JP5211311B2 - Novel lectin and method for producing the same, sugar chain detection method and sugar chain fractionation 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 and a method for producing the same, and a sugar chain detection method and a sugar chain fractionation method using the lectin.

  Lectin is a general term for substances having specific binding activity to sugars 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).
Japanese Patent No. 2630431 Japanese Patent No. 2895962 Japanese Patent Laid-Open No. 10-307138

There are sugar chains that are difficult to recognize with currently known lectins, and there is a growing demand for the development of new lectins. In particular, in the case of known glucose and mannose-specific lectins such as concanavalin A and pea lectin, it is impossible to determine the binding mode of glucose or mannose.
An object of the present invention is to provide a lectin that can specifically detect glucose oligomers or mannose oligomers having an α1 → 6 bond in the binding of glucose or mannose.

The lectin of the present invention has (1) a molecular weight of about 10,000 to 30,000, (2) the amino acid sequence of the N-terminal region is Thr-Ile-Gly-, and (3) Glcα1 It is a lectin derived from basidiomycetes of the genus Hygrophorus that binds to 6Glc and Manα1 to 6Man.
The amino acid sequence of the N-terminal region is preferably Thr-Ile-Gly-X-Ala-Lys-Pro-.
In addition, the lectin of the present invention contains an aqueous medium extract of N. pertussis basidiomycetes, an eluate containing a sugar having a glucose residue and / or a mannose residue, and a glucose residue and / or a mannose residue. It is preferable to be obtained by purification by affinity chromatography using a carrier.

Further, the method for producing a lectin of the present invention comprises an extraction step for obtaining an aqueous medium extract from the fruiting body of the genus Bacillus subtilis, and the aqueous medium extract contains a sugar having a glucose residue and / or a mannose residue. And a purification step for purification by affinity chromatography using a glucose residue and / or a carrier containing a mannose residue.
The basidiomycete basidiomycete is preferably at least one selected from the group consisting of sakura shimeji, hime sakura shimeji, sakura shimeji.

The labeled lectin of the present invention includes the lectin and a labeling means.
The method for detecting a sugar chain compound of the present invention is a method for detecting a sugar chain comprising a step of detecting a sugar chain containing at least one of Glcα1 → 6Glc and Manα1 → 6Man using the lectin or the labeled lectin. .
Moreover, the determination agent of the present invention is a determination agent for determining the presence or absence of a sugar chain containing Glcα1 → 6Glc and / or Manα1 → 6Man containing the lectin.

The lectin immobilization carrier of the present invention includes the lectin and a support carrier.
Furthermore, the method for fractionating a sugar chain compound of the present invention includes a step of fractionating a sugar chain containing at least one of Glcα1 → 6Glc and Manα1 → 6Man using the lectin or the lectin immobilization carrier. It is.

  According to the present invention, it is possible to provide a lectin that can specifically detect glucose oligomers or mannose oligomers having an α1 → 6 bond in the binding of glucose or mannose.

The lectin of the present invention is a lectin derived from a basidiomycete belonging to the genus Numerigasa. Especially, it is preferable that it is a lectin derived from the fruiting body (mushroom) of the genus Numerisasa.
Examples of basidiomycetes of the genus Numeryasa in the present invention can include cherry shimeji, himesakura shimeji, sakura shimeji mushroom, akebonosa shimeji mushroom, turmeric, kineme rimasa, kokei numera, shimofurin rimasa, fukikura shimeji, goattake and the like. Among these, from the viewpoint of lectin sugar recognition specificity and lectin recovery efficiency, at least one selected from cherry shimeji, hime sakura shimeji, and sakura shimeji is preferred, and sakura shimeji is more preferred.

The lectin of the present invention has a molecular weight of 10,000 to 30,000 by SDS electrophoresis, but is preferably 15,000 to 20,000.
The molecular weight can be measured by SDS electrophoresis according to the Laemmi method (Nature, Vol. 227, p. 680, 1976), for example.

