JP5286467B2 - Novel lectin, production method thereof and sugar chain detection method - Google Patents

Description

  The present invention relates to a novel lectin and a method for producing the same, and more particularly to a novel lectin derived from a mushroom extract, a method for producing the same, and a sugar chain detection method using the lectin.

  A lectin is a general term for substances showing specific binding activity to sugar (hereinafter also referred to as “specificity” as appropriate) among proteins or glycoproteins present in plants, animals, fungi and the like. Lectins are widely used as useful sugar chain profilers because they recognize not only monosaccharides but also sugars in sugar chains. In addition, since lectins have high specific binding ability to sugar chains, it is possible to detect target substances using this. In order to detect the target substance more efficiently, new lectins having different specificities and high binding capacities from conventional lectins have been searched and found in various ways (for example, see Patent Documents 1 and 2). ).

On the other hand, with the progress of research on sugar chains, it has become clear that sugar chains play an important role in vivo and in vivo. For example, the role of sugar chains in various diseases such as virus infection and cancer has been regarded as important. Therefore, more detailed sugar chain research is required, and lectins capable of recognizing specific sugar chains have been used as useful tools in sugar chain analysis and cancer diagnosis (for example, patents). References 3 and 4).
Examples of sugar chains having strong antigen activity include type A sugar chains (GalNAc 1-3 (Fuc1-2) Gal 1-3 GalNAc 1-3 Gal 1-4 Glc) belonging to human blood group glycolipids, and Forsman. Antigen sugar chains (GalNAc 1-3 GalNAc 1-3 Gal 1-4 Gal1-4 Glc) are known.

Escargot lectin (HPL) is known as a lectin that recognizes a Forssman antigen sugar chain, but has been reported to bind not only to Forssman antigen sugar chain but also to T antigen (see Non-Patent Document 1). .
Banderia bean lectin (GSL-IA) is known as a lectin that recognizes A-type sugar chains and Forssman antigen sugar chains, but has also been reported to bind to other galactose-containing sugar chains (non-patented). Reference 2).

Japanese Patent No. 2630431 Japanese Patent No. 2895962 JP 2006-149398 A JP-T-2005-515440 Glycobiology, 2007 Oct; 17 (10): pp1077-1083. (Epub 2007 Jul 25) Glycobiology. 1999 Nov; 9 (11): pp.1161-1170

However, none of the above lectins is a lectin that shows high specificity for recognizing only the Forssman antigen sugar chain and the A-type sugar chain. When performing various glycan profiles using lectins, lectins that have a certain degree of binding ability to different sugar chains, even if they have binding ability to the sugar chain to be tested, have an appropriate profile. I can't do it. Some of the currently known lectins have insufficient specificity for sugar chains, and there is a growing demand for the development of new lectins with high specificity.
Therefore, an object of the present invention is to provide a novel lectin having high specificity for Forssman antigen sugar chains and A-type sugar chains.

The present invention is as follows.
[1] A lectin derived from the genus Basidiomycete having the following characteristics:
A lectin derived from the genus Basidiomycete having the following characteristics:
(1) The molecular weight by SDS electrophoresis is 10,000 to 50,000 ,
(2) The molecular weight by gel filtration is 70,000 to 90,000 ,
(3) the amino acid sequence of the N-terminal region is at least one of SEQ ID NO: 1 and SEQ ID NO: 2,
( 4 ) binds to Forssman antigen sugar chain and A-type sugar chain ,
(5) It has an affinity represented by a binding constant of 1.0 × 10 ⁵ M ⁻¹ or more for Forssman antigen sugar chain and A-type sugar chain .

[ 2 ] An aqueous medium extract from the oyster mushroom basidiomycetes, an eluate containing a saccharide having at least one of a galactose residue and an N-acetylgalactosamine residue, and at least one of a galactose residue and an N-acetylgalactosamine residue The lectin according to [1 ] above, which is obtained by purification by affinity chromatography using a carrier containing
[ 3 ] The lectin according to [1] or [ 2] , wherein the basidiomycete basidiomycete is gray shimeji.
[ 4 ] The method for producing a lectin according to any one of [1] to [ 3 ], wherein an aqueous medium extract is obtained from a Bacillus basidiomycete, and the aqueous medium extract is converted into a galactose residue and N A lectin comprising an eluate containing a saccharide having at least one of acetylgalactosamine residues and purification by affinity chromatography using a carrier containing at least one of galactose residues and N-acetylgalactosamine residues Manufacturing method.

[ 5 ] A labeled lectin comprising the lectin according to any one of [1] to [ 3 ] and a labeling compound.
[ 6 ] A lectin immobilization carrier comprising the lectin according to any one of [1] to [ 3 ] and a support carrier.
[ 7 ] Forsman antigen sugar chain and A-type sugar using the lectin according to any one of [1] to [ 3 ], the labeled lectin according to [ 5 ] or the lectin immobilization carrier according to [ 6 ] above A method for detecting a sugar chain, comprising detecting a sugar chain-containing substance containing at least one of the chains.
[ 8 ] The method for detecting a sugar chain according to [ 7 ], further comprising separating the sugar chain-containing product from other compounds.
[ 9 ] A determination drug for determining the presence or absence of at least one of a Forssman antigen sugar chain and an A-type sugar chain, comprising the lectin according to any one of [1] to [ 3 ].

  ADVANTAGE OF THE INVENTION According to this invention, the novel lectin which shows high specificity with respect to the sugar chain containing a Forssman antigen sugar chain and A type sugar chain can be provided.

  The lectin of the present invention is derived from the genus Basidiomycete and has the characteristics (1) to (3) above. A lectin derived from the fruit body (mushroom) of the genus Basidiomycete is preferred.

  As the basidiomycete genus basidiomycetes in the present invention, gray shimeji (Clitocybe nebularis (Batsch: Fr.) Kummer), blue shimeji (Clitocybe odora (Bull .: Fr.) Kummer) oyster (Clitocybe gibba (Pers .: Fr.) Kummer) , White shimeji (Clitocybe clavipes (Pers .: Fr.) Kummer) and the like. Among these, from the viewpoints of sugar recognition specificity of lectin and lectin recovery efficiency, gray shimeji is particularly preferred.

