JPH01110698A - Human placenta-or umbilical cord-derived lectin - Google Patents

Abstract

NEW MATERIAL:A compound having an amino acid sequence expressed by the formula. USE:A remedy for myasthenia gravis and activator for cells, such as leukocyte. PREPARATION:For example, a human placenta is ground in a physiological saline solution buffered with a phosphoric acid containing 2mM EDTA 4mM beta-mercaptoethanol and centrifuged to collect a supernatant, which is then subjected to column chromatography to provide an active fraction. The resultant fraction is passed through a column of immobilized asialofetuin and the adsorbed substance is eluted with the above-mentioned physiological saline solution buffered with the phosphoric acid to collect an active fraction, which is subsequently freeze-dried to afford the aimed human placenta-derived lectin having the amino acid sequence expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to lectins. More specifically, lectins derived from human placenta or umbilical cord are used.

[Prior Art] Lectin is a protein derived from animals, plants, and microorganisms, and is a general term for substances that specifically bind to red blood cells.
Plant-derived hemagglutinin is known. The hemagglutination reaction caused by lectins is said to occur when lectins specifically bind to sugar chains present in red blood cells [
Toshiaki Osawa, Yoshi Mori - ed.; Lectin, 1 page, Kodansha (197
6th year)].

In addition, the lectins are not only for red blood cells, but also for white blood cells,
It is known that it also binds to cancer cells and aggregates them, and this property can be used to suppress the proliferation of cancer cells [Motoo Hozumi; Lectin, p. 141 (1976)] and to inhibit subclasses of blood cells. Separation method [tea, nakano ('l', l'Ja
J.Immun) et al.;
o 1. ) + Kamiyutan, p. 192B (1980)] have been proposed.

The currently known lectins derived from vertebrates include membrane proteinaceous ones with high molecular weight and Ca2+ dependence, and low molecular weight ones that are not Ca2+ dependent and have an affinity for the terminal β-D-galactoside structure. It is broadly divided into those that indicate

A typical example of the latter is the electric eel.
rophorus electricus), which has recently been reported to be effective in the treatment of experimental myasthenia (rabbit), which is a pathological model of myasthenia gravis [Giovann Levy].
I Levi) et al.; Neil, G.I., Imzuru;
(Eur, J, Immuno1.) + -L"-, 5
00 pages (1983)]. However, there are problems with immunogenicity when administering it to humans.

On the other hand, as human-derived low molecular weight lectins with an affinity for β-galactoside, those derived from cardiac muscle, skeletal muscle, and lung are known. Of these, those derived from muscle have not yet been sufficiently studied, and their properties are not clear. In addition, it has been reported that the molecular weight of lung-derived material is approximately 14,500, but its amino acid sequence is unknown [Janet T., Powell
l) Author: Biochem, J.
) +Eee L.

123 pages (1980)].

In addition, lectins derived from human placenta or human fallen cord are disclosed in Japanese Patent Application Laid-open No. 1983
-184020 (published on September 19, 1985), the amino acid sequence is reported along with its collection method and various properties, but there is still no knowledge regarding the amino acid sequence.

[Problems to be solved by the invention] Needless to say, the extraction of polypeptides includes problems such as difficulty in obtaining raw materials, decomposition or denaturation during extraction, and large-scale equipment. Because there are many types of amino acids that can be used as units, problems include selection, removal, and racemization of protecting groups. Furthermore, none of the methods is general. On the other hand, considering that operations on DNA can be easily performed using general methods compared to polypeptides, there are high expectations for the genetic engineering synthesis of lectins. However, at present, even the beginnings of synthesizing lectins through genetic engineering have not been provided.

The purpose of the present invention is to elucidate the amino acid sequence of a lectin derived from the human placenta or abortifacient under the circumstances described above, and to provide a starting point for its genetic engineering synthesis.

[Means for Solving the Problems] The present invention provides a human placenta- or umbilical cord-derived lectin represented by the following amino acid sequence.