The lectin of the present invention includes a protein having an amino acid sequence consisting of Thr-Ile-Gly- (SEQ ID NO: 1) in the N-terminal region. The amino acid residue in the N-terminal region is Thr-Ile- Gly-X-Ala-Lys-Pro- (SEQ ID NO: 2) is preferred. Here, X may be any amino acid residue, but is preferably Thr or Cys.
Among them, the N-terminal region is Thr-Ile-Gly-Thr-Ala-Lys-Pro-Ile-Leu-Ala-Gln-Thr-Ala-Ile-Val-Gly-Gly-Pro-Ser-Val-Pro- Phe-Asp-Asp-Ala-Arg-Glu-Val-Ala-Ser-Trp-Pro-Ala-Lys-Leu-Glu-Ile-Ala-Gln-Asp- (SEQ ID NO: 3), Thr-Ile-Gly- X-Ala-Lys-Pro-Val-Leu-Val-Gln-Asn-Val-Leu-Leu-Gly-Gly-Pro-Ala-Val- (SEQ ID NO: 4) or Thr-Ile-Gly-X-Ala- It is more preferable to include a protein having an amino acid sequence consisting of Lys-Pro-Val-Leu-Val-Gln-Thr-Gly-Ile-Val-Gly-Gly-Pro-X-Val- (SEQ ID NO: 5).

Moreover, the lectin of the present invention is characterized by having high binding specificity for Glcα1 → 6Glc and Manα1 → 6Man.
Here, Glcα1 → 6Glc means an isomaltose structure in which two molecules of glucose are linked by an α1 → 6 bond. In addition, Manα1 → 6Man means an α1 → 6 mannobiose structure in which two molecules of mannose are bonded by an α1 → 6 bond.
The lectin of the present invention has a sugar-binding specificity not known to conventional lectins that can specifically recognize a sugar chain having a partial structure in which glucose or mannose is bound by an α1 → 6 bond.

  The sugar binding specificity of a lectin can be confirmed, for example, by examining the type and concentration of a saccharide that can inhibit the agglutination reaction of erythrocytes using erythrocytes that can be specifically aggregated. .

  The lectin of the present invention can be isolated and produced from a basidiomycete basidiomycete by appropriately combining known extraction methods, separation methods, purification methods, and the like. Especially, it is preferable that it is a lectin obtained with the manufacturing method demonstrated below.

  The method for producing a lectin of the present invention includes an extraction step of obtaining an aqueous medium extract using the fruit body of the genus Bacillus basidiomycetes as a raw material and using an aqueous medium as an extraction solvent.

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, phosphate buffer and the like are 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 physiological saline obtained by further adding sodium chloride to a phosphate buffer solution can be preferably used as the aqueous medium in the present invention.

  The organic solvent can be used without particular limitation as long as it is an organic solvent that can be mixed with water. Among them, acetone, methanol, ethanol, 2-propanol, and acetonitrile are preferable. 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.

  A method for obtaining an aqueous medium extract from an aqueous medium and a fruit body of the genus Bacillus subtilis is not particularly limited as long as the aqueous medium and the fruit body of the genus Bacillus subtilis can be brought into contact with each other. From the viewpoint of extraction efficiency, a method of pulverizing the fruit bodies of the genus Bacillus basidiomycetes in an aqueous medium to form a suspension is preferable. Moreover, as a method to grind | pulverize, normal grind | pulverizing methods, such as a mixer and a homogenizer, can be mentioned.

  The extraction step in the present invention preferably further includes a step of removing an insoluble matter in the aqueous medium from the mixture of the aqueous medium and the fruit body of the genus Basilis. Examples of the method for removing insoluble materials include filtration, centrifugation, and the like, but centrifugation is preferred from the viewpoint of removal efficiency.

  The extraction step of the present invention is particularly preferably a step of obtaining an aqueous medium extract by pulverizing the fruiting bodies of the genus Bacillus basidiomycetes in phosphate buffered saline and removing the insoluble matter by centrifugation.

  In the method for producing a lectin of the present invention, the aqueous medium extract obtained by the extraction step described above is prepared by using an eluate containing a saccharide having a glucose residue and / or a mannose residue, a glucose residue and / or a mannose residue. A purification step of purification by affinity chromatography using a carrier containing a group. Thereby, purification can be performed more efficiently.

