JPH0770186A - Novel lectin and its production - Google Patents

Abstract

PURPOSE:To obtain a novel lectin extracted from a red alga, Hypnea japonica, accelerating the clarification of the structural activity relation of lectin molecule and useful as an important basic information material used for the practical utilization of the lectin. CONSTITUTION:A red alga, Hypnea japonica, is extracted with a wateralcohol mixture solvent, and the extract is subjected to a cold alcohol precipitation treatment, to a gel filtration and subsequently to a reversed phase high performance liquid chromatography to obtain the objective lectin having the primary structure of the formula (the double bond exhibits a disulfide bond). The red alga is preferably collected in the coast of a southwest warm district in Japan, e.g. in the coast of Kagoshima prefecture.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel lectin and a method for producing the same. More specifically, it relates to a novel lectin consisting of 90 amino acid residues and a method for producing the same.

[0002]

2. Description of the Related Art Lectin is a sugar-binding protein (or glycoprotein) other than the product of an immune reaction and is a substance having an action of aggregating cells or precipitating a complex carbohydrate.
Lectins also exhibit various biological activities such as lymphocyte blastogenic activity and tumor cell-specific aggregation activity, and there is much interest in elucidating their physiological significance. Lectin was first found in plants, but it was revealed that it is distributed not only in plants but also in animals and microorganisms, and in recent years it has been found that it is present in many seaweeds. Many seaweed lectins exist as monomers of lower molecular weight than plant lectins, and have the common feature of not recognizing monosaccharides [eg moat.
Naruji, Biology and Chemistry, 27, 210-212 (1989)].

Red alga Hypnea japonica
The lectin from which it was found was found by some of the present inventors and was named "Hypnin A". The basic component of this lectin has been announced as a simple protein with a molecular weight of 4,200. It has a blastogenic activity on mouse spleen-derived lymphocytes and the ability to aggregate mouse tumor cells, and is a complex N-glycoside type. It has been reported to have many features common to seaweed lectins, such as recognition of sugar chains [K. Hori, K. Miyazawa, N. Fusetani,
K. Hashimitomo and K. Ito, Biochem. Biophys. Act
a, 873, 228-236 (1986)]. The molecular weight of "Hypnin A" was announced based on the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 4.
200 (5,700 for gel filtration without organic solvent)
Value is reported to be extremely low and has the lowest molecular weight among lectins obtained from nature, and is considered to be a kind of active fragment of high molecular weight lectins. Moreover, the aggregation activity of "Hypnin A" is 100.
It has been reported that it is not inactivated even if it is heated at 30 ° C for 30 minutes, and it is a protein with extremely strong heat resistance.

"Hypnin A" having the interesting properties as described above is considered to be an excellent material for elucidating the structure-activity relationship of the lectin molecule, but its purification is insufficient, and it is structurally constructed. The characteristics have not been revealed. In addition, the primary structure that is the basis of the molecular structure of proteins has not been elucidated at all.

[0005]

Therefore, the object of the present invention is to promote the separation and purification of the constituents of the lectin derived from the red alga Kalybaranori sufficient for practical use,
It is to provide a lectin whose primary structure is clear. Another object of the present invention is to provide a method for producing the lectin.

[0006]

[Means for Solving the Problems] In view of the above circumstances, the present inventors have earnestly studied and, as a result, have obtained a new lectin derived from the red alga Nostoc communis, and elucidated its primary structure, and also produced the lectin. A method was found and the present invention was completed.

The novel lectin derived from the red alga Nostoc communis of the present invention is characterized by having an amino acid sequence shown in the following sequence listing and having a primary structure represented by the following formula. The new lectin will be referred to as “hypnin A-
2 "is abbreviated.

[0008]

[Chemical 2]

The abbreviations used in the above formula have the following meanings. A Ala Alanine (all L-forms) R Arg Arginine N Asn Asparagine D Asp Aspartic acid C Cys Cysteine Q Gln Glutamine E Glu Glutamate G Gly Glycine I Ile Isoleucine L Leu Leucine K Lys Lysine F Phe Proline Alanine Thr Threonine W Trp Tryptophan Y Tyr Tyrosine V Val Valine

(1) Production of "Hypnin A-2""HypninA-2" can be produced as follows. That is, using the red alga Kagiibara Nori as a raw material, water-
It was purified from the mixed alcohol extract by cold alcohol precipitation and then gel filtration until a single band was obtained on SDS-PAGE, and the purified sample thus obtained was further subjected to reverse phase high performance liquid chromatography (HPLC).
By using the same N,
An active ingredient having a terminal amino acid sequence and exhibiting the same level of hemagglutination activity was obtained as three separated active ingredients. Of these three active ingredients, the active ingredient with the highest yield was selected as "hyphenin A-
Collect as 2 ".

