JPH07100719B2 - Colony-stimulating factor and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel colony stimulating factor isolated from human urine and stimulating colony formation of mammalian monocyte-macrophage cells, and a method for producing the same. is there.

[Prior Art] A colony-stimulating factor (hereinafter abbreviated as CSF) is a hematopoietic factor that stimulates the differentiation and proliferation of hematopoietic stem cells present in mammalian hematopoietic tissues such as bone marrow, and many CSFs are derived from glycoproteins. It is made up. So far, factors that act on monocyte-macrophage stem cells (M-CSF or CSF-1), factors that act on granulocyte-monocyte stem cells (GM-CSF), and factors that act on granulocyte stem cells (G -CSF), and factors that act on pluripotent stem cells common to granulocytes, monocytes, erythrocytes and megakaryocytes (Multi-CSF, interleukin-3 or IL-3)
4 kinds of are known.

Except for Multi-CSF, the above-mentioned three kinds of human-derived CSF have cloned gene cDNAs encoding the respective amino acid sequences, and their protein structures have been clarified [GGWong
Et al., Science, 228, 810-815, 1985; ES Kawasaki
Et al., Science, 230, 291-296, 1985; S. Nagata et al., Na.
ture, 319, 415-418, 1986].

As CSF present in human urine, factors acting on monocyte-macrophage cells (CSF-1 or M-CSF) [SKDa
s & ERStanley, Journal of Biological Chemistry, 2
57, 13679-13684, 1982], HGI-glycoprotein that stimulates granulocyte colony [Japanese Patent Publication No. 60-30291] and CSF.
-HU [K. Motoyoshi et al., Blood, 52, 1012-1020, 1978 and Blood, 60, 1378-1386, 1982] have been reported.

Among them, CSF-1 acting on monocyte-macrophage cells is completely blunted, and the cDNA encoding the protein has been cloned (ESKawa, supra).
saki et al.).

In the structure of blunted CSF-1, two polypeptides containing a sugar chain form a biologically active homodimer by disulphide bond. This dimer produces two identical subunits by cleaving the disulphide bond with a reducing agent. Of this glycoprotein with biological activity,
The molecular weight measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 45,000-60,000 daltons. The molecular weight of the polypeptide subunit forming the dimer, excluding the sugar chains, is 14,000 to 17,000 daltons. The number of amino acids deduced from the cDNA considered to encode the amino acid sequence of this CSF-1 subunit is
It is a polypeptide with 224 molecules and a molecular weight of 26,000 daltons.

[Problems to be Solved by the Invention] Although the present inventors act in human urine in the same manner as CSF-1,
The present invention was found by discovering the existence of a novel glycoprotein CSF, which has not been known so far, which is physicochemically distinguished from CSF-1, and has isolated it and clarified its physicochemical and biological properties. Was completed.

The object of the present invention is to provide a novel CS having a therapeutic effect on leukopenia, immunodeficiency, bone marrow transplantation, etc. due to anticancer drug chemotherapy.
To provide F and its manufacturing method.

That is, the present invention provides a novel CSF consisting of a glycoprotein having the following physicochemical properties and having an action of stimulating colony formation of mammalian monocyte-macrophage cells.

a) Molecular weight It is a homodimer dissociated into two identical subunits by a reducing agent like CSF-1, and has a molecular weight of 70,000 to 90,000 daltons measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the subunit, which had been dissociated with a reducing agent and had lost its biological activity, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 35,000 to 45,000 daltons.

(B) Subunit Amino Acid Sequence The subunit protein constituting the homodimer consists of at least 189 amino acid sequences shown below, The 122 and 140th asparagine (Asn) has a typical N-glycoside binding site represented by asparagine (Asn) -X-threonine (Thr) or serine (Ser), respectively. However, X represents an arbitrary amino acid.

c) Isoelectric point Isoelectric point (pI) measured by polyacrylamide gel isoelectric focusing method and sucrose density gradient isoelectric focusing method.
Is 3.1 to 3.7.

d) Constituent monosaccharide of sugar chain After hydrolysis, analysis was performed by high performance liquid chromatography, and mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid were identified as constituent sugars of the sugar chain.

e) Circular dichroism spectrum The far-UV external CD spectrum measured by the circular dichroism dispersometer has a wavelength of
There are minimal peaks at m and 222 nm, respectively, and an α-helix structure is included.

f) Thermal stability No biological activity is lost by heating at 60 ± 0.5 ° C. for 60 minutes; g) Infrared absorption spectrum It has the infrared absorption spectrum shown in FIG.