Ala-Cys-Gly-Leu-Val-Ala
-Set-Asn-Leu-Asn -Leu-Lys
-Pro-Gly-Glu -Cys-Leu-Arg
-Vat-Arg-Gly-Glu-Vat-Ala-
Pro-Asp-Ala-Lys-5er-Phe
-Val-Leu-Asn-Leu-Gly -Lys
-Asp-9er-Asn-Asn-Leu-Cys-
Leu-His-Phe-Asn-Pro-Arg-
Phe-Asn-Ala-His-Gly-Asp-A
la-Asn-Thr-11e-Val-Cys-A
sn-Ser-Lys-Asp-Gly-Gly-A
la-Trp-Gly-Thr-Glu-Gln-A
rg-Glu-Ala-Val-Phe-Pro-Ph
e-Gln-Pro-Gly-3er-Val-Ala
-Glu-Vat-CVs -11e-Thr
-Phe-Asp-Gln-Ala-Asn -Le
u-Thr-Vat-Lys-Leu-Pro-A
sp-Gly-Tyr-Glu-Phe-Lys-P
be-Pro-Asn-Arg-Leu-Asn-L
eu-Glu-Ala-11e-Asn-Tyr-M
et -Ala-Ala-Asp-cly-A
sp -Phe-Lys-1ie-Lys-Cys
-Val-Ala-Phe-Asps Lectin derived from human placenta or umbilical cord can be collected as follows.

Human placenta or umbilical cord is triturated in an aqueous saline solution containing a protein protectant and then centrifuged. The separated cell debris is extracted with a solution prepared by adding a compound having a galactose residue to an aqueous salt solution containing the same kind of protein protecting agent as described above, and then purified.

The above-mentioned crushing treatment can be carried out according to a conventional method using a mixer, a homogenizer, etc.

Examples of the aqueous salt solution include phosphate buffered saline, Tris-hydrochloric acid buffer, borate buffer, Good's buffer, etc., and I) H is preferably 6 to 8.

The protein protecting agent added to the aqueous salt solution is ethylenediaminetetraacetic acid rIi (hereinafter referred to as rEDTAJ).
It is necessary to use a metal chelating agent such as a sodium salt or a potassium salt thereof in combination with a compound having a mercapto group such as β-mercaptoethanol, glutathione, dithiothreitol, dithioerythritol, cysteine, or thioglycolic acid.

Examples of compounds having galactose residues include lactose, melibiose, raffinose, phenyl β-D-galactoside, thiodigalactoside, asialofetuin, and the like. The total concentration of these compounds having galactose residues added to the extract is preferably 0.01-1M, more preferably 0.02-0.2M.

Although the extract obtained as described above can be purified by conventional methods, it is particularly preferable to purify by affinity chromatography using the aforementioned compound having a galactose residue as a ligand.

The polypeptide of the present invention has few problems with immunogenicity, and is expected to have excellent effects in the treatment of myasthenia gravis in humans, as well as in activating leukocytes and other cells, which have been observed in conventional lectins. .

It is also known to induce the release of cytotoxins from mouse macrophages and human peritoneal monocytes [T., Kajikawa et al.; Life Sciences + Uni+1].
177-1.181 (1986)].

Next, the present invention will be explained in more detail using Examples and Experimental Examples. The following Examples and Experimental Examples do not limit the scope of the present invention in any way.

・ Fresh human placenta (wet weight 409 g) that had been washed with water and refluxed to remove blood was mixed with 2mM sodium EDTA and 4mM
Phosphate-buffered saline (20 mM sodium phosphate, pH 7, 2, 1) containing β-mercaptoethanol
50mM sodium chloride) (rEDTA-MEPB)
The mixture was ground for 1 minute using a Waring blender in approximately 4 times the volume (referred to as SJ), centrifuged at 6000 rpm for 30 minutes, and then washed. E containing 0.1M lactose in the sediment
DTA-MEPBS was added in the same volume as that used in the trituration, and the mixture was shaken and extracted at 4°C for 30 minutes. 30 at 6000rpm
Centrifuge for 1 minute, remove the supernatant, and remove the supernatant.
l) P-6 (50-1OO mesh) [Polyacrylamide gel manufactured by Bio-Lad]
A column filled with EDTA-MEPBS and equilibrated with EDTA-MEPBS (
4.3 x 920 mm) and collected active fractions. Next, Sepharose on which asialofetuin was immobilized was collected.
Se) (agarose gel manufactured by Pharmacia) was placed on a column (φ 22 x 160 mm), and EDTA-M
After washing with EPBS, E containing 100 mM lactose
Both active fractions were collected by elution with DTA-MEPBS. Through these operations, the degree of purification was approximately 5301B compared to the extract, and the activity yield was approximately 13%.