As the carrier in the present invention, any carrier containing a glucose residue and / or a mannose residue can be used without particular limitation. Among these, a carrier containing a glucose residue having an α1 → 6 bond is preferable. Examples of the carrier containing a glucose residue having an α1 → 6 bond include a dextran carrier.
The dextran carrier is preferably a carrier containing a crosslinked dextran, more preferably a carrier having a fractional range of 100,000 Da or less, and more preferably a carrier having 1,500 to 80,000 Da. . Specifically, for example, Sephadex carrier (manufactured by GE Healthcare Biosciences) can be mentioned. Of these, Sephadex G-50 or Sephadex G-75 is particularly preferably used 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 a glucose residue and / or a mannose residue as a packing material. The aqueous medium extract is adsorbed on the column, and separated and fractionated using an eluate containing a saccharide having a glucose residue and / or a mannose residue, thereby further efficiently purifying the lectin of the present invention. be able to.
In the present invention, after the aqueous medium extract is adsorbed on the column, it is washed with an appropriate washing solution, for example, the above-mentioned buffer solution, and then has a glucose residue and / or a mannose residue. It is more preferable to separate and fractionate using an eluate containing saccharides.

The eluate can be used without particular limitation as long as it is a solution containing a saccharide having a glucose residue and / or a mannose residue. Examples of the saccharide having a glucose residue and / or a mannose residue include isomaltoligosaccharide, glucose, mannose, methyl-α-mannoside, 2α mannobyose, 3αmannobiose, 4αmannobiose, and 6αmannobiose. Etc. Among these, from the viewpoint of the sugar specificity of the lectin of the present invention, it is preferable to use isomaltoligosaccharide, glucose and mannose.
Although there is no restriction | limiting in particular in the density | concentration of the saccharide contained in the said eluate, For example, as content of the saccharide in the eluate containing an isomaltoligosaccharide, it is 0.1 mass%-10 mass% from the point of purification efficiency. It is preferable that there is 0.5% by mass to 2% by mass. The concentration of saccharide in the eluate containing glucose or mannose is preferably 10 mM to 1000 mM, more preferably 10 mM to 500 mM, from the viewpoint of purification efficiency.
In addition, commercially available glucose, mannose and isomaltoligosaccharides can be preferably used.

  As a purification step in the present invention, the aqueous medium extract is adsorbed on a column having a dextran 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 10 mM to 500 mM of at least one selected from mannose, glucose and isomaltoligosaccharide.

The method for producing a lectin of the present invention can include a step of dialysis treatment of the fraction containing lectin obtained in the purification step, and a step of lyophilizing the lectin solution after the dialysis treatment. Thereby, a lectin can be isolated.
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 it specifically recognizes α1 → 6 bond of glucose and mannose, it can be used as a test reagent, an immunomodulator, a specific adsorbent for separation analysis of carbohydrates, and the like.

The labeled lectin of the present invention includes at least the lectin and a labeling means, and is characterized by being detectably labeled. As the labeling means, known labeling methods can be applied without particular limitation, and examples thereof include labeling with a radioisotope, binding of a labeling compound, and the like.
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 sugar chain detection method of the present invention includes a step of detecting a sugar chain containing Glcα1 → 6Glc and / or Manα1 → 6Man using the lectin or the labeled lectin. Since the lectin is capable of specifically recognizing and binding Glcα1 → 6Glc and Manα1 → 6Man, sugar chain compounds such as polysaccharides, glycolipids, and glycoproteins containing sugar chains including Glcα1 → 6Glc and / or Manα1 → 6Man Can be specifically detected.
The lectin or the labeled lectin can be used as a determination agent for determining the presence or absence of a sugar chain containing Glcα1 → 6Glc and / or Manα1 → 6Man.

Detection of the sugar chain compound to which the lectin is bound can be easily performed by, for example, labeling the lectin in advance by a labeling means. 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 means used. For example, it can be performed by absorbance measurement, emission intensity measurement, fluorescence intensity measurement, visual observation, or the like.

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 the organic material include polyvinyl polymers such as rubber, latex, polystyrene and polypropylene, polyamides such as polyacrylate, polyacrylamide, polyester and gelatin, polysaccharides such as cellulose, agar and agarose. it can.
Moreover, there is no restriction | limiting in particular as a structure of a support carrier, For example, a porous structure, a fibrous structure, a gel-like structure etc. can be mentioned.