Human urine containing colony stimulating factors having the above physicochemical properties was adjusted to pH 8 to 9 to precipitate insoluble matter, and the supernatant was desalted with an ultrafiltration membrane to obtain at least 20
After concentrating to 0 times or more, adjust the pH to 6.5-7.5 and
After heat treatment for 0 hours, the resulting precipitate was removed by centrifugation, adsorbed on an anion exchanger, eluted with 0.2-0.4M buffer solution, and then gel-filtered in 1-4M buffer solution to give a fraction having a molecular weight of 70,000 daltons or more. The fraction was collected, the fraction was adsorbed to a hydrophobic affinity substance and eluted with 0.5 to 1 M buffer, and the eluate was subjected to high performance liquid gel filtration to collect a fraction having a molecular weight of 70,000 to 150,000 daltons. The method is a method for producing a colony stimulating factor, characterized in that the active ingredient is eluted by adjusting the pH to 1 to 2 and performing reverse phase high performance liquid chromatography.

[Detailed Description of the Invention] (1) Production of CSF The CSF of the present invention is produced as follows.

Adjust the urine of healthy people to pH 8.0 to 9.0, precipitate and remove the viscous substances in urine, and concentrate and desalt the supernatant using an ultrafiltration membrane that passes a molecular weight of 10,000 to 50,000 daltons. .

Concentrate at least 200 times (1% protein concentration (w
/ v) or more), adjust the pH to 6.5 to 7.5, and heat at 60 ℃ for 10 hours (inactivate viruses, etc.). The precipitate formed is removed by centrifugation and the active ingredient is adsorbed on an anion exchanger such as DEAE-cellulose.

Next, the ion exchange is performed with 0.05 to 0.1 M buffer (pH 6.5 to 7.5).
After washing the body, use 0.2-0.4M buffer (pH 6.5-7.5)
Elute the active ingredient. If necessary, use the ultrafiltration membrane for the eluate
Concentrate and contain 1M-4M salts such as ammonium sulfate, salt, etc.
Gel filtration agent equilibrated with buffer solution (pH 6.5 to 7.5), eg
By Sephacryl Gel filtration with S-300 (Pharmacia)
The fraction with a molecular weight range of 70,000 to 150,000 daltons.
To collect. The fractions were then equilibrated with the above 1M-4M salt-containing buffer.
Derivatized hydrophobic affinity, for example Phenyl-Sepharose (P
harmacia), 0.5-1.0M salt-containing buffer
Elute at (pH 6.5-7.5). The eluate was concentrated with an ultrafiltration membrane.
High-performance liquid gel filtration column, such as TSKG-3000SW
(Made by Toyo Soda Co., Ltd.) gel filtration, molecular weight range 70,000-15
Collect a fraction of 0,000 Daltons. Concentrate the fraction again
With 0.1% trifluoroacetic acid (TFA) solution (pH 1-2)
Equilibrated high-performance liquid reverse-phase column, such as Hi-Pore RP
Adsorbed on -304 (BioRad) and contains 0.1% TFA
A solvent such as acetonitrile or isopropanol
Elute by the linear gradient elution method. Obtained this way
CSF has a specific activity of 1 x 10⁸More than unit / mg protein
It is a pure substance.

(2) Physicochemical properties of CSF The CSF of the present invention produced as described above has the following physicochemical properties. In this physicochemical property test, CSF purified by the method of Example 1 was used.

(A) Molecular weight In the absence of a reducing agent, Laemmli (Nature, Vol. 227, 680-685)
Page, 1970), the molecular weight was 70,0 when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
It was between 00 and 90,000 Daltons.

