JP2009209071A - New lectin, method for producing the same and method for detecting sugar chain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new lectin showing a high specificity against a glycolipid-based sugar chain (Gb4). <P>SOLUTION: This lectin originated from a basidiomycete belonging to the genus Clitocybe is provided by having the following characteristics. (1) Having 5,000 to 40,000 molecular weight by an SDS electrophoresis, (2) Having 5,000 to 40,000 molecular weight by a gel filtration method, and (3) Having the characteristics of bonding with a Globoside-based sugar chain. <P>COPYRIGHT: (C)2009,JPO&INPIT

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). Reference 3 and Patent Document 4).

It is a globoside sugar chain, and a sugar chain having a GalNAc1-3Gal1-4Gal1-4Glc structure (Gb4 sugar chain) is also known as a bacterial receptor sugar chain. Escherichia coli that causes human urinary tract infection also binds to glycolipids such as globotetraosylceramide (Gb4).
Further, in β-hexosaminidase A and B in lysosomal diseases, particularly Sandhoff disease, globotetraosylceramide (Gb4) cannot be decomposed into globotriosylceramide (Gb3), and globotetraosyl. It is known that ceramide accumulates in body fluids and in many organs / cells (see, for example, Non-Patent Document 1).

Japanese Patent No. 2630431 Japanese Patent No. 2895962 JP 2006-149398 A JP-T-2005-515440 PNAS, April 3, 2007, vol.104, no.14, pp.5977-5982

However, when performing various sugar chain profiles using lectins, even if the sugar chains to be tested have specificity, lectins that exhibit a certain degree of specificity for different sugar chains, Proper profile is not possible. 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.
Moreover, there was no lectin that could be used for profiling of globoside glycolipid sugar chains.
Therefore, an object of the present invention is to provide a novel lectin exhibiting high specificity for globoside sugar chains.

The present invention is as follows.
[1] A lectin derived from the genus Basidiomycete having the following characteristics:
(1) The molecular weight by SDS electrophoresis is 5,000 to 40,000,
(2) The molecular weight by gel filtration is 5,000 to 40,000,
(3) Binding to globoside sugar chains.
[2] The lectin according to [1], which has affinity for a globoside sugar chain containing N-acetylgalactosamine.
[3] The lectin according to any one of [1] or [2], which has an affinity represented by a binding constant of 1.0 × 10 ⁵ M ⁻¹ or more for a globoside sugar chain.

[4] 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 any one of [1] to [3], which is obtained by purification by affinity chromatography using a carrier containing
[5] The lectin according to any one of the above [1] to [4], wherein the basidiomycete basidiomycete is gray shimeji.
[6] The method for producing a lectin according to any one of [1] to [5], 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.

[7] A labeled lectin comprising the lectin according to any one of [1] to [5] and a labeling compound.
[8] A lectin immobilization carrier comprising the lectin according to any one of [1] to [5] and a support carrier.
[9] Using the lectin according to any one of [1] to [5], the labeled lectin according to [7] or the lectin immobilization carrier according to [8], a globoside sugar chain-containing product is detected. A method for detecting a sugar chain.
[10] The method for detecting a sugar chain according to [9], further comprising separating the globoside-based sugar chain-containing product from other compounds.
[11] A determination drug for determining the presence or absence of a globoside sugar chain, comprising the lectin according to any one of [1] to [5].

  ADVANTAGE OF THE INVENTION According to this invention, the novel lectin which shows high specificity with respect to a globoside 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 viewpoint of lectin sugar recognition specificity and lectin recovery efficiency, gray shimeji is particularly preferred.

The lectin of the present invention has a molecular weight by SDS electrophoresis of 5,000 to 40,000, and preferably 8,000 to 40,000 from the viewpoint of high sugar chain recognition ability.
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 a boiling treatment under the general electrophoresis conditions at 25 ° C. under SDS (0.5% by mass), a single amount of 8,000 to 15,000 is obtained after boiling. It is preferably present as a body.
The molecular weight of the lectin not subjected to boiling treatment is preferably 5,000 to 40,000 as a result of estimation using a standard protein for molecular weight estimation under general measurement conditions when gel filtration is used. More preferably, it is 10,000 to 40,000.