The obtained purified product was freeze-dried and stored at -80°C until use.

1 Extract human umbilical cord lectin in exactly the same manner as in Example 1.
Purified. The degree of purification was about 530 times, and the activity yield was about 13%, which were also the same as the results of Example 1.

Experimental example 1 ゛ ・(1) Isoelectric point 3 W/V% Servalyt
Using a thin layer polyacrylamide gel containing [Manufactured by Serva, pH range 3 to 6],
d-ola) method [Rado
ra B, J.); Electrophoresis (Elec
trophoresis), supra, pp. 43-56 (198
(year 0). pI mom--proteins include amyloglucosidase (p I 3.5), glucose oxidase (4,15), soybean trypsin inhibitor (4,55), and bovine β-lactoglobulin (5,20).
).

Bovine carbonic anhydrase (5°85) was used.

5DS-polyacrylamide gel electrophoresis appeared to be uniform, but multiple bands were observed around pH 5. 1st band is p
It was observed in H4.9 (Fig. 1).

(2) Under molecular weight non-denaturing conditions, an Asahi Back G5-510 column equipped with a 6000A pump and a 440A detection device manufactured by Waters [manufactured by Asahi Kasei Corporation,
7.6×150 mm], and the molecular weight was determined by high performance gel chromatography.

Chromatograms were recorded on a Waters 730 data module.

That is, lyophilized lectin was dissolved in 150 mM NaC at pH 7.2.
ff1 (or PBS) in 20 m m sodium phosphate or 10 m m lactose in PB
It was dissolved in S at a concentration of 1 mg/mff1.

After 0, 8, and 28 hours at 4°C, 10 μg of each sample was loaded onto the column. The equilibration buffer was PBS and the flow rate was 1 m! /minute.

The human placental lectin of the present invention is mainly a dimer (2800
0), but a small amount was eluted at the position corresponding to the monomer (14
000) position. When the solution was allowed to stand for several hours, the ratio of monomer to dimer increased (Figure 2).

In Fig. 2, a%b1C with an arrow indicates chicken ovalbumin (molecular weight 45,000) and soybean trypsin inhibitor (molecular weight 220) as marker proteins, respectively.
00) indicates the elution position of bovine ribonuclease A (13700).

(3) Ultraviolet absorption spectrum Jasco's UVID
Measurements were made using an EC)-50 spectrophotometer under non-denaturing and denaturing conditions. i.e. 150mM NaCL.

at a concentration of 0.08 mg/mg in 20 mM sodium phosphate, pH 7.2, containing 2 mM EDTA and 4 mM 2-mercaptoethanol, and in 6 M hydrochloric acid, pH 6.5, containing 20 mM sodium phosphate. Identical results were obtained when measuring in guanidine at concentrations of 0 and 2 mg/ml (Fig. 3).

Although many shoulders were observed between 250 and 270 nm,
This tendency has been reported for lectins derived from electric eel [Levi G, et al.; J. Biol, Chem.
), pp. 256.5735-5740 (1981)]
This is thought to be because there are more bephenylalanine residues than tyrosine residues.

(4) Thiol group 5,5-dithio-bis-(2-nitrobenzoic acid) (DT
NB), and the Ellman method [Ellman G, L, (author); Arc, Biochem, Biophys, (Arch.

Biochem, Biophys, ) 82, 70
-77 pages (1969).

That is, human placental lectin (0, 25M) Lysu HC
1,0 ml of 8M urea (pH 8,0) in ff1
and a 25 molar excess of DTNB was added.

The reaction was monitored for 15 minutes by absorbance at 412 nm,
The number of thiol groups was calculated based on the molar extinction coefficient of the chromogen 2-nitro-5-mercaptobenzoic acid (t 3600 M-' at 412 nm).

4.2 thiol groups were found per subunit. Amino acid analysis of the reduced carboxymethylated product also confirmed the presence of 4.7 cysteine residues per subunit.