Next, it was reduced with 0.2 M mercaptoethanol, and when measured by the same method, it was dissociated into subunits having a molecular weight of 35,000 to 45,000 daltons (Fig. 1).

FIG. 1 is a electrophoresis diagram of CSF of the present invention by sodium dodecylsulfate / polyacrylamide electrophoresis, in which A to E are non-reducing (dimers), F and G are molecular weight marker proteins, and H to L.
Indicates reduction (subunit), and the number on the vertical axis indicates molecular weight (× 10 ³ dalton).

(B) Sabuyunitsuto protein amino acid sequence of the NH ₂ - terminal amino acid sequence was analyzed by a conventional method in the gas phase amino acid sequencer purified CSF. The purified CSF was then denatured with 6M guanidine, alkylated with monoiodoacetic acid, desalted and digested with trypsin. The trypsin-treated peptide was fractionated by Vydac C-18 reverse phase high performance liquid chromatography to obtain 23 peptide fractions. Each fraction was analyzed by a gas phase amino acid sequencer to analyze the amino acid sequence of the peptide fragment. The amino acid primary structure of the subunit protein was determined from the amino acid sequence of the tryptic digested peptide fragment and the nucleotide sequence of mRNA cloned by the present inventors. The results are shown in Table 1.

NH ₂ - From glutamate, amino terminal to the 149th glutamine is identical to the known CSF-1, 0.99
The 40 amino acids from position 1 to position 189 were completely different from the known ones. Also, at least next to the COOH-terminal amino acid leucine, The existence of three amino acids of α, β, and γ was qualitatively determined, and it was predicted that α is threonine, β is tryptophan, and γ is glutamic acid. The 122nd and 140th asparagine had a typical N-glycoside bond structure of Asn-X-Ser / Thr, and it was presumed that sugar chains were bonded at this site. Here, X represents an arbitrary amino acid.

(C) Constituent Monosaccharide of Sugar Chain The constituent monosaccharide of the sugar chain bound to the polypeptide was hydrolyzed and released, and then analyzed by high performance liquid chromatography. Aldose, sialic acid for anion exchange column,
Hexosamine was fractionated by a cation exchange column by a borate buffer gradient elution method, post-column labeled with cyanoacetamide or arginine, and then identified by a fluorescent method. Although the sugar chains contained in the CSF molecule were heterogeneous and difficult to quantify, mannose, galactose, N-acetylglycosamine and N- were used as constituent monosaccharides.
Acetylneuraminic acid was identified. The molar ratio was about 2.5: 1.4: 2.5: 2.1.

(D) Isoelectric point The isoelectric point was measured by polyacrylamide gel isoelectric focusing method and sucrose density gradient isoelectric focusing method. As a result, pI was 3.1 to 3.7.

(E) Circular dichroism (CD) spectrum The CD spectrum in the far ultraviolet region was measured with a circular dichroism dispersion meter (J-600 manufactured by JASCO) (Fig. 2).

FIG. 2 shows the CD spectrum of the CSF of the present invention, the horizontal axis shows the wavelength (nm), and the vertical axis shows the ellipticity (mdeg). Wavelength 208nm and 2
A minimal peak was observed at 22 nm, and it was presumed that the secondary structure of this CSF contains an α-helix structure.

(F) Thermostability This CSF was dissolved in a dilute buffer (pH 7.0) at a concentration of 1 μg / ml and heated at 60 ± 0.5 ° C. for 60 minutes to measure its colony stimulating activity (described later). Almost no decrease in activity was observed.

(G) Infrared absorption spectrum An infrared absorption spectrum of the freeze-dried powder of the present CSF measured by a Fourier transform infrared spectroscope (5D × C manufactured by Nicolet Co.) by a transmission measurement method (KBr window) is as shown in. . The horizontal axis of FIG. 3 represents the wave number (cm ⁻¹ ) and the vertical axis represents the transmittance.

This CSF is strong absorption in 1650cm ^-1, 1201cm ^-1 and 1133cm ^-1, 1
It showed moderate absorption at 537 cm ^-1 , 1432 cm ^-1 and 1068 cm ^-1 .