In the present invention, the red alga Kagibaranori used as a raw material can be collected on the southwestern coast of Japan, such as the coast of Kagoshima Prefecture. As the algal cells, either fresh algal cells or freeze-dried powder may be used.

As the alcohol used in the extract and the alcohol precipitation, any of methanol, ethanol and propanol can be used, but ethanol is preferable. The proportion of alcohol in the extract is 20% to 55%
The degree is preferable, and an optimum ratio is selected between them. 20
% Or as low as 30%, including chlorophyll,
Unwanted protein-binding substances also tend to be extracted together, and if the mixing ratio is too high, the desired protein will not be extracted sufficiently. In addition, about 4 degreeC is illustrated as a preferable temperature at this time. The final alcohol concentration during cold alcohol precipitation may be higher than 70% and selected in the range of about 85%. The temperature of the cold alcohol at this time is -2.
The temperature is, for example, about 0 ° C to -40 ° C. The degree of purification can be increased by repeating a series of operations from extraction to cold alcohol precipitation twice or more.

The obtained protein is adjusted to pH 5 or pH.
During 8 hours, it is more preferably suspended in a buffer solution having a pH of 5 to 7 and applied to a gel filtration column equilibrated with the same buffer, and the active ingredient is fractionated by the absorption at 280 nm and the hemagglutination activity as indicators. Then, apply SDS-PAGE,
Make sure to give a single band. This is the above-mentioned "hypnin A".

Next, this "hypnin A" is further subjected to reverse phase HPLC. This process is called "Hypnin A-
It is indispensable to obtain 2 ”. Octadecylsilane (ODS) filler is suitable as the stationary phase for reverse phase HPLC, but C ₈ alkylsilane fillers can also be used. "Hypnin A" is separated into three active components by the reverse phase HPLC treatment. Of these, the component with a high yield, which accounts for 60% or more of the whole, is fractionated as a new lectin "Hypnin A-2" and used for the following primary structure analysis. The three active ingredients were determined to have a molecular weight of about 6,000 by the gel filtration method, and the hemagglutination activity was almost the same.

(2) Analysis of the primary structure of "Hypnin A-2" The analysis of the primary structure can be carried out as follows. First, regarding the amino acid composition analysis of proteins, various known methods can be used. Post-column method using ninhydrin, O-phthalaldehyde, or fluoresamine, or preliminarily converting amino acid to phenylisothiocyanate (PITC), 4-fluoro-7
-Nitrobenzo-2-oxa-1,3-diazole (N
BD-F), 4-dimethylaminoazobenzene 4'-
A pre-column method in which a derivatizing reagent such as sulfonyl chloride (dabsyl chloride) is reacted and then this is separated and detected by reverse phase liquid chromatography can also be applied. The precolumn method enables highly sensitive analysis using a small amount of sample, and the analysis time is short.

For the hydrolysis of proteins, an acid hydrolysis method is devised so that ordinary protein-constituting amino acids containing tryptophan, cysteine modified by reductive alkylation, etc. can be separated and quantified simultaneously and in a short time. Have been announced in recent years and can be efficiently achieved by applying them.

Regarding the analysis of the entire primary structure of "hypnin A-2" in the present invention, in order to identify cysteine and make it susceptible to hydrolysis by the subsequent enzymatic reaction,
First, it is preferable to cut and modify the disulfide bond. Although several methods are known for this purpose, the reductive alkylation method, the reductive carboxymethylation method, the reductive pyridylethylation method and the like are suitable for the present invention, and the reductive pyridylethylation method is particularly preferable. . The thus modified "hypnin A-2" is cleaved in several ways with high specificity for a particular amino acid residue. Each cleaved peptide fragment is separated and purified, and the amino acid sequence of each peptide fragment is determined.

As the highly specific cleavage method, there are a chemical cleavage method and an enzymatic cleavage method. In the present invention, a hydrolysis method using an enzyme, particularly a highly specific protease, is suitable. Highly specific proteases include trypsin, α-chymotrypsin, Staphyrococcus
aureus V8 protease and the like. Purified products of these enzymes are commercially available and can be easily used.

Separation and purification of peptide fragments is carried out by reversed-phase liquid chromatography, for example, HPLC using a reversed-phase system column.
Effectively achieved by. The achievement of separation and purification can be confirmed by, for example, the uniformity on thin layer chromatography.