Examples of the support carrier in the present invention include polyacrylic acid ester, agarose, vinyl polymer and the like, and agarose is preferable from the viewpoint of sugar chain fractionation efficiency.
The lectin can be immobilized on the support carrier using a conventional method corresponding to the support carrier. Further, a linking group (spacer) may be provided between the lectin and the support carrier.

  The method for fractionating sugar chains of the present invention comprises the step of fractionating a sugar chain containing Glcα1 → 6Glc and / or Manα1 → 6Man using the lectin or the lectin immobilization carrier. Since the lectin is capable of specifically recognizing and binding Glcα1 → 6Glc and Manα1 → 6Man, sugar chain compounds such as polysaccharides, glycolipids and glycoproteins containing sugar chains containing Glcα1 → 6Glc and / or Manα1 → 6Man Can be specifically separated.

  The separation of the sugar chain compound to which the lectin binds can be easily performed by using, for example, a carrier on which the lectin is immobilized. 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.

  In the sugar chain detection method and sugar chain fractionation method of the present invention, the lectin of the present invention is used, but from the viewpoint of sugar chain detection sensitivity and sugar chain fractionation efficiency, mutual interaction with sugar chains including Glcα1 → 6Glc and Manα1 → 6Man The action strength is preferably 10 times or more the interaction strength with a sugar chain not containing Glcα1 → 6Glc and Manα1 → 6Man. The interaction strength between the lectin and the sugar chain can be measured using, for example, a hemagglutination inhibition test.

  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
100 ml of 10 mM phosphate buffered saline (pH 7.4, hereinafter sometimes referred to as “PBS”) is added to 100 g of fruit body of Hygrophorus russula collected from the mountain forest in Yamanashi Prefecture, and is added to the mixer. And crushed. This was left standing at 4 ° C. day and night, and then centrifuged (8500 × g, 20 minutes, 4 ° C.), and the supernatant was used as a crude extract (aqueous medium extract).
The obtained crude extract was applied to a Sephadex G-75 affinity column (GE Healthcare Biosciences; φ5 × 15 cm) equilibrated with PBS. The non-adsorbed fraction was washed with PBS (about 700 ml) and then eluted with a 300 mM mannose solution to obtain a lectin-containing fraction. The fraction containing lectin was identified by measuring the absorbance at 280 nm and the hemagglutination activity.
The obtained lectin-containing fraction was dialyzed by a conventional method and freeze-dried to obtain 6 mg of a lectin (Hygrophorus russula lectin: HRL) derived from basidiomycetes.

<Evaluation of lectins>
The following evaluation was performed on the lectin (HRL) obtained in Example 1.
[SDS-polyacrylamide electrophoresis]
According to the method of Laemmli et al., An acrylamide slab gel of 10% SDS containing concentrated gel 5% T and separation gel 15% T was prepared.
10 μg of lectin was dissolved in 31.25 μl of distilled water, then 10 μl of 10% SDS, 6.25 μl of 0.5M Tris buffer (pH 6.8) and 2.5 μl of 2-mercaptoethanol were added to make a total volume of 50 μl. After boiling this for 10 minutes, 5 μl each of 0.05% bromophenol blue and 70% glycerol was added to prepare a sample solution. As a molecular weight marker, XL-Ladder Low range (manufactured by APRO) was used.
Electrophoresis was performed using Compact-PAGE (manufactured by ATTO). Both 5 μl of sample solution and molecular weight marker were used. As a buffer solution for the electrophoresis tank, a buffer solution containing 25 mM Tris containing 0.1% SDS and 192 mM glycine was used. After completion of the electrophoresis, the gel was immediately immersed in a staining solution (0.1% CBB R-250, 30% methanol, 10% acetic acid) and stained while gently shaking at 60 ° C. for 30 minutes. Subsequently, it was immersed in a decolorizing solution (30% methanol, 10% acetic acid) and similarly decolorized at 60 ° C. with occasional replacement of the decolorizing solution. After sufficiently decolorizing, the gel was dried on a filter paper with a gel dryer, and the molecular weight was estimated from the relative mobility. The results are shown in FIG.