  The lectin of the present invention binds to a globoside sugar chain, and preferably has affinity for a globoside sugar chain (globotetraose: Gb4) containing N-acetylgalactosamine. This globotetraose (Gb4) is an antigen sugar chain known as GalNAc 1-3Gal1-4 Gal 1-4 Glc, in particular, GalNAcβ1-3Galα1-4 Galβ1-4 Glc. The lectin of the present invention recognizes this sugar chain. Furthermore, the lectin of the present invention recognizes a glycolipid in which ceramide is further bound to the terminal Glc.

Thus, the lectin of the present invention shows high specificity for globotetraose (Gb4), which is a globoside sugar chain, and does not show specificity for other sugar chains or below the detection limit. It is a very weak bond. For this reason, even in an environment where other sugar chain sequences exist, only globotetraose (Gb4), which is a globoside sugar chain, can be identified. A lectin exhibiting such high selectivity has not been known so far.
This makes it possible to use globoside sugars in various glycolipid sugar chains such as blood group glycolipids, gangliosides, globosides, galactolipids, galas, lactos, neolactos, moles, and arsulos. Lectins can be provided that can be used for strand profiling.

  “High specificity” in the present invention refers to selectively recognizing only globotetraose (Gb4), which is a globoside sugar chain, from sugar chains composed of a combination of 49 or more kinds of sugar chains. Means that it does not exhibit binding properties to glycans, and preferably only globotetraose (Gb4), which is a globoside sugar chain, among glycans composed of a combination of 100 or more glycans This means that it does not show very weak binding or binding ability below the detection limit for other sugar chains.

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 ⁴ M ⁻¹ or more, more preferably 1 for a globoside sugar chain, particularly a sugar chain having a GalNAc1-3Gal1-4Gal1-4Glc structure. 0.0 × 10 ⁵ M ⁻¹ or more. 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 ordinary lectins, 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 basidiomycete basidiomycetes, an eluate containing a saccharide having at least one of a galactose residue and an N-acetylgalactosamine residue, -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 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, globotetraose (Gb4), which is a globoside sugar chain, can be recognized with high selectivity, so that it can be used for testing reagents, immunomodulators, and carbohydrate separation analysis for the purpose of detecting these sugar chains. It can be used as a specific adsorbent or the like.

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 the affinity column, elution is performed using a desorbing agent such as sodium chloride or hapten sugar in a neutral non-denaturing buffer solution 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 globoside-based sugar chain-containing substance using the lectin, the labeled lectin or the lectin immobilization carrier (hereinafter referred to as a detection step). Since the lectin of the present invention recognizes only a globoside sugar chain with high specificity, a sugar chain-containing product containing globotetraose (Gb4), which is a globoside sugar chain, can be detected with high accuracy. Here, the sugar chain-containing material includes polysaccharides, glycolipids, glycoproteins, cells, cell fragments and the like.

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 to globotetraose (Gb4), which is the globoside sugar chain, with high specificity, these sugar chain-containing substances can be separated from other substances with high accuracy.

Separation of the sugar chain-containing product to which the lectin binds can be easily performed 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 drug of the present invention contains the lectin and determines the presence or absence of a globoside sugar chain. Since the lectin of the present invention can recognize globoside sugar chains with high accuracy as described above, the presence or absence of a trace amount of globoside sugar chains in a sample can be determined by using this determination agent. .

For this reason, it can be suitably used for diagnosis and treatment of lysosomal diseases, in which globoside sugar chains, especially globotetraose (Gb4) are known to be detected at high frequency, and research of infectious diseases. .
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.