The amino acid sequence of each peptide fragment is conveniently determined by an automatic Edman degradation analyzer, that is, a protein sequencer having a phenylthiohydantoin (PTH) -amino acid analyzer. By thus overlapping the amino acid sequence of each peptide fragment thus obtained with the amino acid sequence of each peptide fragment hydrolyzed by another specific protease, it is possible to determine the entire amino acid sequence of "hypnin A-2". it can.

The determination of the N-terminal amino acid sequence is carried out by "Hypnin A-" by the protein sequencer described above.
2 ”has already been achieved by determining the amino acid sequence of itself, but for determining the C-terminal amino acid sequence, the C-terminal peptide is selectively isolated using an immobilized anhydrotrypsin column, and the amino acid is determined by Edman degradation. It is preferred to determine the sequence from the N-terminus. Besides, a gas phase hydrazine decomposition method or the like can also be used.

Thus, "hypnin A-2" consists of 90 amino acid residues, the N-terminal is Phe (phenylalanine), the C-terminal is Asn (asparagine), and G
It was found to be a protein containing 16 residues of ly (glycine) and 16 residues of Ser (serine).

The existence state of the cysteine residues at positions 5, 12, 62 and 89 can be clarified by using a fluorescent thiol reagent. 7-Fluoro-4-sulfamoyl-2,1,3-benzoxydiazole (ABD
By using -F) and the like under reducing and non-reducing conditions, the presence or absence of a free SH group can be investigated relatively easily.
Furthermore, by using tri-n-butylphosphine and ABD-F, the amino acid sequence of a peptide containing a disulfide bond can be determined. Thus, the total primary structure is determined.

According to the result of hydropathic analysis in which 19 amino acid residues are sequentially set from the end and the amino residues are shifted one by one, the results show that only a slight hydrophobic region exists on the N-terminal side. It has been found that there are many hydrophilic regions around the ends.

The molecular weight of the novel lectin "Hypnin A-2" of the present invention calculated from the primary structure is 9,109.4. On the other hand, as a result of mass spectrometry by the plasma desorption (PD) method, which is one of the soft ionization methods, a monovalent molecular ion peak with a mass number of 9,163.0 is detected.
It was in agreement with the molecular weight calculated from the primary structure. They are,
Molecular weight obtained from SDS-PAGE (4,000),
It is different from the molecular weight (6,000) obtained from gel filtration. Therefore, the novel lectin of the present invention is SDS-PAG.
It is considered that E and gel filtration show abnormal behavior.

Regarding the relationship between disulfide bond and activity,
The hemagglutination activity disappeared when the disulfide group was cleaved and cysteine was reductively pyridylethylated. From this, it is considered that two disulfide bonds are necessary for the activity expression of the novel lectin. When two sulfide bonds are represented by Cys as Cys, Cy is as described above.
It has been found to exist between s ⁵ and Cys ⁶² and between Cys ¹² and Cys ⁸⁹ .

[0027]

According to the present invention, it is possible to provide a novel primary structure of a lectin which has hitherto been unknown and a method for producing the same. In addition, the determined primary structure of the novel lectin promotes elucidation of the structure-activity relationship of the lectin molecule and serves as important basic information that contributes to the practical use of the lectin.

[0028]

EXAMPLES Examples will be given to explain the present invention in more detail, but the present invention is not limited thereto.

(1) Manufacture of "Hypnin A-2" Fresh algal bodies of Scutellaria barnori collected in Kagoshima Prefecture 9.6
Pour kg into 50% ethanol, leave at 4 ° C,
Stirring was repeated occasionally to obtain an extract. The obtained extract was concentrated about 10 times and the active fraction was recovered by precipitation with 50-83% cold ethanol. The collected fraction was suspended in a 20 mM phosphate buffer solution (pH 5.5) and dialyzed sufficiently against the same solution. The crude fraction thus obtained was used for purification by gel filtration column Toyopearl HW40S column (φ2.5 ×
95 mg) and eluted with the above buffer at a flow rate of 20 ml / h to purify 33 mg of "hypnin A".
SDS-PAGE on the purified "Hypnin A"
When its unity was investigated by, the molecular weight was about 4,000.
0 single band was given. Then, a part of the obtained "Hypnin A" was reversed phase HPLC YMC-Pack.
It was subjected to R & D (φ4.6 × 250 mm) and eluted with a gradient of acetonitrile to obtain three separated fractions. All of these three fractions contained a hemagglutination active component, which was analyzed in the order of elution by "hypnin A-
1 "," Hypnin A-2 "," Hypnin A- "
3 ”. The yields were 330, 996 and 249 μg, respectively. The molecular weights of these three elution fractions were determined as ribonuclease (MW: 13.5 kDa), insulin (MW: 5.8 kDa), and insulin B chain (M:
W: 3.4 kDa), bacitracin (MW: 1.4 kDa)
Gel filtration column TSKge with a) as a molecular weight marker
When determined by gel filtration using a 1 G2000SW column, all were judged to be about 6,000 monomers.
"Hypnin A-2" with the highest yield
Was subjected to the following primary structural analysis.