(5) Effect of Thiol 1@Decorative Agent on Sugar Chain Binding Activity Similar to other lectins, human placental lectin also loses hemagglutination activity in the absence of 2-mercaptoethanol.

In order to investigate the role of thiol groups in this sugar chain bond, these groups were combined with p-chloromercuribenzoic acid (pCM
B) and N-ethylmaleimide (NEM) 1:1
The remaining activity against asialofetuin (prepared by treating fetuin obtained from Gibco with closed acid at pH 2 and 80° C. for 1 hour) was measured by a solid-phase cutin binding ability assay. The results are shown in Figure 4.

In FIG. 4, the graph connecting the dots shows the residual activity after 30 minutes when the reaction was carried out without using any modifier. The graph connecting O is 20 under the same conditions.
Shows residual activity after time.

The graph connecting the groups shows the residual activity after 30 minutes when the reaction was performed after modification with NEM.

The graph connecting Δ indicates the residual activity after 20 hours under the same conditions. The graph connected with ■ indicates the residual activity after 30 minutes when the reaction was carried out after modification with pCMB. The closed graph shows the residual activity after 20 hours under the same conditions.

pCMB completely inactivated lectin activity within 30 minutes, but the effect of NEM was incomplete even after 30 minutes (residual activity was 25% of control).
In the absence of any modifier, approximately 50% of the original activity remained after 20 hours.

1) Production of reduced/carboxymethylated polypeptides Since the polypeptide of the present invention lacks an N-terminal amino group, it is necessary to cleave it into fragments before applying it to an amino Shun analyzer.

5.6 mg of human placental lectin was added to IOM's urea and 3 mM.
of EDTA and 5.6 mff1 of 0.55 M Tris-HCL (pH 8.8), and then 10 μm of 2-mercaptoethanol was added.

The reaction mixture was aged at 37° C. for 4 hours under a nitrogen atmosphere. Then 22 mg of recrystallized monoiodoacetic acid was added,
The reaction was allowed to proceed for 20 minutes at 37°C under a nitrogen atmosphere. Bio-Gel P-6 [Bio-Rad Laboratories] equilibrated with 100 mM ammonium bicarbonate in the dark.
manufacturers), 50-100 mesh, 4.3
Excess reagent was removed by gel filtration on a X92 cm column.

2) Bitterness. - The obtained reduced/carboxymethylated product (110 nanomoles) was mixed with about 200-fold molar excess of cyanogen bromide (BrCN) in 1.0 mff1 of 70 V/V% formic acid for 24 hours at room temperature.
) was processed. Then, 5me of water was added and freeze-dried.

The running sample was redissolved in 1.0 ml of 100mM ammonium bicarbonate and Sephadex equilibrated with 100mM ammonium bicarbonate.
G, -50 fine column [manufactured by Pharmacia, 1.8
CN- consisting of 120 residues
1 fragment and a CN-2 fragment consisting of 14 residues were isolated and purified.

3) 1 mol of iodine, 110 nanomoles of the carboxymethylated product in a dark place,
3 mg of 0-iodosobenzoic acid (IB
A). The reaction was stopped by the addition of lOμt of 2-mercaptoethanol and 300μi of 5M NaOT(
It was neutralized.

Sephadex G-25 fine equilibrated with 100 mM ammonium bicarbonate [Pharmasia, 1.4X]
Excess reagent was removed by gel filtration on a 135 cm] column. Both the spilled parts were collected and dried.

1.0ml of dry sample! Sephadex G-50 redissolved in 100mM ammonium bicarbonate and equilibrated with 100mM aqueous ammonia.
Fine column [manufactured by Pharmacia, 1.8X135c
IB-1 fragment consisting of 68 residues and 66
The IB-2 fragment consisting of these residues was isolated and purified. Next, Deverosil OD S
The product was further purified by reverse phase chromatography on a -5 (manufactured by Nomura Chemicals) column (linear gradient elution was performed with increasing amounts of acetonitrile in 0.IV/V% trifluoroacetic acid). The results are shown in Figure 6. A is I BA-1
, B represents purification of IBA-2.