  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-II 20 ml of 0.02% NaN ₃ / PBS was added to 0.5 mg of powdered fruit body (Clitocybe nebularis) fruit body powder (manufactured by Nippon Mushroom Laboratory), and shaker (CS- (450 HITACHI) and shaken at 7 ° C. for 2 hours. Next, the column was centrifuged at 15,000 rpm for 20 minutes in a centrifuge (CS-450 HITACHI), and the supernatant was used as a crude extract (aqueous medium extract) C column (GE Healthcare Biosciences; φ10 × 100 mm) ) Was filled with 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.
The lectin-containing fractions Nos. 43 to 46 obtained in (1) above confirm the hemagglutination activity as the hemagglutination titer (titer) 64, titer 512, titer 256, and titer 32, respectively. I was able to.
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 200μ 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 of the gel filtration collection liquid were measured, and SDS-polyacrylamide gel electrophoresis was performed to identify fractions in which both the hemagglutination activity and OD 280 nm were confirmed. Recovered. 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 lectin-containing fraction showing the hemagglutination activity, a single band was confirmed at a molecular weight of about 15,000, and this was determined to be CNA-II.

[Example 2]
(1) Purification of lectin CNA-II 100 g of fruit body (Clitocybe nebularis) fruit body (collected in Fukushima Prefecture) was freeze-dried using lyophilization (FD-5N, manufactured by EYELA), A powder was obtained. To 20 g of this lyophilized powder, 200 ml of 0.02% NaN ₃ / 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 (CS-450, 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-II was 8.37 mg. Furthermore, when the rabbit hemagglutination activity was measured, the titer was 64 (1 mg / ml) and the minimum aggregation concentration was 5.3 (μg / ml).

(3) SDS polyacrylamide electrophoresis The lectin CNA-II obtained in (2) above was boiled (sample A) and subjected to SDS polyacrylamide electrophoresis in the same manner as described above. It was. The results are shown in FIG. In FIG. 1, lane 1 indicates sample A, and M indicates a marker.
As a result, in the case of Sample A, a band could be confirmed at 15,000 (see FIG. 1).
(4) Molecular weight measurement by gel filtration About lectin CNA-II 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 When the molecular weight was estimated using (molecular weight: 29,000) and Ribonuclease B (molecular weight: 13,700), it was about 12,000.

[Example 3]
The lectin CNA-II obtained in Example 2 was evaluated as follows.
(1) MALDI-TOF mass spectrometry 10 μg of lectin CNA-II 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-II 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-II was measured in the LP mode. As a result, the molecular weight of CNA-II was 15,142.

(2) Temperature stability 50 μg of CNA-II 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 50 ° C., but becomes unstable above 60 ° C.

(3) pH stability 50 μg of CNA-II 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-IIA of the present invention is stable at pH 5 to pH 11, and particularly shows good stability at pH 6 to pH 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 phosphoric 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: 20 mM glycine-NaOH buffer Each buffer contains 0.15 M sodium chloride.

(4) Rabbit hemagglutination activity test Rabbit stored blood (manufactured by Nippon Biotest Co., Ltd.) was centrifuged (3000 × g, 3 min, normal temperature) to remove plasma, leukocytes and impurities, and only red blood cells were obtained. 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.

(5) Rabbit Hemagglutination Inhibition Test 32 types of glycoproteins and sugars shown in Tables 2 and 3 were weighed in an appropriate amount, and PBS was added to prepare the concentrations shown in Tables 2 and 3. The rabbit hemagglutination activity of CNA-II 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, the lectin (CNA-II) obtained in Example 2 is galactose, N-acetylgalactosamine, melibiose, raffinose, lactose, mucin (pig), asialomucine (pig), mucin (bovine). ) Was found to bind specifically. Glycoprotein was found to bind to mucin.

[Example 4]
(6) Specificity test (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 of front eluate with respect to standard oligosaccharide (PA-M3, number 003): V-V0] is measured. did.
As a result of evaluating the specificity of the sugar chain group composed of 49 types, as shown in FIG. 7, it was confirmed that CNA-II had a delay in elution that was bound only to globotetraose (Gb4).