The lectin of the present invention has a molecular weight by SDS electrophoresis of 10,000 to 60,000, and preferably 10,000 to 50,000 from the viewpoint of high sugar chain recognition ability. The molecular weight by gel filtration method is 60,000 to 100,000, preferably 70,000 to 90,000.
Measurement of molecular weight by SDS electrophoresis (SDS-PAGE) and measurement of molecular weight by gel filtration can be performed according to conventional methods.

When the lectin of the present invention is subjected to boiling conditions under general electrophoresis conditions at 25 ° C. under SDS (0.5% by mass) conditions, those subjected to boiling treatment are 13,000 and 17,000, respectively. It exists as a monomer of a seed | species, It is preferable to exist as a multimer of about 40,000-41,000 as a whole in the thing which does not perform a boiling process.
The molecular weight of the lectin that is not subjected to boiling treatment is preferably 60,000 to 100,000 as a result of estimation using a standard protein for molecular weight estimation under general measurement conditions when gel filtration is used. 70,000 to 90,000 are more preferable.

  In addition, the lectin of the present invention has two types of subunits as described above. In this case, the amino acid sequence of the N-terminal region of each unit is preferably SEQ ID NO: 1 or SEQ ID NO: 2. Lectins having these amino acid sequences have not been known so far, and thus Met-Leu-Tyr-Leu-Val-Ser-Ile-Gly-Glu-Thr-Ala-Glu-Leu-Gln-Ser-Pro- Phe-Thr-Thr-Asp (SEQ ID NO: 1) or Thr-Ile-Asp-Asn-Thr-Ala-Phe-Asn-Leu-Ser-Asp-Leu-Gly-Gly-Asn-Val-Leu-Asn- A lectin having an N-terminal region represented by His-Ile (SEQ ID NO: 2) is novel.

The lectin of the present invention shows high specificity for Forssman antigen sugar chains and A-type sugar chains.
Forssman antigen sugar chain is GalNAc 1-3 GalNAc 1-3 Gal 1-4 Gal1-4 Glc, especially GalNAc α1-3 GalNAc β1-3 Gal α1-4 Galβ1-4 Glc or GalNAc 1-3 GalNAc 1 -3, in particular, an antigen sugar chain known as GalNAc α1-3 GalNAc β1-3 Gal, and the lectin of the present invention recognizes this sugar chain. Preferably, the lectin of the present invention recognizes a glycolipid in which ceramide is further bound to the terminal Glc.
A type sugar chain means blood group A type sugar chain, GalNAc 1-3 (Fuc1-2) Gal 1-4 Glc or GalNAc 1-3 (Fuc1-2) Gal 1-4 GlcNAc 1-3 Gal 1- 4 Glc is a sugar chain composed of tetrasaccharide or hexasaccharide, especially GalNAcα 1-3 (Fucα1-2) Galβ 1-4 Glc and GalNAcα 1-3 (Fucα1-2) Galβ 1-4 GlcNAcβ It is preferably 1-3 Gal β1-4 Glc.

  Thus, the lectin of the present invention exhibits high specificity for Forssman antigen sugar chains and A-type sugar chains, and does not exhibit specificity for other sugar chains or is very low below the detection limit. It is a weak bond. For this reason, even in an environment where other sugar chain sequences exist, only the Forssman antigen sugar chain and the A-type sugar chain can be identified. A lectin exhibiting such high selectivity has not been known so far.

  “High specificity” in the present invention refers to selectively recognizing only the Forssman antigen sugar chain and the A-type sugar chain from a group of sugar chains composed of a combination of 49 or more sugar chains. It means that the sugar chain does not show very weak binding or binding ability below the detection limit, and preferably, a Forssman antigen sugar from the sugar chain group composed of a combination of 100 or more sugar chains This means that only the chain and the A-type sugar are selectively recognized, and the other sugar chains do not show very weak binding or binding properties below the detection limit.

Such high specificity can be evaluated, for example, based on affinity due to a binding constant. The lectin of the present invention preferably exhibits an affinity of 1.0 × 10 ⁵ M ⁻¹ or more, more preferably 1.0 × 10 ⁷ M ^{− to} the Forssman antigen sugar chain or the A-type sugar chain. ^One or more can be indicated. Although the upper limit of this binding constant is not particularly limited, the binding constant shows a maximum value of the binding constant shown in the case of a normal lectin, for example, an affinity of 1.0 × 10 ¹⁰ M ⁻¹ or less. In addition, the affinity of the lectin of the present invention for other sugar chains is low, and for example, the binding constant is preferably 1.0 × 10 ⁴ M ⁻¹ or less.
The binding constant used here can be measured by, for example, the frontal affinity chromatography (FAC) method and the like, and in the case of the present invention, it means that measured as follows.

Sugar chain has been found not to interact with the lectin of the present invention, based on the elution front of (standard sugar chain) (V _0), the elution front end of the detection target sugar chains (V) delay (V-V ₀₎ Is measured as the interaction strength. Based on the following criteria type FAC, determine binding constants of the sugar chain and the lectin from _{V-V 0} and the effective ligand amount (Bt) (Ka).
In the following reference formula, A is the substance used for elution, A ₀ is the initial concentration of substance A, B is the immobilized ligand, V is the elution volume, V ₀ is the elution front volume of the standard sugar chain, Bt is the effective ligand amount, Kd means dissociation constant (reciprocal of association constant). In the present invention, since the initial concentration of the substance A is considered to be sufficiently small, the following reference formula (3) is adopted.