After dissolving the reduced/carboxymethylated product (110 nanomoles) in 0.15mff1 (10mM) Lys-HCff1 buffer (pH 8,8), the amount of Achromobacter prosthesis was 1/200 times in molar ratio! was added and digested at 37°C for 12 hours. The digested material was subjected to reverse phase chromatography (Deverosil 0DS-5 column, 4.6 x 250 mm, 0
．． Fractionation using 1% trialifluoroacetic acid/acetonitrile) was performed to purify eight fragments (AP-1 to AP-8).

The reduced carboxymethylated product (79 nanomoles) was
．． 2 mL of 0.1 M) Lis HCl buffer (pH 8,
submaxillaris protease was added in an amount of 1755 times by weight, and the mixture was digested at 37°C for 16 hours. The digested product was fractionated by reverse phase chromatography under the same conditions as in 4) above, and 11 fragments (SMI-11
) was purified.

6) Standing Enni 2-de 1゛-47OA
Sequencer [Applied Biosystems (App
The amino acid sequence was analyzed using a Co., Ltd. manufactured by Lied Biosystems, and the underlined amino acid sequence (displayed using one-letter symbols) was determined.

Fragment name iVc I TFDQANLTVKLPDG Y E
F K F P N RL N L E A I N
The amino acid sequence was analyzed using a YMAADGDFK IKCVAFD 477A sequencer [manufactured by Applied Biosystems], and the underlined amino acid sequence shown in FIG. 7 (indicated by one-letter symbol) was determined.

Since the amino acid sequence of the N-terminal fragment 5M-8 is blocked at the N-terminus, the acetyl group was first identified by the dansylhydrazine decomposition method, and then the following procedure was performed.

Ten nanomoles of 5M-8 were digested with 0.05 units of the acylated amino acid free radical AARE for 16 hours at 37°C under a nitrogen stream. Acylated amino acids, remaining cleaved fragments, and undigested fragments were collected using reverse phase chromatography (ODS).
807M column, manufactured by TOS OH, 4.6X150
mm, 0.1% trifluoroacetic acid/acetonitrile), and the digested fragments were separated using the 477A sequencer.
The amino acid sequence was determined. Acylated amino acids were analyzed using reverse phase chromatography (ODS 807M column,
Made by TOS OHif, 4.6X 150mm, 0°1
% trifluoroacetic acid).

As shown in FIG. 8, the entire amino acid sequence of the human placental lectin of the present invention was determined using the overlapping portions of the fragments whose amino acid sequences had been determined as clues.

When the physicochemical properties of the human umbilical cord lectin obtained in Example 1 were measured in the same manner as in Experimental Example 1, the isoelectric point, molecular weight, ultraviolet absorption spectrum, thiol group, and sugar chain binding activity The effects of the thiol modifier on the results were all the same as in Experimental Example 1.

The product was hydrolyzed in a vacuum tube at 110'C for 20 hours in an eMHCffi and analyzed using a Hitachi 835 amino acid analyzer.

The results are shown in Table 2 (numbers indicate mole fractions) together with the results of amino acid analysis of human placental lectin. The values for methionine and tyrosine are the results of amino acid analysis after hydrolysis in the presence of 4% thioglycolic acid. Cystine and tryptophan values are pending.

Table 2 Human placental lectin Human umbilical cord lectin Asp
19.5 19.4Thr 1.2
1. l5er 4.2
4.0 Glu 8.6 8
．． 5Pro 4.8 4.7Gl
y 9.0 9.4Ala
11.5 11.5 Val
7.7 7.7Met
O,90,81ie
3.1 3.2 Leu
9. 2
9. 3Tyr 1. 1
1. 0 Phe 7
．． 8 7. 9Lys
8. 0B,
0His 1. 5
1. 5Arg 3.9
From the results of 1L,0 Experimental Example 3.4, it was concluded that human placental lectin and human umbilical cord lectin are the same substance.

look! and 1) To isolate the lectin cDNA, the C-terminal side of the lectin amino acid sequence (Met-Ala-Ala-Asp
-Gly-Asp-Phe-Lys -11e-Lys
-Cys-Va I) was added to ABO381A of Abride Biosystems Japan Co., Ltd.
It was produced as follows using a model DNA synthesizer.