From FIG. 1, it can be seen that the molecular weight of HRL is 15,000 to 20,000.
In the preparation of the sample solution, electrophoresis was performed in the same manner as described above using a sample solution prepared in the same manner except that 33.75 μl of distilled water was used and 2-mercaptoethanol was not added. The same result as above was obtained. From this, it can be seen that the lectin of the present invention is not a protein in which subunits are linked by disulfide bonds.

[MALDI-TOF mass spectrometry]
10 μg of HRL was dissolved in TA (a mixture of 0.1% TFA 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 HRL at a volume ratio of 4: 1 onto the target plate. The molecular weight of HRL was measured in LP mode using Autoflex manufactured by Bruker Dartonics as a mass spectrometer. As a result, the molecular weight of HRL was about 18,500.

[Gel filtration chromatography]
As a gel filtration column, TSK-gel BioAssist G3SWXL (φ7.8 × 300 mm, manufactured by Tosoh Corporation) was used, and gel filtration chromatography by HPLC was performed as follows, and the molecular weight of unmodified HRL was measured. .
The sample solution was applied to a column that had been equilibrated in advance with PBS. Elution was performed at a flow rate of 0.5 ml / min, and the molecular weight was estimated from the elution time of the sample. Detection was carried out by absorbance at 280 nm, and molecular weight markers for gel filtration chromatography (manufactured by Sigma) were used to prepare a calibration curve for molecular weight estimation.

  From the results of gel filtration chromatography, it was found that the molecular weight of HRL in the native state was 74 kDa. Therefore, it can be estimated that the lectin of the present invention is a tetramer of subunits having a molecular weight of 18,500.

[Amino acid sequence of N-terminal region]
When the amino acid sequence of the N-terminal region of HRL was analyzed using Protein Peptide Sequencer PPSQ-21 System (manufactured by Shimadzu Corporation), it was found that it was the following amino acid sequence (SEQ ID NO: 3).
Thy-Ile-Gly-Thr-Ala-Lys-Pro-Ile-Leu-Ala-Gln-Thr-Ala-Ile-Val-Gly-Gly-Pro-Ser-Val-
Pro-Phe-Asp-Asp-Ala-Arg-Glu-Val-Ala-Ser-Trp-Pro-Ala-Lys-Leu-Glu-Ile-Ala-Gln-Asp

[Isoelectric focusing]
Isoelectric focusing was performed using a commercially available gel (PastGel IEF 3-10; manufactured by GE Healthcare Bioscience) according to the attached instruction manual.
A sample obtained by dissolving 10 μg of HRL in 10 μl of distilled water was used, and a load pI calibration kit (manufactured by GE Healthcare Bioscience) was used as an isoelectric point marker.
The isoelectric point of the lectin of the present invention was pI6.55.

[Temperature stability]
50 μg of HRL was dissolved in 1 ml of physiological saline and incubated 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.

  FIG. 2 shows that the lectin of the present invention exhibits temperature stability at 0 ° C. to 70 ° C.

[PH stability]
50 μg of HRL 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.

-PH buffer solution-
pH 2.0-3.5: 20 mM KCl-HCl buffer pH 4.0-5.5: 20 mM acetate buffer pH 6.0-7.5: 20 mM phosphate buffer pH 8.0-8.5: 20 mM Tris ( Tris (hydroxymethyl) aminomethane) buffer pH 9.0-10.5: 20 mM glycine-NaOH buffer Each buffer solution contains 0.15 M sodium chloride.

[Protein content]
The protein content was measured by the Bradford dye binding method using IgG as a standard. An IgG solution of 10 μg / ml to 1.25 μg / ml was prepared. To 800 μl of each solution, 200 μl of Bio-Rad Protein Assay Staining Solution (manufactured by Nippon Bio-Rad Laboratories) was added and stirred vigorously. After 30 minutes, the absorbance at 595 nm was measured to prepare a calibration curve.
A 20 to 5 μg / ml HRL sample solution was prepared, the absorbance was measured in the same manner as described above, and the protein content was estimated from a calibration curve.
The protein content of HRL was 79.6%.