  Moreover, the lectin of the present invention exhibits galactose binding properties. For this reason, in order to obtain the lectin of this invention by the manufacturing method mentioned later, it is preferable to use the support | carrier containing at least one of a galactose residue and an N-acetylgalactosamine residue. That is, the lectin of the present invention is obtained by using an aqueous medium extract obtained from basidiomycetous basidiomycetes, an eluate containing a saccharide having at least one of a galactose residue and an N-acetylgalactosamine residue, and a galactose residue and N -It may be obtained by purifying by affinity chromatography using a carrier containing at least one of acetylgalactosamine residues.

The lectin of the present invention can be isolated and produced from the oyster mushroom basidiomycetes by appropriately combining known extraction methods, separation methods, purification methods, and the like.
Among them, obtaining an aqueous medium extract from the basidiomycete basidiomycetes (extraction process), elution liquid containing the saccharide having at least one of galactose residue and N-acetylgalactosamine residue, and galactose A lectin obtained by a method for producing a lectin comprising purification by affinity chromatography using a carrier containing at least one of a residue and an N-acetylgalactosamine residue (purification step) is preferable.

Examples of the aqueous medium include various buffer solutions, mixtures of various organic solvents that can be mixed with water, and water or buffer solutions. It is preferable to use a buffer solution or a mixture of an organic solvent and a buffer solution.
The buffer solution is not particularly limited, and a known buffer solution can be used. Especially, what has a buffer capacity in the range of pH3-pH10 is preferable, and the buffer solution which has a buffer capacity in the range of pH6-pH8 is more preferable.
Examples of the buffer used in the present invention include a phosphate buffer, a citrate buffer, an acetate buffer, and a Tris buffer. Among these, a phosphate buffer is preferable from the viewpoint of extraction efficiency.
Although there is no restriction | limiting in particular about the salt concentration of a buffer solution, From the point of extraction efficiency and buffer capacity, it is preferable that it is 1 mM-100 mM, and it is more preferable that it is 5 mM-20 mM.
The buffer solution can further contain various salts. For example, phosphate buffered saline (PBS) obtained by further adding sodium chloride to a phosphate buffer solution can be preferably used as the aqueous medium in the present invention. .

Moreover, as an organic solvent, if it is an organic solvent which can be mixed with water, it can be especially used without a restriction | limiting, Among these, acetone, methanol, ethanol, 2-propanol, and acetonitrile can be mentioned preferably. The content of the organic solvent in the case of mixing the organic solvent with water or a buffer is preferably 10% by mass to 40% by mass.
There is no particular limitation on the method for obtaining the aqueous medium extract from the aqueous medium and the oyster mushroom basidiomycetes. From the viewpoint of extraction efficiency, a method of pulverizing oyster mushroom basidiomycetes in an aqueous medium to form a suspension is preferable. Examples of the pulverization method include ordinary pulverization methods such as a mixer and a homogenizer.

The extraction step in the present invention preferably further includes a step of removing an insoluble matter with respect to the aqueous medium from the mixture of the aqueous medium and the oyster mushroom basidiomycete. Examples of the method for removing insoluble materials include filtration, centrifugation, and the like, but centrifugation is preferred from the viewpoint of removal efficiency.
Therefore, in the extraction step in the present invention, from the viewpoint of the purity of the obtained lectin, the oyster mushroom basidiomycetes are pulverized in phosphate buffered saline, and the insoluble matter is removed by centrifugation to obtain an aqueous medium extract. A process is particularly preferred.

The aqueous medium extract obtained by the extraction step then uses an eluate containing galactose or N-acetylgalactosamine and a carrier containing at least one of galactose residues and N-acetylgalactosamine residues in the purification step. Purify by affinity chromatography.
As the carrier in the present invention, any carrier containing at least one of a galactose residue and an N-acetylgalactosamine residue can be used without particular limitation, and includes a lactose-agarose carrier, an acid-treated sepharose carrier, and any galactose. Examples thereof include a carrier on which sugar, glycoprotein, glycolipid and the like are immobilized, and these may be used alone or in combination.
Specifically, for example, a lactose-agarose carrier (manufactured by J-Oil Mills) or a sepharose carrier (manufactured by GE Healthcare Bioscience) treated with an acid such as hydrochloric acid (acid-treated sepharose carrier) can be mentioned. it can. Among them, it is particularly preferable to use a lactose-agarose carrier from the viewpoint of purification efficiency.

  In the affinity chromatography in the present invention, it is preferable to use a column using a carrier containing at least one of a galactose residue and an N-acetylgalactosamine residue as a packing material. Thereby, the lectin in the aqueous medium extract is adsorbed on this column. Next, the lectin adsorbed on the column can be purified more efficiently by separation and fractionation using an eluate containing a saccharide having at least one of a galactose residue and an N-acetylgalactosamine residue. . The elution of the lectin from the column may be performed under the conditions usually used.

  The eluent can be used without particular limitation as long as it is a solution containing a saccharide having at least one of a galactose residue and an N-acetylgalactosamine residue. Examples of the saccharide having a galactose residue or N-acetylgalactosamine residue include galactose, N-acetylgalactosamine, methyl β-galactose, methyl α-galactose, galactosucrose, N-acetylgalactosamine, lactose, lactosamine, lactulose, melibiose , Raffinose, neolactose, galactobiose and the like, and these may be used alone or in combination. Among these, galactose, methyl β-galactose, methyl α-galactose, N-acetylgalactosamine, and lactose are preferably used from the viewpoint of the sugar specificity of the target lectin, and lactose is particularly preferable from the viewpoint of cost in purification.

  The concentration of the saccharide contained in the eluate is not particularly limited. For example, in the case of an eluate containing lactose, the saccharide content in the eluate is the total mass of the eluate from the viewpoint of purification efficiency. It is preferable that it is 0.1 mass%-10 mass%, and it is more preferable that it is 0.5 mass%-2 mass%. The concentration of saccharide in the eluate containing lactose is preferably 10 mM to 1000 mM, more preferably 10 mM to 500 mM, from the viewpoint of purification efficiency. In addition, a commercially available thing can be used suitably for lactose.

  In the purification step, the aqueous medium extract is adsorbed on a column having a lactose-agarose carrier as a packing material, and a non-adsorbed component is washed with a buffer solution, particularly phosphate buffered saline. Particularly preferred is a step of purification using an eluate containing 100 mM to 200 mM of lactose.