5'-CACGCATTTAATCTTGAAGTC
TCCATCCGCCGCCAT-3'2) Next, c
DNA library [Clontic
ch Laboratories Inc.)! ! ]
Roll up I
Cultured at 7°C. The phage plaques were then directly filtered onto two nitrocellulose filters (G S N
-6, 138 mmφ).

Next, this replica nitrocellulose was cultured on an agar medium at 37°C for 4 hours. Thereafter, the nitrocellulose filter was soaked in 0.5N NaOH solution, 1.
Soaked in 5M NaCff1 solution for 5 min at room temperature, then IM
) Lith-HC1 (pH 8,0), neutralized by immersion in a 1.5M NaCl solution at room temperature for 5 minutes. Furthermore, it was immersed in a 2XSSPE solution for 5 minutes at room temperature, and then air-dried.

After air-drying, this nitrocellulose filter was dried under vacuum.
The DNA was fixed on the nitrocellulose filter by treatment at 80°C for 2 hours.

3) Synthetic NA (100mg) 05 shown in 1) above
' end with 3000 Ci/mmol γ-32pA'rp
[Amerciam? ! ].

100 μCi of T-4 polynucleotide kinase (manufactured by Takara) and 50 m Tris-H
CQ (pH 7,6), 1mM spermidine, 5mM
Dithiothreitol, 0.1mM EDTA, 10mM
gCff1. The cells were labeled by reacting for 1 hour at 37°C. This labeled synthetic NA was purified by Sephadex G-50 column chromatography in a conventional manner. By this operation, labeled DNA having a specific activity of 2×10'cpm/1100n was obtained.

4) The nitrocellulose filter prepared in 2) above and the labeled DNA were mixed with 50mM) Lis-HCL (pH
7,5) , IMNaCl, 10mM EDTA
, 0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% BSA, 0.1% SDS, and 1 mg DNA at 42° C. overnight. Then, a nitrocellulose filter
2 X S S C (0, 3M Na Cl, 0.0
3M sodium citrate) and incubated at 42°C for 5 minutes.
The temperature was repeated three times. Thereafter, the nitrocermise filter was dried and an autoradiogram was performed.

5) Plaques that specifically hybridized with the synthetic NA obtained by autoradiogram were selected. The selected clones were further made into a single clone by repeating the operations described above from 2) above to obtain one clone. DNA was obtained from this clone and used as EcoR.
After digestion with I and agar gel electrophoresis, Southern method was performed. As a result, this clone contains D that hybridizes with the synthetic NA synthesized in 3) above.
It was confirmed that NA was indeed included.

[Effects of the Invention] As described above, the present invention provides, for the first time, the beginnings of genetically engineering synthesis of human-derived lectins. The significance of the present invention is extremely large because operations on DNA can be more easily performed using general methods than on polypeptides.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the results of isoelectric point measurement of the polypeptide of the present invention. FIG. 2 is a diagram showing the results of molecular weight measurement of the polypeptide of the present invention. FIG. 3 is a diagram showing the ultraviolet absorption spectrum of the polypeptide of the present invention. FIG. 4 is a diagram showing the influence of a thiol modifier on the sugar chain binding activity of the polypeptide of the present invention. FIG. 5 is a diagram showing the separation of fragments obtained by treating the polypeptide of the present invention with cyanogen bromide and 0-iodosobenzoic acid. FIG. 6 is a diagram showing the behavior when the fragment obtained by treating the polypeptide of the present invention with 0-iodosobenzoic acid was further purified by reverse phase chromatography. FIG. 7 shows human fetal lectin AP-2 to 4.7.8.
Amino a sequences of 5M-2, 8, 10, 11 fragments are shown. Figure 8 shows the amino acid sequence of human placenta or umbilical cord lectin. Patent applicant Ken Kasai (and 5 others) Fig. 2 Elution amount (mA) 3rd wavelength (nm) Ikuo Kobe Fig. 5 0 1oo 200 Elution amount (m) Procedural amendment May 1988 l hustle and bustle

Claims (1)

[Claims] A lectin derived from human placenta or umbilical cord, represented by the following amino acid sequence. [There is a gene sequence]