(2) Amino acid composition analysis "Hypnin A-2" was added to 7M guanidine hydrochloride and 1
3% in sealed tube after pyridylethylation in 0.5 M Tris-HCl buffer (pH 8.5) containing 0 mM EDTA
(W / V) Phenol-6N HCl at 166 ° C. for 20
After hydrolysis for minutes, the degradation product was dabsylated and the amino acid composition was analyzed by an amino acid analyzer (Hitachi 835 type).

(3) Determination of the total primary structure of "Hypnin A-2" (i) Cleavage and modification of disulfide bond 900 µg of "Hypnin A-2" was added to 300 µl of guanidine hydrochloride and 0.5 mM containing 10 mM EDTA.
It was dissolved in Tris-hydrochloric acid buffer solution (pH 8.5). The inside of the container was replaced with nitrogen, 1 μl of 4-vinylpyridine was added and mixed, and then 2 μl of tri-n-butylphosphine was further added and left overnight in the dark to allow sufficient reaction. Then, it was dialyzed against water to remove salts and excess reagents to prepare "pyridylethyl (PE) -hypnin A-2".

(Ii) Enzymatic Degradation For enzymatic degradation of "PE-hypnin A-2", trypsin, α-chymotrypsin, Staphyrococcus
Using aureus V8 protease, the procedure was as follows. (Decomposition by trypsin) 100 μg of “PE-hypnin A-2” was dried to dryness under reduced pressure, 90 μl of 50 mM ammonium hydrogencarbonate buffer (pH 8.5) was added to dissolve it, and 10 μl of trypsin solution (0.2 μg / ml) was added and the mixture was decomposed at 37 ° C. for 12 hours. (Decomposition by α-chymotrypsin) 100 μg of “PE
Chemically-hypnin A-2 "was similarly dried under reduced pressure to 90 μm.
l of 50 mM ammonium hydrogen carbonate buffer (pH 8.
3) was added and dissolved, 4 μl of α-chymotrypsin solution (1 μg / ml) was added, and the mixture was decomposed at 37 ° C. for 6 hours. ( Decomposition by Staphyrococcus aureus V8 protease) 100 μg of “PE-hypnin A-2” was similarly dried under reduced pressure, and 90 μl of a phosphate buffer solution (pH 7.8) containing 2 mM EDTA was added and dissolved. To this, Stap
hyrococcus aureus V8 protease solution (1 μg / m
1) was added and the mixture was decomposed at 37 ° C. for 20 hours.

(Iii) Separation of peptides by reverse phase HPLC column and determination of primary structure First, trypsin, α-chymotrypsin, Staphyrococcus
Each degradation product decomposed by each enzyme of aureus V8 protease was used as a reverse phase HPLC column TSKgel O.
It was subjected to a reversed phase HPLC column using a DS-120T column, and each peptide fragment was separated (Fig. 1, Fig. 2, Fig. 3). After confirming the unity of each separated peptide fragment by TLC, a peptide sequencer (477A) and PTH-amino acid analyzer (1) manufactured by ABI were used.
20A) analyzed the amino acid sequence. The entire amino acid sequence of "hypnin A-2" was determined by overlapping the amino acid sequences thus obtained. The C-terminal amino acid sequence was subjected to affinity chromatography using immobilized anhydrotrypsin of 100 μg of PE-hypnin A-2 trypsin degradation product (FIG. 4), and non-adsorbed fractions were collected to obtain peptide fragments thereof. Was confirmed. On the other hand, the N-terminal amino acid sequence was confirmed from the same sequence of PE-hypnin A-2 itself. As a result, "Hypnin A-2" is a simple peptide consisting of 90 amino acid residues having the amino acid sequence described in the sequence listing and the primary structure shown in Chemical formula 1, and the N-terminal is It was revealed that phenylalanine, asparagine at the C-terminus, contains 16 residues of glycine, 16 residues of serine, and 7 residues of prilone.