Further, in the production method of the present invention, the eluate obtained by the purification step using affinity chromatography may be used again, and affinity chromatography may be repeated, or further purification by other chromatography may be performed. Good.
Examples of such other chromatography include gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography and the like. From the viewpoint of accurately separating lectins from contaminating components, gel filtration chromatography is more preferable.

  As a filler used for gel filtration chromatography, Sephadex, Sephacryl, Superose, Superdex, Toyopearl and the like can be used. In order to purify the lectin of the present invention, Toyopearl and Superdex are particularly preferred from the viewpoint of resolution. As conditions for gel filtration chromatography, the conditions usually used may be applied as they are, and as the mobile phase, the same aqueous medium as described above, for example, a phosphate buffer or the like can be used. When gel filtration chromatography is used, the fraction containing the target lectin can be identified based on hemagglutination activity and the like.

The lectin production method can include a step of dialysis treatment of the fraction containing lectin obtained in each purification step, and a step of freeze-drying the lectin solution after the dialysis treatment. Thereby, a lectin can be isolated with high purity.
The step of dialysis treatment and the step of freeze-drying can be performed by a commonly used known method.

  The lectin of the present invention is a novel lectin having different physicochemical properties and biochemical properties such as binding specificity to sugar chains from conventionally known lectins. In particular, since a sugar chain containing at least one of a Forssman antigen sugar chain and an A-type sugar chain can be recognized with high selectivity, a test reagent, an immunomodulator, a sugar for the purpose of detecting these sugar chains It can be used as a specific adsorbent for quality separation analysis.

The labeled lectin of the present invention includes at least the lectin and a labeled compound, and is characterized by being detectably labeled. As a labeling compound, a known labeling method can be applied without particular limitation.
The labeling compound can be applied without particular limitation as long as it is usually used for this purpose, and examples thereof include a direct or indirect labeling compound, an enzyme, and a fluorescent compound. Specific examples include biotin, digoxigenin, horseradish peroxidase, fluorescein isothiocyanate, and CyDye. These labeling compounds can be bound to lectins by a conventional method.

The lectin immobilization carrier of the present invention includes at least the lectin and a support carrier. There is no restriction | limiting in particular as a material which comprises a support carrier, For example, an inorganic material, an organic material, and these composite materials can be mentioned. Examples of the inorganic material include metals, semiconductors, metal compounds such as metal oxides, ceramics, glass, silica, and the like. Examples of organic materials include polyvinyl polymers such as rubber, latex, polystyrene, and polypropylene, polyamides such as polyacrylate, polyacrylamide, polyester, and gelatin, polysaccharides such as cellulose, agar, agarose, dextran, and starch. From the viewpoint of sugar chain fractionation efficiency, agarose and the like are preferable.
The structure of the support carrier is not particularly limited, and examples thereof include a porous structure, a fibrous structure, a gel structure, and a membrane structure.

The lectin can be immobilized on the support carrier using a conventional method corresponding to the support carrier. The amount of lectin to be immobilized is usually 0.0001 mg to 100 mg / ml, preferably 0.01 mg to 20 mg / ml. When the carrier is an agarose gel, it can be activated with CNBr and then coupled to the lectin.
A lectin may be immobilized on a gel into which an activation spacer has been introduced. Further, the lectin may be immobilized on a gel into which a formyl group has been introduced and then reduced with NaCNBH ₃ . A commercially available activated gel such as NHS-Sepharose (manufactured by GE Healthcare Bioscience) may also be used.

After the sugar chain sample is applied to the column, a buffer solution is flowed for washing and equilibration purposes. An example of the buffer is a molar concentration of 5 mM to 500 mM, preferably 10 mM to 500 mM, a pH of 4.0 to 10.0, preferably 6.0 to 9.0, and a NaCl content of 0 to 0.00. 5M, preferably _0.1M~0.2M, CaCl _2, MgCl 2 or MnCl ₂ content 0 to 10 mm, a buffer preferably 0-5 mM.
After washing of the affinity column, elution is performed using a desorbing agent such as sodium chloride or hapten sugar in a neutral non-denaturing buffer that can effectively elute the sugar chain. This buffer may be the same as described above. The concentration of the desorbing agent is preferably 1 mM to 500 mM, particularly preferably 10 mM to 200 mM.

  The sugar chain detection method of the present invention includes detecting a sugar chain-containing product containing at least one of a Forssman antigen sugar chain and an A-type sugar chain using the lectin, the labeled lectin, or the lectin immobilization carrier. It is a waste. Since the lectin of the present invention recognizes only a sugar chain containing at least one of a Forsman antigen sugar chain and an A-type sugar chain with high specificity, the lectin of the present invention contains a sugar chain containing a Forsman antigen sugar chain and an A-type sugar chain. Can be detected with high accuracy. Here, the sugar chain-containing material includes polysaccharides, glycolipids, glycoproteins, cells, cell fragments and the like. The detection method of the present invention uses the lectin or the labeled lectin to detect a sugar chain-containing substance containing at least one of the target Forsman antigen sugar chain and the A-type sugar chain (hereinafter referred to as “detection step”). As long as it performs the above, it may include other steps included in a normal detection method for detecting sugar chains using lectins.

Detection of a sugar chain-containing product to which a lectin is bound can be easily performed by, for example, previously labeling the lectin with a labeling compound. Among these, from the viewpoint of sensitivity and ease of detection, it is preferable that the labeling compound is previously bound to the lectin to label the lectin, and the labeling compound is detected.
The means for detecting the labeled lectin is appropriately selected according to the labeling compound used. For example, absorbance measurement, emission intensity measurement, fluorescence intensity measurement, visual observation and the like can be performed, and a frontal affinity chromatography (FAC) method or the like can be preferably used.

