JPH0834801A - Polysaccharide sulfate and production thereof - Google Patents

Abstract

PURPOSE:To obtain a polysaccharide sulfate having a specific affinity for the growth factor of liver parenchima cells and a consequent improved pharmacological effect on the cells by selecting a polysaccharide sulfate containing specified sugar unit structures. CONSTITUTION:This sulfate is a polysaccharide sulfate at least partially consisting of structural units of an octasaccharide or higher saccharides having an affinity for the growth factor of liver parenchima cells. This is obtained for example by bringing a mixture of heparin or heparan sulfate, products of their partial decomposition or products of their limited decomposition into contact with an affinity support to which the growth factor of liver parenchima cells is bonded as a ligand and separating the product into a fraction having an affinity for the growth factor and a fraction having no affinity therefor. The structural unit comprises hexuronic acid and hexosamine or an N-acetyl- substituted product thereof and contains at least four sulfated disaccharide unit structures.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a sulfated polysaccharide having a specific affinity for hepatocyte growth factor (hereinafter abbreviated as "HGF").

[0002]

PRIOR ART AND PROBLEMS TO BE SOLVED BY THE INVENTION HG
F is a protein growth factor showing a growth-promoting action at a concentration of only a few ng / ml on hepatocytes that control a specific function of the liver. This HGF has an action of not only proliferating hepatocytes but also enhancing synthesis of proteins specifically produced in the liver such as serum albumin. Further HG
It has been clarified that F plays an important role in the development, differentiation and tissue construction of the living body. Interestingly, HGF is a factor (Tumor Cy) having a cytotoxic activity against cancer cells.
It has been clarified that it is the same molecule as totoxic factor (TCF). These things are HG
It has been shown that F acts as various cytokines in vivo.

In recent years, in vivo pharmacological effects have been verified using HGF. As a result, it has been reported that the use of HGF improves the regeneration rate of excised liver tissue and further improves various liver and renal dysfunctions. By the way, since HGF is purified by a heparin column, it is known as a growth factor having an affinity for heparin. On the other hand, a large number of heparan sulfates having a structure similar to that of heparin are present on the cell surface, and HGF is trapped on the cell surface as a kind of low affinity receptor for HGF. Furthermore, HGF bound to these receptors is rapidly transferred into cells, decomposed and released outside the cells. Therefore, HG in vivo
The blood half-life of F is extremely short, and the current pharmacological effect of HGF cannot be sufficiently obtained.

Therefore, various sulfated polysaccharides (heparin, dextran sulfate, chondroitin sulfate, etc.) having a structure similar to that of heparan sulfate existing on the cell surface are HG.
An attempt was made to mix with F. The added sulfated polysaccharide binds to HGF to suppress the binding between heparan sulfate existing on the cell surface and HGF. As a result,
It has been found that the degradation of HGF is remarkably suppressed, and the activity of HGF in vitro and the pharmacological effect in vivo are improved (Japanese Patent Laid-Open No. 5-3018).
No. 24).

On the other hand, among the sulfated polysaccharides in the living body, glycosaminoglycan is one of the substances having the most structural diversity and having a great influence on various cell activities. Examples of this molecule include heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate. Heparin and heparan sulphate are made up of repeating disaccharide units containing uronic acid and glucosamine residues.
Uronic acid has α-D-glucuronic acid (GlcUA) and β-L-iduronic acid (IdoA) depending on the configuration of C-5.
And a part of IdoA is a 2-O-sulfate group (Id
oA (2S)). Glucosamine residues are N-acetyl group (GlcNAc) or N-sulfate group (Glc
NS). Some glucosamine residues have a sulfate group at C-6. Compared to these, although the content is low, 2-O-sulfated α-D-glucuronic acid (GlcU
A (2S)) and 3-O-sulfated GlcNS (Glc
NS (3S)) or 3-O, 6-O-disulfated Gl
cNS (GlcNS (3,6S) is present. Heparin and heparan sulphate have a repeating structure of tens to hundreds of disaccharides composed of these residues. The combination of different disaccharide units forms the structural diversity of heparin and heparan sulfate.