(3) Biological activity of CSF In the same manner as in the above item (2), CSF purified by the method of Example 1 was used for this colony stimulating activity and specific activity test.

(A) Colony stimulating activity and specific activity The colony stimulating activity of the CSF of the present invention was measured by a colony formation test method using a mouse bone marrow cell on a monolayer soft agar gel. The CSF sample was mixed with 1 ml of McCoy's 5A medium containing 0.3% agar, 20% fetal calf serum (FCS) and 1 × 10 ⁵ mouse bone marrow cells, and cultured at 37 ° C. under 7.5% CO ₂ aeration for 7 days. After culturing, 50 or more cell aggregates were determined as colonies, and the number of formed colonies was counted. The colony stimulating activity was expressed in units, and one unit was defined as the amount of CSF required to form one colony. The specific activity was represented by the number of colonies (unit) formed per mg of CSF protein. As a result, the
CSF had a specific activity of 1.4 × 10 ⁸ units / mg protein. When the formed colonies were morphologically classified by staining with hematoxylin-eosin, 95% or more of the colonies were formed from monocytes-macrophages.

(B) in vitro and mouse bone marrow monocytes in in vivo - in macrophages stem cell promoting effect on the growth of (CFU-M) b-1 ) in vitro test C ₅₇ BL mouse bone marrow cells flat adsorption method, Non-adsorbed bone marrow cells in McCoy's 5A medium containing 20% FCS 1 × 10 ⁶ cells / m
CSF of the present invention, 0 (control), 100 units /
ml, 500 units / ml, 1,000 units / ml and 2,000 units / ml, respectively, and the mixture was incubated at 37 ° C. for 24 hours under aeration of 7.5% CO ₂ . After culturing, each bone marrow cell was washed by centrifugation, diluted 5 times with the same medium, and added to each four groups of scare.
McCoy's 5A with 1,000 units of CSF, 0.3% agar and 20% FCS
0.1 ml was added to 1 ml of the medium, and the mixture was cultured at 37 ° C. for 7 days under aeration of 7.5% CO ₂ . After culturing, aggregate 50 or more cells into monocytes-
The cells were determined to be macrophage stem cells (CFU-M), and the number of CFU-M formed was counted. The results are shown in Table 2.

As shown in Table 2, the number of CFU-M in mouse bone marrow cells increased depending on the added concentration of CSF.

b-2) In vivo test 0 (physiological saline) per 1 kg of body weight, 80 × 10 ⁴ units, 160 × 10 ⁴ units and 320 × 10 ⁴ units CSF for C ₅₇ BL mice (5 / group) 1 CSF It was administered intraperitoneally once a day for 3 consecutive days. On the day after the end of administration, the femur bone marrow and spleen were removed from each mouse, and the number of monocyte-macrophage stem cells (CFU-M) in the bone marrow and spleen was adjusted to 1,000 units of CSF as a stimulator. It was measured by a colony formation test by the method. The results are shown in Tables 3 and 4.

As shown in Tables 3 and 4, an increase in CFU-M in the bone marrow and spleen was observed by the administration of 80 × 10 ⁴ units / kg body weight of CSF, and it was remarkable in the administration of 160 × 10 ⁴ units / kg body weight or more. An increase was observed.

The CSF of the present invention having the above physicochemical properties and biological activities is compared with the known similar substances as follows.

The CSF of the present invention is composed of a biologically active homodimer in which two polypeptides containing a sugar chain are disulphide-bonded like CSF-1, and has a molecular weight of 70,000 to 90,000 daltons. , Which is larger than that of CSF-1. Further, the subunit polypeptide constituting the CSF of the present invention consists of at least 189 amino acids and has a molecular weight of 35,00.
0-45,000 Daltons, 14,000-1 of that of CSF-1
Greater than 7,000 Daltons. When the amino acid sequence of the subunit was compared with that of CSF-1, the amino acid sequences from 1 to 149th at the NH ₂ -terminal were the same, but the amino acid sequence from 150th to 189th was the cDNA of CSF-1. It was not encoded on the CSF-1 gene, which was different from that deduced from Therefore, the CSF of the present invention is partially CSF.
-1 has a commonality, but genetically and structurally
It was found to be a separate factor from the known CSF-1.