  The detection method of the present invention may further include separating the detected sugar chain-containing product from other substances (sorting step). Such a separation method is preferably performed using the lectin or the lectin-immobilized carrier. Since the lectin can bind with high specificity to at least one of the Forssman antigen sugar chain and the A-type sugar chain, these sugar chain-containing substances can be separated from other substances with high accuracy.

Separation of the sugar chain-containing material to which the lectin binds can be performed more easily by using, for example, a carrier on which the lectin is immobilized as described above. The method for fractionating the sugar chain compound is appropriately selected according to the carrier on which the lectin is immobilized, and may be performed by column chromatography, thin layer chromatography, B / F separation (bound / free separation) or the like.
Note that the separation step is not necessarily a step independent of the detection step, and the detection and the separation may be performed simultaneously or continuously.

  In the sugar chain detection method of the present invention, from the viewpoint of sugar chain detection sensitivity and sugar chain sorting efficiency, the interaction strength between the lectin of the present invention and the sugar chain recognized by this lectin is the interaction with other sugar chains. The strength is preferably 10 times or more, more preferably 100 times or more, and even more preferably 1000 times or more. The interaction strength between lectin and sugar chain is, for example, a hemagglutination reaction inhibition test, comparison of time to elution, binding amount (weight), etc. It can be used without being particularly limited.

  For detection of sugar chains in the lectin production method and sugar chain detection method of the present invention, other than the above, chromatography, lectin chip, enzyme immunoassay (ELISA) method, aggregation method, surface plasmon resonance apparatus, electrophoresis, etc. Can also be used in a known manner.

  The determination agent of the present invention contains the lectin and determines the presence or absence of at least one of a Forssman antigen sugar chain and an A-type sugar chain. Since the lectin of the present invention can recognize the Forsman antigen sugar chain and the A-type sugar chain with high accuracy as described above, a trace amount of Forsman antigen sugar chain or The presence or absence of the A-type sugar chain can also be reliably determined.

For example, because a Forssman antigen sugar chain becomes a heterologous antigen for humans, for example, it is determined whether or not this heterologous antigen is contained in an object, for example, a sample, or a sample containing a heterologous antigen. It can be used to determine whether or not there is. The type A sugar chain can be widely used for blood type determination, determination of whether or not this sugar chain is contained in a sample, determination of whether or not the sample includes this sugar chain, and the like. . Therefore, by using the determination agent of the present invention, for example, it is possible to easily select a sample that lacks or is compatible with a predetermined target species or type.
In addition, a lectin of the present invention is brought into contact with a liquid sample such as blood of a subject or a dry sample that is spotted and immobilized on a substrate, and a substance that can bind to the lectin is detected by this contact. Such detection methods are also included in the present invention.

  Note that the ABO blood type (antigen) is expressed not only in blood but also in various organs. Therefore, the lectin of the present invention can be used not only as the determination drug, but also as a temporary blocking agent or auxiliary agent at the time of ABO blood type incompatibility, and further, blood using its synthetic product or the like It can also be used as a blocking agent or an adjuvant for type A sugar chain antigens in organs and organs.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, “%” is based on mass.

[Example 1]
(1) Purification of lectin CNA-I 20 ml of 0.02% NaN ₃ / PBS is added to 0.5 mg of powdered fruit body (Clitocybe nebularis) fruit body powder (manufactured by Mushroom Research Institute of Japan) and shaker (CS- (450 HITACHI) and shaken at 7 ° C. for 2 hours. Subsequently, the column was centrifuged at 15,000 rpm for 20 minutes with a centrifuge (CF-51 HITACHI), and the supernatant was used as a crude extract (aqueous medium extract) C column (manufactured by GE Healthcare Biosciences; φ10 × 100 mm) ) Was filled with 5 ml of lactose-agarose (manufactured by J-Oil Mills) and equilibrated with PBS, and then the crude extract obtained above was used. The non-adsorbed portion was washed with PBS (about 26 ml), then eluted with 0.1 M lactose solution as an eluent at 25 ° C. and 0.5 ml / min. Lectin-containing fraction no. 43 to 46 were obtained (total adsorption rate 68%).
Each fraction containing lectin was identified by measuring the absorbance at 280 nm (OD280) and the hemagglutination activity described below.

(2) Rabbit hemagglutination activity test Rabbit stored blood (manufactured by Nippon Biotest Co., Ltd.) was centrifuged (3000 × g, 3 min, normal temperature) to obtain only red blood cells. PBS was added to this about 3 times the amount of erythrocytes, mixed well, centrifuged under the same conditions, and the supernatant was removed. By repeating this three times, plasma and white blood cells or impurities were completely removed, and this was used as washed red blood cells. The washed erythrocytes were diluted with PBS to obtain a 2% rabbit erythrocyte solution.
20 μl each of PBS was placed in one lane of a 96-well titer plate, and 20 μl of the lectin solution or the fraction collected in each chromatography was placed in the well to prepare a serial 1/2 dilution series. 40 μl each of the 2% rabbit erythrocyte solution obtained above was added, and after standing at room temperature for about 60 minutes, the hemagglutination activity was observed with the naked eye. For the hemagglutination activity, the reciprocal of the maximum dilution at which aggregation was observed was used as the aggregation titer of the stock solution. In addition, the maximum dilution ratio of lectin in which erythrocytes aggregate was determined, and the minimum aggregation concentration was calculated.
Lectin-containing fraction No. obtained in (1) above. 43 to 46 were able to confirm the hemagglutination activity as the hemagglutination titer (titer) 64, titer 512, titer 256, and titer 32, respectively.
When the non-adsorbed component was collected and applied again to the column filled with lactose-agarose, the non-adsorbed component contained almost no lactose-agarose adsorbed component (0.2% adsorption rate).

(2) SDS-polyacrylamide electrophoresis The lectin-containing fraction obtained in the above (1) was subjected to a Phassystem (manufactured by GE Healthcare Biosciences) as the electrophoresis apparatus, and to the gene as Homogeneous 20 (GE Health). SDS-polyacrylamide electrophoresis using Care Bioscience). Using 1 μl of both the sample solution and the molecular weight marker, electrophoresis was performed according to the product protocol and the usual method, and the molecular weight was estimated from the relative mobility, suggesting that a plurality of lectins exist in the vicinity of about 17,000.
The lectin-containing fraction was then further purified as described below.