Furthermore, the number of sugar chains was increased, and the fluorescence intensity (excitation wavelength: 310 nm and fluorescence wavelength: 380 nm) of the PA sugar chain eluate was monitored, and the interaction strength [standard oligosaccharide (PA-M3, number 003) A similar experiment was conducted to measure the difference: V−V0].
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).

Paranitrophenyl (pNP-)-labeled βGalNAc (No. 1004) was prepared in a TBS buffer so as to be 0 μM to 3.5 μM. The analysis was performed in the same manner as described above, and the interaction strength [standard oligosaccharide (pNP-Fuc, No. 1011)] at each concentration was measured to determine the difference in frontal eluate: V−V ₀ ]. (Bt) was determined. As a result, the binding constant (Ka) = 5.56 × 10 ⁵ M ⁻¹ and the amount of effective ligand (Bt) = 0.045 nmol. The interaction strength and binding constant of the bound sugar chain were calculated.
Paranitrophenyl (pNP-)-labeled αGalNAc (No. 1003) was prepared with TBS buffer so as to be 0 μM to 7.0 μM. The analysis was performed in the same manner as described above, and the interaction strength [standard oligosaccharide (pNP-Fuc, number 1011)] at each concentration was measured to determine the difference in front eluate: V−V ₀ ], and the binding constant (Ka) and effective ligand were determined. The amount (Bt) was determined. As a result, the binding constant (Ka) = 2.70 × 10 ⁵ M ⁻¹ and the effective ligand amount (Bt) = 0.039 nmol. The interaction strength and binding constant of the bound sugar chain were calculated. The results are shown in Table 4 and FIG.

  As shown in Table 4 and FIG. 8, also in the sugar chain group composed of 151 types, the lectin CNA-II of the present invention binds only to globotetraose (Gb4) which is a globoside sugar chain of No. 716. did.

  Therefore, since the lectin CNA-II shows high specificity for the globoside sugar chain, it can be used for accurately detecting and separating only the sugar chain-containing substances containing these sugar chains, The presence or absence of these sugar chains can be accurately determined.

It is a figure which shows the result of the molecular weight measurement by SDS-PAGE of lectin CNA-II concerning the Example of this invention. It is a figure which shows the temperature stability of the lectin CNA-II concerning the Example of this invention. It is a figure which shows the pH stability of the lectin CNA-II 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 the lectin CNA-II concerning the Example of this invention. It is a conceptual diagram which shows the measurement result of the sugar chain specific binding property of the lectin CNA-II concerning the Example of this invention.

Claims (11)

A lectin derived from the genus Basidiomycete having the following characteristics:
(1) The molecular weight by SDS electrophoresis is 5,000 to 40,000,
(2) The molecular weight by gel filtration is 5,000 to 40,000,
(3) Binding to globoside sugar chains.   The lectin according to claim 1, having affinity for a globoside sugar chain containing N-acetylgalactosamine. 3. The lectin according to claim 1, wherein the lectin has an affinity represented by a binding constant of 1.0 × 10 ⁵ M ⁻¹ or more for a globoside 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 any one of claims 1 to 3, which is obtained by purification by affinity chromatography using a carrier containing sucrose.   The lectin according to any one of claims 1 to 4, wherein the basidiomycete basidiomycete is gray shimeji mushroom.   It is a manufacturing method of the lectin of any one of Claims 1-5, Comprising: Obtaining an aqueous-medium medium extract from a cabbage genus basidiomycete, The said aqueous-medium medium extract is 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 5 and a labeled compound.   A lectin immobilization carrier comprising the lectin according to any one of claims 1 to 5 and a support carrier.   Detection of a globoside sugar chain-containing substance using the lectin according to any one of claims 1 to 5, the labeled lectin according to claim 7, or the lectin immobilization carrier according to claim 8. A sugar chain detection method comprising:   The sugar chain detection method according to claim 9, further comprising separating the globoside-based sugar chain-containing product from other compounds.   The determination drug which determines the presence or absence of a globoside type sugar chain containing the lectin of any one of Claims 1-5.