Next, examples of the present invention will be described.

Example 1 200 l of urine of a healthy person was adjusted to pH 8.5, the precipitate was removed by filtration, and concentrated and desalted with an ultrafiltration membrane (Amicon, H10 × 50) having a cutoff molecular weight of 50,000 daltons. Next, p
It was adjusted to H7.0 and sterilized by heating in a sealed container at 60 ° C for 10 hours. After sterilization, the precipitate was removed by centrifugation (5,000 xg for 30 minutes), and then mixed with DEAE-cellulose equilibrated with 0.02M phosphate buffer (pH 7.2) to adsorb. DEAE-cellulose was washed with 0.02 M phosphate buffer and 0.05 M salt-added 0.02 M phosphate buffer (pH 7.2), and then eluted with 0.25 M salt-added buffer (pH 7.2). The eluate was concentrated with an ultrafiltration membrane (H1P10 manufactured by Amicon) and Sephacryl S-300 (Pharmacia, φ4 × 80 cm) was used to add a 1 M ammonium sulfate addition buffer (pH 7.
Gel filtration was performed in 2). Molecular weight range for gel filtration 70,000-1
A phenyl-Sepharose 4B column (Falmatia,
φ2 × 20 cm) and then eluted with 0.5 M ammonium sulfate addition buffer (pH 7.2). The eluate was concentrated with an ultrafiltration membrane (Asahi Kasei, NM-3) and subjected to high performance liquid chromatography on a TSKG-3,000SW column (Toyo Soda, φ4 × 600mm × 2), molecular weight range 70,000-150,000 Daltons. Was obtained. This fraction was concentrated again and the Hi-Pore RP-304
0.1% on reverse phase column (Bio-Rad, φ4 × 150mm)
Acetonitrile containing trifluoroacetic acid 0-100% (p
H2.0) was subjected to high-performance liquid chromatography using a linear concentration gradient to elute CSF to obtain purified CSF having a specific activity of 1.4 × 10 ⁸ units / mg protein. The degree of purification of CSF in each step of the above production process is shown in Table 5.

Example 2 Sephacryl S-300 was added to TSKG-3,000SW by the method of Example 1.
(Toyo Soda, HLC-837), phenyl-Sepharose 4B in TSKph
Instead of enyl-5pw (Toyo Soda), it was purified by high performance liquid chromatography. The obtained CSF has a specific activity
It was 1.5 × 10 ⁸ units / mg, and the recovery rate was similar to that in Example 1.

[Brief description of drawings]

FIG. 1 is a migration diagram of CSF of the present invention by sodium dodecyl sulfate / polyacrylamide gel electrophoresis (SDS-PAGE), and FIGS. 2 and 3 are far-far ultraviolet C of the CSF of the present invention.
The D spectrum and the infrared absorption spectrum are shown. In Fig. 1, A to E ... non-reduced products (dimers) F, G ... molecular weight marker proteins H to L ... reduced products (subunit)

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Nobuya Yanagiuchi 5-21-10 Minamidai, Nakano-ku, Tokyo Morinaga Milk Industry Co., Ltd. Nakanosha's house 401 (72) Inventor, Muneo Yamada 752, Kuji, Takatsu-ku, Kawasaki-shi, Kanagawa Terrace 205 (72) Inventor Hajime Yokota Hajime 2-5-1-406 Shirokane, Minato-ku, Tokyo (56) References International Publication 87/6954 (WO, A)

Claims (2)