(3) Fractionation by gel filtration 1 ml (Fr. 44) of the lectin-containing fraction obtained in (1) above was concentrated to 200 μl by ultrafiltration, filtered, and 100 μl was equilibrated with PBS. Using a gel filtration column (TSK-GEL G3000XL (manufactured by TOSOH; φ7.8 × 300 mm)), gel filtration was performed twice at 25 ° C. under a flow rate of 0.5 ml / min. In the same manner as described above, the absorbance at 280 nm and the hemagglutination activity were measured for the gel filtration collection liquid, and SDS-polyacrylamide electrophoresis was performed to identify and collect fractions having both high hemagglutination activity and OD 280 nm. . The collected fractions were concentrated with an ultrafiltration membrane (Ultrafree, manufactured by Millipore; MWCO 10,000), and then each fraction was freeze-dried with a freeze dryer (FD-5N, manufactured by EYELA). Was measured.
In the above-described lectin-containing fraction showing high hemagglutination activity, bands were confirmed at molecular weights around 13,000 and 17,000, and this was determined to be CNA-I.

[Example 2]
(1) Purification of lectin CNA-I Approximately 400 g of fruit body (Clitocybe nebularis) fruit body (collected in Fukushima Prefecture) was freeze-dried with a freeze dryer (FD-5N, manufactured by EEYLA), and then freeze-dried gray body fruit body. A powder was obtained. To 20 g of this lyophilized powder, 200 ml of 0.02% NaN3 / PBS was added, and the mixture was shaken at 7 ° C. for 2 hours with a shaker (CS-450, manufactured by HITACHI). Subsequently, it was centrifuged at 15,000 rpm for 20 minutes with a centrifuge (CF-51, manufactured by HITACHI), and the supernatant was used as a crude extract (aqueous medium extract).
A column (GE Healthcare Bioscience, Inc .; φ16 × 200 mm) was filled with 25 ml of lactose-agarose (J-Oil Mills) and equilibrated with PBS, and then the crude extract obtained above was used. After washing the non-adsorbed portion with PBS (about 100 ml), elution was performed at 7 ° C. and 1 ml / min using a 0.1 M lactose / PBS solution as an eluent. A lectin-containing fraction was identified based on the peak hemagglutination activity of OD280, and a fraction showing an OD280 peak and high hemagglutination activity was obtained as a lectin-containing fraction.
The collected liquid from which the active fraction was collected was placed in Spectra / Por Membrane MWCO: 6-8,000 (planar width 14.6 mm), and dialyzed at 7 ° C. with 100 times the amount of distilled water three times.
The lectin-containing fraction was then further purified as described below as in Example 1.

(2) Fractionation by gel filtration A gel filtration column (glass column (GE Healthcare Bio) was equilibrated with PBS to a sample solution obtained by dissolving 80 mg of the lectin-containing fraction obtained in (1) above in 2 ml of PBS. Using a Superdex G200 (manufactured by GE Healthcare Bioscience Co., Ltd.) in a diameter of 26 mm × 800 mm), gel filtration was performed at 7 ° C. under a flow rate of 1 ml / min. In the same manner as described above, the absorbance and erythrocyte agglutination activity were measured with respect to the gel filtration collection liquid, and SDS-polyacrylamide electrophoresis was performed to identify and collect fractions with high erythrocyte agglutination activity and OD 280 nm. . The collected fractions were concentrated with an ultrafiltration membrane (Ultrafree, manufactured by Millipore; UFC3LGC00, MWCO 10,000), and then each fraction was freeze-dried with a freeze dryer (FD-5N, manufactured by EYELA). The amount of purification was measured. The purified CNA-I was 11.86 mg. Further, when the rabbit hemagglutination activity was measured, the titer was 10240 (1 mg / ml) and the minimum aggregation concentration was 0.033 (μg / ml).

(3) SDS polyacrylamide electrophoresis The lectin CNA-I obtained in (2) above was subjected to boiling treatment (sample A) and not subjected to boiling treatment (sample B). Except that, SDS polyacrylamide electrophoresis was performed in the same manner as described above. The results are shown in FIG. In FIG. 1, lane 1 indicates sample A, lane 2 indicates sample B, and M indicates a marker.
As a result, bands were confirmed at 13,000 and 17,000 in the case of sample A and 40,000 and 41,000 in the case of sample B, respectively (see FIG. 1).
(4) Molecular weight measurement by gel filtration About lectin CNA-I obtained as described above, standard sample Transferrin (molecular weight: 80,000), Bovine serum albumin (molecular weight: 67,000), Ovalbumin (molecular weight: 47,000), Carbonic anhydrase (Molecular weight: 29,000) and molecular weight estimated using Ribonuclease B (molecular weight: 13,700) were about 80,000.

[Example 3]
The lectin CNA-I obtained in Example 2 was evaluated as follows.
(1) MALDI-TOF mass spectrometry 10 μg of lectin CNA-I was dissolved in TA (a mixture of 0.1% trifluoroacetic acid and acetonitrile in a volume ratio of 2: 1). A sample was prepared by dropping 1.0 μl of a mixture of a saturated matrix dissolved in TA and a TA solution of CNA-I at a volume ratio of 4: 1 onto the target plate. Using Autoflex (manufactured by Bruker Daltonics) as a mass spectrometer, the molecular weight of CNA-I was measured in the LP mode. As a result, the molecular weights of CNA-I were 16,260 and 11,042.

(2) Amino acid sequence of N-terminal region The amino acid sequence of the N-terminal region of CNA-I was analyzed using PPSQ-21 (manufactured by Shimadzu Corporation) as a protein sequencer. It was found that (SEQ ID NO: 2). When the obtained two amino acid sequences were subjected to homology search with FAST and BLAST, there was no protein showing homology, and thus the lectin CNA-I was found to be a novel lectin.