[Neutral sugar content]
The neutral sugar content was measured by the phenol-sulfuric acid method using glucose as a standard. A 0-30% glucose solution was prepared and 100 μl of glucose solution was dispensed into a colorimetric tube. 0.5 ml of 5% phenol and then 2.5 ml of concentrated sulfuric acid were quickly added and shaken vigorously. And after cooling to room temperature, the light absorbency of 485 nm was measured and the analytical curve was created.
Using a 1 mg / ml aqueous solution of HRL as a sample, the absorbance was measured in the same manner as glucose, and the neutral sugar content was estimated from the calibration curve.
The neutral sugar content of HRL was 3.5%.

[Rabbit hemagglutination activity test]
After collecting 10 ml of rabbit blood, it was immediately mixed well in a test tube to which 2.0 ml of a 3.8% sodium citrate solution was added to prevent coagulation of the blood. Next, centrifugation (3000 × g, 3 min, room temperature) was performed to remove plasma and white blood cells, 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 rabbit 3% erythrocyte suspension.

-Measurement of hemagglutination activity-
20 μl of PBS was placed in one stage of a 96-well titer plate, 20 μl of lectin solution (20 μg / ml) was placed in the well, and a 1/2 dilution series was sequentially prepared. 20 μl of each 3% 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 agglutination was observed was defined 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.

[Sheep hemagglutination activity test]
In the rabbit hemagglutination activity test, a sheep hemagglutination activity test was conducted in the same manner except that sheep blood was used instead of rabbit blood. The results are shown in Table 1.

[Human hemagglutination activity test]
After collecting 10 ml of A-type, B-type and O-type human blood, it was immediately mixed well in a test tube to which 2.0 ml of 3.8% sodium citrate solution was added to prevent blood coagulation. Next, centrifugation (3000 × g, 3 min, room temperature) was performed to remove plasma and white blood cells, 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 3% erythrocyte suspensions of A type, B type, and O type. Next, the following treatment was performed to prepare an actinase E-treated erythrocyte suspension, a trypsin-treated erythrocyte suspension, and a neuraminidase-treated erythrocyte suspension.

-Preparation of actinase E-treated erythrocyte suspension 1 ml of the washed erythrocyte obtained above was suspended in 9 ml of PBS in which 4 mg of actinase E was dissolved, and the mixture was reacted by shaking at 45 ° C for 30 minutes. After the reaction, centrifugation (3,000 × g, 3 min, room temperature) was performed, and washing with only red blood cells by removing the supernatant was repeated three times. The red blood cells were diluted with PBS to give a 3% actinase E-treated red blood cell suspension.

-Preparation of trypsin-treated erythrocyte suspension 1 ml of the washed erythrocyte obtained above was suspended in 9 ml of PBS in which 1 mg of trypsin was dissolved, and the mixture was reacted by shaking at 37 ° C for 90 minutes. Furthermore, 1 mg of trypsin was added, and the reaction was performed by shaking at 37 ° C. for 90 minutes. After the reaction, centrifugation (3,000 × g, 3 min, room temperature) was performed, and washing with only red blood cells by removing the supernatant was repeated three times. The erythrocytes were diluted with PBS to give a 3% trypsinized erythrocyte suspension.

-Preparation of neuramidase-treated erythrocyte suspension 1 ml of the washed erythrocytes obtained above was suspended in 9 ml of PBS in which 1 U of neuramidase was dissolved, and reacted by shaking at 37 ° C for 60 minutes. After the reaction, centrifugation (3,000 × g, 3 min, room temperature) was performed, and washing with only red blood cells by removing the supernatant was repeated three times. The red blood cells were diluted with PBS to obtain a 3% neuramidase-treated red blood cell suspension.

  A human hemagglutination activity test was carried out in the same manner as in the rabbit hemagglutination activity test except that the A, B, and O type actinase E-treated erythrocyte suspension, trypsin-treated erythrocyte suspension, and neuraminidase-treated erythrocyte suspension obtained above were used. Hemagglutination activity was measured. The results are shown in Table 1.

