were cultured on a cell culture dish (φ10 cm) until they reached 80% confluence. The cells were washed three times with chilled PBS, and the cells were scraped off and collected using a cell scraper. Cells were collected by centrifugation at 2,000g for 5 minutes at 4°C, and left standing in 4% paraformaldehyde for 8 minutes at room temperature to fix the cell membrane. After washing with PBS, blocking was performed with PBE (PBS containing 0.5% BSA and 5mM EDTA). Next, the recombinant sialidase prepared in Example 1 was added at a concentration of 19.2 μg/ml, and the mixture was reacted at 37° C. for 30 minutes with shaking. After washing with PBS, 10 μg/ml anti-polysialic acid antibody (12E3) was added as a primary antibody, and the mixture was left standing on ice for 30 minutes. After further washing with PBS, anti-mouse IgM antibody-Alexa488 was added as a secondary antibody, and the mixture was left standing on ice for 30 minutes. After washing with PBS, the number of cells to which the anti-polysialic acid antibody was bound to the cell surface was counted using a flow cytometer Gallios (Beckman Coulter, Tokyo). In order to observe the changes caused by sialidase, a cell group that had not been treated with sialidase, and in order to observe the specific binding of the anti-polysialic acid antibody, a cell group to which only the secondary antibody had been added was also carried out at the same time. The results are shown in FIG.

As shown in FIG. 8, the results of flow cytometry revealed that sialidase treatment reduced the number of cells to which the primary and secondary antibodies bound. That is, it was found that the sialidase treatment of cells liberated sialic acid residues on the cell surface, and the primary antibody no longer bound to them. From the above results, it was found that the present sialidase effectively acts on sugar chains presented on the cell surface.

Claims (9)

The following (a) to (c);
(a) A protein having an amino acid sequence represented by SEQ ID NO: 1 or 2. (b) A protein having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, including Neu5Ac, Neu5Gc and KDN sialic acid, a protein having sialidase activity that releases the sialic acid bound to a sugar chain-containing compound through an α(2→9) glycosidic bond (c) In the amino acid sequence represented by SEQ ID NO: 1 or 2. The sialic acid of Neu5Ac, Neu5Gc and KDN has one to 30 amino acids substituted, inserted, deleted and/or added, and has a sugar chain with an α(2→9) glycosidic bond. Containing one or more proteins selected from the group consisting of proteins having a sialidase activity that releases the sialic acid bound to the containing compound,
A sugar chain treating agent for releasing the sialic acid bound to the sugar chain-containing compound through an α(2→9) glycosidic bond. The protein further comprises the sialic acid bound by one or more α-glycosidic bonds selected from α(2→3) bonds, α(2→6) bonds, and α(2→8) bonds. The sugar chain processing agent according to claim 1, which has a sialidase activity that releases . Furthermore, the sugar chain treating agent according to claim 1 or 2, wherein the optimum pH for the sialidase activity is 6.5 or more and 7.5 or less. A treatment step of treating a sugar chain-containing compound with the sugar chain treating agent according to any one of claims 1 to 3,
A method for treating sugar chains, in which in the sugar chain-containing compound, any one or more of Neu5Ac, Neu5Gc, and KDN, or two or more types of sialic acid bound by an α(2→9) glycosidic bond, is released. 5. The processing method according to claim 4, wherein the sugar chain-containing compound is selected from glycoproteins and glycolipids. 6. The treatment method according to claim 4, wherein the treatment step is a step of treating the sugar chain-containing compound presented on the cell surface with the sugar chain treating agent . A treatment step of treating a sugar chain-containing compound with the sugar chain treating agent according to any one of claims 1 to 3;
Detecting one or more sialic acid residues of Neu5Ac, Neu5Gc and KDN bound by an α(2→9) glycosidic bond to the sugar chain-containing compound released by treatment with the sugar chain treating agent. a detection step;
A method for determining glycan properties, comprising: A method for producing a glycan degradation product, the method comprising:
A treatment step of treating a sugar chain-containing compound with the sugar chain treating agent according to any one of claims 1 to 3;
Any or two or more sialic acid residues of Neu5Ac, Neu5Gc, and KDN bonded to the sugar chain-containing compound produced by the treatment step through an α(2→9) glycosidic bond, and/or residues of the sialic acid. a step of recovering the desorbed material;
A production method comprising: A sugar chain processing kit,
The following (a) to (c);
(a) A protein having an amino acid sequence represented by SEQ ID NO: 1 or 2. (b) A protein having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, including Neu5Ac, Neu5Gc and KDN sialic acid, a protein having sialidase activity that releases the sialic acid bound to a sugar chain-containing compound through an α(2→9) glycosidic bond (c) In the amino acid sequence represented by SEQ ID NO: 1 or 2. The sialic acid of Neu5Ac, Neu5Gc and KDN has one to 30 amino acids substituted, inserted, deleted and/or added, and has a sugar chain with an α(2→9) glycosidic bond. comprising a sialidase expression vector comprising DNA encoding one or more proteins selected from the group consisting of proteins having sialidase activity that release the sialic acid bound to the containing compound;
A kit for releasing any one or more of Neu5Ac, Neu5Gc, and KDN bound by an α(2→9) glycosidic bond in the sugar chain-containing compound.

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