(Iv) Mass Spectrometry by Plasma Desorption (PD) Method The molecular weight of "Hypnin A-2" was applied to a mass spectrometer by PD method (BILON, ABI). Adsorb "Hypnin A-2" on nitrocellulose paper,
The mass of the "hyphenin A-2" molecular ion desorbed by the collision of energetic particles generated by the decay of the ion source was measured. As a result, a monovalent molecular ion peak with a mass number of 9,163.0 was detected (FIG. 5).

(V) Hydropathy analysis The hydropathy analysis is GENE as a database.
Using TYX, 19 amino acid residues were sequentially set from the end as a set and examined by shifting the amino acid residues one by one. It was found that there is only a slight hydrophobic region on the N-terminal side and there are many hydrophilic regions centering on the C-terminal.

(Vi) Examination of Existence State of Cysteine Residues It was found by the above-mentioned primary structural determination that one molecule of the novel lectin of the present invention has four cysteine residues. Since they construct a higher-order structure by binding them with a disulfide bond in the protein, the existence state thereof was examined. First free SH
ABD-F solution with or without a group at 60 ° C under reducing and non-reducing conditions
The fluorescence intensity was measured by the fluorescence method in which each of the four residues was involved in the disulfide bond. Therefore, untreated "Hypnin A-2" 10
0 .mu.g of .alpha.-chymotrypsin degradation product was fractionated by reverse phase HPLC, and 0.1 .mu.M boric acid containing 1 mM EDTA and 0.5% SDS was added to 20 .mu.l of the fractionation solution in the presence of 10 .mu.l of reducing agent tri-n-butylphosphine. After reacting with a 1 mM ABD-F solution prepared with a sodium buffer solution (pH 8.0) at 60 ° C. for 1 hour, 0.6 ml of 0.1N was added after cooling.
Add HCl, excitation wavelength 385 nm, fluorescence wavelength 520
The fluorescence intensity was measured in nm and the fraction containing the disulfide bond was searched. Fractions with high fluorescence intensity were subjected to TLC, and after confirming the unity, the disulfide-bonded peptide fragment was subjected to amino acid composition and amino acid sequence analysis.
The disulfide bond between ^5- Cys ⁶² was confirmed and identified. In addition, since there is no free SH group in the lectin, the remaining disulfide bond was identified as Cys ¹² -Cys ⁸⁹ .

[0037]

[Sequence list]

Sequence length: 90 Sequence type: Amino acid Topology: Both forms Sequence type: Peptide Origin Biological name: Red alga Hypnea japonica Sequence features 5th and 62nd, 12th and 89th Cy
s forms a disulfide bond. Sequence Phe Gly Pro Gly Cys Gly Pro Ser Thr Phe Ser Cys Thr Ser Pro Gln 5 10 15 Lys Ile Leu Pro Gly Ser Ser Val Ser Phe Pro Ser Gly Tyr Ser Ser 20 25 30 Ile Tyr Leu Thr Thr Glu Ser Gly Ser Ala Ser Val Tyr Leu Asp Arg 35 40 45 Pro Asp Gly Tyr Trp Val Gly Gly Ala Asp Ser Lys Gly Cys Ser Asn 50 55 60 Phe Gly Gly Phe Ser Gly Asn Gly Asp Ser Lys Val Gly Asn Trp Gly 65 70 75 80 Asp Val Pro Val Ala Ala Trp Ala Cys Asn 85 90

[Brief description of drawings]

FIG. 1 shows a chromatogram pattern when a trypsin degradation product of “PE-hypnin A-2” was subjected to reverse phase HPLC.

FIG. 2 shows a chromatogram pattern obtained by subjecting an α-chymotrypsin degradation product of “PE-hypnin A-2” to reverse phase HPLC.

[3] "PE of - Haipunin A-2" Staphyroco of
Reversed phase HPLC of ccus aureus V8 protease degradation product
The chromatogram pattern when exposed to water is shown.

FIG. 4 shows a chromatogram pattern when a peptide fragment of an affinity chromatography non-adsorbed fraction using anhydrotrypsin immobilized with a trypsin degradation product of “PE-hypnin A-2” was subjected to reverse phase HPLC. .

FIG. 5 shows a chart of “Hypnin A-2” when subjected to PD mass spectrometry.

Claims (2)

[Claims] 1. A novel lectin having the following primary structure: 2. The red alga Hypnea japonic
The novel lectin according to claim 1, which comprises a step of extracting a ) with a water-alcohol mixture solution, cold alcohol precipitation, gel filtration of this, and subjecting it to reverse phase high performance liquid chromatography. Manufacturing method.