[Sugar binding specificity]
The sugar binding specificity of lectins was evaluated by a hemagglutination inhibition test using various monosaccharides, oligosaccharides, polysaccharides and glycoproteins. A 2-fold dilution series of 10 μl sugar solution was prepared on a 96-well U-bottom microtiter plate. 10 μl of lectin solution that had been adjusted to a titer of 4 in advance was added to each well. After sensitizing by allowing to stand at room temperature for 1 hour, 20 μl of 3% red blood cell suspension was added to each hole, and the mixture was further allowed to stand at room temperature for 1 hour. Thereafter, the dilution rate of the sample solution that completely inhibits erythrocyte aggregation was determined with the naked eye. The lowest concentration showing inhibition was taken as the minimum inhibitory concentration. The smaller this minimum inhibitory concentration, the higher the specificity for lectins. The results are shown in Tables 2 and 3 below.
In addition, Table 2 and Table 3 below show the results of evaluating these specificities for concanavalin A lectin (ConA) and pea lectin (PSA), which are commercially available glucose mannose specific lectins.

Although not shown in Table 2, galactose, fucose, L-fucose, arabinose, L-arabinose, ribose, raffinose, L-rhamnose, saccharose, lactose, lactitol, N-acetylglucosamine, N-acetylgalactosamine, N -Acetyl lactosamine, methyl β-glucoside, methyl α-galacside, methyl β-galacside, melibiose, xylose, galacuronic acid, gluctosamine hydrochloride, galactosamine hydrochloride, mannosamine hydrochloride, guluronic acid, 2-deoxyribose, chitobiose, and Maltose showed no hemagglutination inhibitory activity at 100 mM.
Furthermore, N-acetylneuraminic acid and N-glycolylneuraminic acid did not show hemagglutination inhibitory activity at 40 mM.

Although not shown in Table 3, α ₁ -acid glycoprotein, transferrin, hyaluronic acid, and bovine albumin did not show hemagglutination inhibitory activity at 1 mg / ml.

  From the above results, it can be seen that the lectin (HRL) obtained in Example 1 specifically binds to Glcα1 → 6Glc and Manα1 → 6Man.

[Mitogenic activity]
Spleen lymphocytes were prepared by a conventional method using F344 rats (male, 8 weeks old). RPMI1640 (10% FCS, glutamine, 2-mercaptoethanol, penicillin / streptomycin, amphotericin B added) was used as a medium, and the culture solution of spleen lymphocytes was adjusted to 1 × 10 ⁶ cells per well. The HRL was added so that the final concentrations were 0 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, and 25 μg / ml. The cells were cultured at 37 ° C. for 38 hours in a 5% carbon dioxide atmosphere. The culture supernatant was collected, and the contents of cytokines (IL-6 and IFN-γ) in the culture supernatant were measured by ELISA. In addition, 4 μg / ml of ConA was used instead of HRL, and the amount of satokine in the culture supernatant was measured in the same manner. The results are shown in FIGS.

  4 and 5 show that HRL has IFN-γ and IL-6 production-inducing activities. Since IFN-γ production is induced depending on the HRL concentration, it can be seen that HRL has mitogenic activity. In addition, unlike ConA, HRL was found to significantly improve IL-6 production.

(Example 2)
In Example 1, 1 mg of lectin (HCL) derived from basidiomycetes basidiomycetes was obtained in the same manner as in Example 1, except that instead of cherry shimeji mushroom, Hygrophorus capreolarius was used.
The results of SDS-polyacrylamide electrophoresis are shown in FIG. It can be seen from FIG. 1 that the molecular weight of HCL is 15,000 to 20,000.

[Amino acid sequence of N-terminal region]
When the amino acid sequence of the N-terminal region of HCL was analyzed in the same manner as in Example 1, it was found that it was the following amino acid sequence (SEQ ID NO: 4).
Thr-Ile-Gly-X-Ala-Lys-Pro-Val-Leu-Val-Gln-Asn-Val-Leu-Leu-Gly-Gly-Pro-Ala-Val-
X is expected to be Cys because it could not be determined by N-terminal amino acid analysis.

[Hemagglutination activity]
Rabbit, sheep, and human hemagglutination activities were measured in the same manner as in Example 1 except that HCL was used instead of HRL. The results are shown in Table 1.
From the above results, it can be seen that HCL exhibits hemagglutination activity in rabbits and sheep.

[Sugar binding specificity]
The sugar-binding specificity of HCL was evaluated in the same manner as in Example 1 except that HCL was used instead of HRL. The results are shown in Tables 2 and 3.
From the above results, it can be seen that HCL specifically binds to Glcα1 → 6Glc and Manα1 → 6Man.