[Claims] 1. A colony-stimulating glycoprotein characterized by having a colony-forming stimulatory effect on mammalian monocyte-macrophage cells and having the following physicochemical properties: a) From two subunits having the same molecular weight A homodimer consisting of
The molecular weight measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 70,000 to 90,000 daltons, and the molecular weight measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the subunit dissociated with a reducing agent to eliminate biological activity is , 35,000 to 45,000
It is Dalton; b) Amino acid sequence of subunit The subunit protein that constitutes the homodimer consists of at least 189 amino acid sequences shown below, and the 122nd and 140th asparagine (Asn) are asparagine (Asn), respectively. ) -X-threonine (Thr) / serine (Ser) has an N-glycoside binding site, wherein X represents an arbitrary amino acid; c) Isoelectric point Isoelectric point (pI) measured by polyacrylamide gel isoelectric focusing method and sucrose density gradient isoelectric focusing method
Is from 3.1 to 3.7; d) Constituent monosaccharide of sugar chain The constituent monosaccharide of sugar chain identified by analysis by high performance liquid chromatography after hydrolysis is mannose, galactose, N-acetylglucosamine and N-acetyl neu. It is laminic acid; e) Circular dichroism spectrum The far-UV CD spectrum measured by a circular dichroism disperser is 208n
It has a minimum peak at m and 222 nm, and contains an α-helix structure; f) Thermal stability No biological activity is lost by heating at 60 ± 0.5 ° C for 60 minutes; g) Infrared absorption spectrum Infrared absorption in the spectrum, 1650 cm ^-1, strong absorption at 1201cm ^-1 and 1133cm ^-1, 1537cm ^-1, 1432cm ^-1 and 1068cm
^-1 shows moderate absorption; h) Specific activity 1.4 × 10 ⁸ units / mg. 2. Human urine is adjusted to pH 8 to 9 to precipitate insoluble matter, and the supernatant is desalted with an ultrafiltration membrane to obtain at least 200
After concentrating more than twice, adjust the pH to 6.5-7.5 and
After heat treatment for an hour, the precipitate is removed by centrifugation, adsorbed on an anion exchanger and eluted with a 0.2 to 0.4 M buffer, and then 1 to 4
Gel filtration in M buffer was performed to collect a fraction having a molecular weight of 70,000 daltons or more, and the fraction was adsorbed on a hydrophobic affinity substance,
Elute with ~ 1M buffer, subject the eluate to high performance liquid gel filtration to collect a fraction having a molecular weight of 70,000 to 150,000 daltons, adjust the fraction to pH 1-2 to perform reverse phase high performance liquid chromatography, Method for producing colony-stimulating glycoprotein having the following physicochemical properties, which has a colony-forming stimulatory action on mammalian monocyte-macrophage lineage cells, characterized in that an active ingredient is eluted: a) Subunits having the same molecular weight A homodimer consisting of two,
The molecular weight measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 70,000 to 90,000 daltons, and the molecular weight measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the subunit dissociated with a reducing agent to eliminate biological activity is , 35,000 to 45,000
It is Dalton; b) Amino acid sequence of subunit The subunit protein that constitutes the homodimer consists of at least 189 amino acid sequences shown below, and the 122nd and 140th asparagine (Asn) are asparagine (Asn), respectively. ) -X-threonine (Thr) / serine (Ser) has an N-glycoside binding site, wherein X represents an arbitrary amino acid; c) Isoelectric point Isoelectric point (pI) measured by polyacrylamide gel isoelectric focusing method and sucrose density gradient isoelectric focusing method
Is from 3.1 to 3.7; d) Constituent monosaccharide of sugar chain The constituent monosaccharide of sugar chain identified by analysis by high performance liquid chromatography after hydrolysis is mannose, galactose, N-acetylglucosamine and N-acetyl neu. It is laminic acid; e) Circular dichroism spectrum The far-UV CD spectrum measured by a circular dichroism disperser is 208n
It has a minimum peak at m and 222 nm, and contains an α-helix structure; f) Thermal stability No biological activity is lost by heating at 60 ± 0.5 ° C for 60 minutes; g) Infrared absorption spectrum Infrared absorption in the spectrum, 1650 cm ^-1, strong absorption at 1201cm ^-1 and 1133cm ^-1, 1537cm ^-1, 1432cm ^-1 and 1068cm
^-1 shows moderate absorption; h) Specific activity 1.4 × 10 ⁸ units / mg.