(3) Temperature stability 50 μg of CNA-I was dissolved in 1 ml of physiological saline and heated at each test temperature for 30 minutes. Immediately after ice cooling, the hemagglutination activity was measured 10 minutes later as described above. The results are shown in FIG.
From FIG. 2, it was found that the lectin of the present invention is relatively stable from 4 ° C. to 60 ° C., and particularly hardly affected by temperature at 4 to 60 ° C. However, the activity decreased rapidly at 70 ° C. or higher.

(4) pH stability 50 μg of CNA-I was dissolved in 1 ml of physiological saline and dispensed into a test tube. An equal amount of each of the following pH buffer solutions was added and incubated at 4 ° C. for 24 hours, and then hemagglutination activity was measured in the same manner as described above. The results are shown in FIG. From FIG. 3, it was found that the lectin CNA-I of the present invention is stable at pH 3-11, and particularly shows good stability at pH 4-11.
-PH buffer solution-
pH 2.0: 20 mM potassium chloride-hydrochloric acid buffer pH 3.0: 20 mM potassium chloride-hydrochloric acid buffer pH 4.0: 20 mM acetic acid-sodium acetate buffer pH 5.0: 20 mM acetic acid-sodium acetate buffer pH 6.0: 20 mM phosphorus Acid buffer pH 7.0: 20 mM phosphate buffer pH 8.0: 20 mM Tris (tris (hydroxymethyl) aminomethane) buffer pH 9.0: 20 mM glycine-NaOH buffer pH 10.0: 20 mM glycine-NaOH buffer pH 11 0.0: 20 mM glycine-NaOH buffer Each buffer contains 0.15 M sodium chloride.

(5) Rabbit hemagglutination activity test Rabbit stored blood (manufactured by Nippon Biotest Co., Ltd.) was centrifuged (3000 × g, 3 min, room temperature) to remove plasma and leukocytes and impurities to obtain only red blood cells. PBS was added to this about 3 times the amount of erythrocytes, mixed well, centrifuged under the same conditions, and the supernatant was removed. By repeating this three times, plasma and white blood cells were completely removed, and this was used as washed red blood cells. The washed erythrocytes were diluted with PBS to obtain a 2% rabbit erythrocyte solution.

a) Measurement of hemagglutination activity 20 μl of PBS was placed in one stage of a 96-well titer plate, and 20 μl of lectin solution (0.6 mg / ml) was placed in the well to prepare a serial 1/2 dilution series. 40 μl each of the 2% rabbit erythrocyte solution obtained above was added, and after standing at room temperature for about 60 minutes, the hemagglutination activity was observed with the naked eye. The reciprocal of the maximum dilution at which aggregation was observed was taken as the aggregation titer of the stock solution, and the logarithm with the aggregation titer of 2 as the base was shown in Table 1 as the hemagglutination activity. In addition, the maximum dilution ratio of lectin in which erythrocytes aggregate was determined, and the minimum aggregation concentration was calculated.

b) Sheep, pig, human hemagglutination activity test In the rabbit hemagglutination activity test, sheep preservation blood (manufactured by Nippon Biotest), pig preservation blood (manufactured by Nippon Biotest), human preservation blood (instead of rabbit preservation blood) Sheep, pig and human hemagglutination activity tests were performed in the same manner except that Biotest) was used. The results are shown in Table 1.

(6) Rabbit Hemagglutination Inhibition Test Appropriate amounts of the 32 types of glycoproteins and sugars shown in Tables 2 and 3 were weighed, PBS was added, and the concentrations shown in Tables 2 and 3 were prepared. The rabbit hemagglutination activity of CNA-I obtained in the above (Example 2) was measured, and these were diluted with PBS so that the minimum dilution ratio at which erythrocytes aggregate was 2 to 4 times. Next, 25 μl of PBS was placed in each well of a 96-well plate (manufactured by Falcon), and 50 μl of the lectin solution was placed in the well to prepare a serial 1/2 dilution series. 25 μl of the above diluted sample was added to each well, left at room temperature for 60 minutes, then added with 25 μl of 4% rabbit erythrocyte solution, and allowed to stand at room temperature for 1 hour. The results are shown in Tables 2 and 3 below.

  From the results of Tables 2 and 3, it was found that the lectin (CNA-I) obtained in Example 2 specifically binds to N-acetylgalactosamine, galactose, melibiose, and raffinose. Glycoprotein was found to bind to mucin.

[Example 4]
(7) Specificity test (sugar chain detection)
(A) Preparation of oligosaccharide The pyridylaminated (PA-) sugar chain used in the examples is shown in FIG. 4 and FIG. PA sugar chains were purchased from Takara Bio, Biochemical Biobusiness, Masuda Chemical Co., etc. Alternatively, unlabeled sugar chains and sugar chains obtained by enzymatic digestion of sugar chains were pyridylaminated with GlycoTAG (registered trademark, manufactured by Takara Bio Inc.).
Other labeled sugar chains are shown in FIG. The para-nitrophenyl (pNP-) sugar chain and the paramethoxyphenyl (pMP-) sugar chain used were purchased from Seikagaku Biobusiness, Tokyo Kasei, Sigma, Toronto Research Chemical, Calbiochem. .

(B) Preparation of lectin column The lectin was dissolved in 0.2 M NaHCO ₃ buffer (pH 8.3) containing 0.5 M NaCl, and the NHS-activated Sepharose (manufactured by GE Healthcare Biosciences) Combined according to manufacturer's manual. Lectin-Sepharose was suspended in 10 mM Tris-HCl buffer (pH 7.4, TBS buffer) containing 0.8% NaCl and packed in a miniature column (φ2 mm × 10 mm, 31.4 μl).