(Example 3)
In Example 1, 1 mg each of lectin (HPL) derived from the basidiomycete genus Bacillus was obtained in the same manner as in Example 1 except that instead of cherry shimeji mushroom, Hygrophorus purpurascens was used.
The results of SDS-polyacrylamide electrophoresis are shown in FIG. It can be seen from FIG. 1 that the molecular weight of HPL is 15,000 to 20,000.

[Amino acid sequence of N-terminal region]
When the amino acid sequence of the N-terminal region of HPL was analyzed in the same manner as in Example 1, it was found that it was the following amino acid sequence (SEQ ID NO: 5).
Thr-Ile-Gly-X-Ala-Lys-Pro-Val-Leu-Val-Gln-Thr-Gly-Ile-Val-Gly-Gly-Pro-X-Val-
X is expected to be Cys because it could not be determined by N-terminal amino acid analysis.

[Hemagglutination activity]
Rabbit, sheep and human hemagglutination activities were measured in the same manner as in Example 1 except that HPL was used instead of HRL. The results are shown in Table 1.
From the above results, it can be seen that HPL exhibits hemagglutination activity in rabbits and sheep.

[Sugar binding specificity]
The sugar binding specificity of HPL was evaluated in the same manner as in Example 1 except that HPL was used instead of HRL. The results are shown in Tables 2 and 3.
From the above results, it can be seen that HPL specifically binds to Glcα1 → 6Glc and Manα1 → 6Man.

  From the above, the lectin derived from the basidiomycetes of the present invention has a molecular weight of 10,000 to 30,000 as determined by SDS electrophoresis, and the amino acid sequence of the N-terminal region is Thr-Ile-Gly-. Thus, it can be seen that it binds to Glcα1 → 6Glc and Manα1 → 6Man.

It is a figure which shows the result of the molecular weight measurement by SDS-PAGE. It is a figure which shows the temperature stability of HRL. It is a figure which shows the pH stability of HRL. It is a figure which shows the IFN-gamma induction activity of HRL. It is a figure which shows IL-6 induction activity of HRL.

Claims (10)

Lectins derived from Hygrophorus basidiomycetes having the following characteristics:
(1) The molecular weight by SDS electrophoresis is 15,000-20,000 ,
(2) The amino acid sequence of the N-terminal region is Thr-Ile-Gly-X-Ala-Lys-Pro-
(3) It binds to Glcα1 → 6Glc and Manα1 → 6Man. The lectin according to claim 1, wherein the basidiomycete basidiomycete is at least one selected from the group consisting of sakura shimeji, hime shimeji, sakura shimeji. Affinity chromatography using an aqueous medium extract of Bacillus subtilis belonging to the genus Numerisa using an eluate containing a saccharide having a glucose residue and / or a mannose residue and a carrier containing a glucose residue and / or a mannose residue The lectin according to claim 1 or 2, which is obtained by purifying by the method. It is a manufacturing method of the lectin of any one of Claims 1-3 , Comprising: The extraction process which obtains an aqueous medium extract from the fruiting body of a genus Basilis genus, and the aqueous medium extract are made into glucose residue. And a purification step for purification by affinity chromatography using a carrier containing a saccharide having a group and / or a mannose residue and a carrier containing a carrier containing a glucose residue and / or a mannose residue. A method for producing lectins. The method for producing a lectin according to claim 4 , wherein the basidiomycete basidiomycete is at least one selected from the group consisting of sakura shimeji, hime shimeji, sakura shimeji. A labeled lectin comprising the lectin according to any one of claims 1 to 3 and a labeling means. A glycan comprising a step of detecting a glycan containing Glcα1 → 6Glc and / or Manα1 → 6Man using the lectin according to any one of claims 1 to 3 or the labeled lectin according to claim 6. Detection method. 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 or the lectin immobilization carrier according to claim 8 , the method includes a step of fractionating a sugar chain containing Glcα1 → 6Glc and / or Manα1 → 6Man. Glycan fractionation method.   The determination agent which determines the presence or absence of the sugar chain containing Glc (alpha) 1-> 6Glc and / or Man (alpha) 1-> 6Man containing the lectin of any one of Claims 1-3.