(C) Frontal Affinity Chromatography Frontal affinity chromatography was performed using an FAC automatic analyzer (FAC-1, manufactured by Shimadzu Corporation). Specifically, the lectin column prepared above was inserted into a stainless steel holder and connected to the FAC-1 apparatus. The flow rate and column temperature were kept at 0.125 ml / min and 25 ° C., respectively. After equilibrating the miniature column with the TBS buffer, an excess volume (0.5 ml to 4 ml) of PA-glycan (3.75 nM or 7.5 nM) and pNP-glycan and pMP-glycan (1 μM) were automatically Continuous injection into the column using a sampling device.

The fluorescence intensity (excitation wavelength: 310 nm and fluorescence wavelength: 380 nm) of the eluate of PA sugar chain is monitored, and the interaction intensity [difference in front eluate with respect to standard oligosaccharide (PA-M3, No. 003): V−V ₀ ] is obtained. It was measured.
As a result of evaluating specificity with the sugar chain group composed of 49 types, as shown in FIG. 7, CNA-I is an elution that is bound only to the Forssman antigen sugar chain and the A-type sugar chain (hexasaccharide). The delay was confirmed.

Furthermore, the number of sugar chains was increased, and the fluorescence intensity (excitation wavelength: 310 nm and fluorescence wavelength: 380 nm) of the sugar chain eluate was monitored, and the difference between the front-end eluate and the interaction intensity [standard oligosaccharide (PA-M3, No. 003)] : V-V ₀ ] was measured.
Moreover, pNP-sugar chain and pMP-sugar chain were monitored by UV absorption intensity (detection wavelength 280 nm), and the interaction intensity [difference in front-end eluate with respect to standard oligosaccharide (pNP-Fuc, number 1011): V−V ₀ ] Was measured.

Based on the following criteria type FAC, it was determined binding constant (Ka) between the sugar chain and the lectin from _{V-V 0} and the effective ligand amount (Bt).

A paramethoxyphenyl (pMP-) labeled Forssman antigen sugar chain (No. 1107) was prepared in a TBS buffer so as to be 0 μM to 0.9 μM. The analysis was performed in the same manner as described above, and the interaction strength at each concentration [difference in frontal eluate with respect to standard oligosaccharide (pNP-Fuc, No. 1011): V−V ₀ ] was measured, and the binding constant (Ka) and the amount of effective ligand ( Bt) was determined. As a result, the binding constant (Ka) = 2.44 × 10 ⁶ M ⁻¹ and the effective ligand amount (Bt) = 0.015 nmol. The interaction strength and binding constant of the bound sugar chain were calculated. The results are shown in Table 4 and FIG.

  As a result of evaluating the specificity of the 151 sugar chain groups, as shown in Table 4 and FIG. 8, CNA-I was identified as No. 717 Forssman antigen sugar chain, No. 719 type A sugar chain (tetrasaccharide ), Only the A-type sugar chain of number 720 (hexasaccharide) showed binding properties.

  Therefore, since lectin CNA-I shows high specificity for Forssman antigen sugar chains and A-type sugar chains, it is used to accurately detect and separate only those sugar chain-containing substances containing these sugar chains. The presence or absence of these sugar chains in the sample can be determined with high accuracy.

It is a figure which shows the result of the molecular weight measurement by SDS-PAGE of lectin CNA-I concerning the Example of this invention. It is a figure which shows the temperature stability of the lectin CNA-I concerning the Example of this invention. It is a figure which shows the pH stability of the lectin CNA-I concerning the Example of this invention. It is a figure which shows the oligosaccharide (PA-) used for the measurement of the sugar chain specific binding property of this invention. It is a figure which shows the oligosaccharide (PA-) used for the measurement of the sugar chain specific binding property of this invention. It is a figure which shows the oligosaccharide (pNP-, pMP-) used for the measurement of the sugar chain specific binding property of this invention. It is a conceptual diagram which shows the measurement result of the sugar chain specific binding property of lectin CNA-I concerning the Example of this invention. It is a conceptual diagram which shows the measurement result of the sugar chain specific binding property of lectin CNA-I concerning the Example of this invention.

Claims (9)

A lectin derived from the genus Basidiomycete having the following characteristics:
(1) The molecular weight by SDS electrophoresis is 10,000 to 50,000 ,
(2) The molecular weight by gel filtration is 70,000 to 90,000 ,
(3) the amino acid sequence of the N-terminal region is at least one of SEQ ID NO: 1 and SEQ ID NO: 2,
( 4 ) binds to Forssman antigen sugar chain and A-type sugar chain ,
(5) It has an affinity represented by a binding constant of 1.0 × 10 ⁵ M ⁻¹ or more for Forssman antigen sugar chain and A-type sugar chain . An aqueous medium extract obtained from the basidiomycete basidiomycetes, an eluate containing a saccharide having at least one of a galactose residue and an N-acetylgalactosamine residue, and at least one of a galactose residue and an N-acetylgalactosamine residue The lectin according to claim 1, which is obtained by purification by affinity chromatography using a carrier comprising The lectin according to claim 1 or 2, wherein the basidiomycete basidiomycete is gray shimeji mushroom. It is a manufacturing method of the lectin of any one of Claims 1-3 , Comprising: Obtaining an aqueous medium extract from the genus Basidiomycete, The aqueous medium extract is made into galactose residue and N-acetyl. Production of a lectin comprising: elution containing a saccharide having at least one of galactosamine residues and purification by affinity chromatography using a carrier containing at least one of galactose residues and N-acetylgalactosamine residues Method. A labeled lectin comprising the lectin according to any one of claims 1 to 3 and a labeled compound. A lectin immobilization carrier comprising the lectin according to any one of claims 1 to 3 and a support carrier. Using the lectin according to any one of claims 1 to 3 , the labeled lectin according to claim 5 , or the lectin immobilization carrier according to claim 6 , the Forsman antigen sugar chain and the A-type sugar chain A method for detecting a sugar chain, comprising detecting a sugar chain-containing material containing at least one. The sugar chain detection method according to claim 7 , further comprising separating the sugar chain-containing product from other compounds. The determination drug which determines the presence or absence of at least one of a Forssman antigen sugar chain and A type sugar chain containing the lectin of any one of Claims 1-3 .