Glycosaminoglycans are known to exert a strong influence on the physical and chemical environment of the extracellular matrix. Glycosaminoglycans, for example, utilize their specific structure to regulate the permeability of various molecules to the cell basement membrane and to regulate key regulators such as growth factors, hormones, or neurotransmitters. Access to the cell surface can be controlled. Furthermore, glycosaminoglycans including heparin are known to have an affinity for HGF. However, until now, the specific structure of glycosaminoglycan showing affinity with HGF has not been known at all.

[0007]

Means for Solving the Problems The present inventors analyzed the binding between HGF and heparin or heparan sulfate by using an HGF affinity column. That is, as a result of analyzing the binding between an oligosaccharide fragmented with heparin or heparan sulfate and an HGF affinity column, it was found that each oligosaccharide binds to the HGF affinity column in a size-dependent manner. For the first time, it was found that the binding rate of each oligosaccharide to sucrose increased sharply from an oligosaccharide composed of 8 sugars, and that such an oligosaccharide was a sulfated sugar at a specific position. Has been completed.

That is, the gist of the present invention resides in a sulfated polysaccharide having an affinity for hepatocyte growth factor and composed of at least 8 or more constituent sugars. Hereinafter, the present invention will be described in detail. The HGF used in the present invention is derived from mammals including human, and is not particularly limited as long as it is a proteinaceous factor having an activity of promoting the proliferation of hepatocytes. For example, as HGF, any one obtained by purifying from plasma or the like and one obtained by expressing its cDNA by a gene recombination method can be used. Specifically, according to the method described in JP-A-63-22526, by protein-chemically separating and purifying from the plasma of a patient with fulminant hepatitis, or according to the method described in JP-A-3-2855693. It can also be obtained by constructing an expression vector containing a cDNA encoding HGF obtained from a human placenta-derived cDNA library and expressing it in a host such as CHO cells.

Human-derived HGF is a non-reducing SDS.
-Molecular weight of about 76,000-9 by PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)
Give a band to 2,000. In addition, SDS under reduction
-By PAGE, a molecular weight of about 56,000-650,000
It is separated into a large subunit giving a band of 00 and a small subunit giving a band of a molecular weight of about 32,000-35,000. And this HGF is 80
It has the property of losing the activity of promoting the proliferation of hepatocytes by heat treatment at 10 ° C for 10 minutes or by digestion with trypsin or chymotrypsin. Further, the HGF has a strong affinity for heparin. Generally, the H used in the present invention
GF begins to show hepatocyte proliferation activity at a concentration of about 0.5-2 ng / ml and good proliferation activity at a concentration of about 5-10 ng / ml.

In the present invention, "polysaccharide" is defined to include not only polysaccharides in the narrow sense but also "oligosaccharides" composed of several monosaccharides. The sulfated polysaccharide of the present invention can be obtained, for example, as follows. First, known sulfated polysaccharides such as heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate are decomposed with an enzyme, nitrous acid or the like to obtain oligosaccharides (partially decomposed products or limited decomposition products) of various molecular weights. On the other hand, after contacting HGF in the presence of heparin or the like, the HGF is bound to a carrier, and then the heparin or the like is removed to prepare an HGF affinity column. The various oligosaccharides described above are added to this column. Then, by selecting a column having a high binding rate between the column and each oligosaccharide, preferably a binding rate of 5% or more, more preferably 10% or more, a structural unit portion having a high affinity for HGF is contained. The sulfated polysaccharide of the present invention can be obtained.

The sulfated polysaccharide of the present invention contains such a structural unit portion, and as long as it shows an affinity for HGF, not only the above-mentioned decomposed product but also the sulfated polysaccharide before the decomposition has a large amount of the structural unit portion. It also includes what is contained. Furthermore, oligosaccharides that can bind to the HGF affinity column were purified by anion exchange column chromatography, and an enzyme specific to each oligosaccharide (eg, heparitinase I, II, III) was used.
Etc.) to form a disaccharide unit, and the disaccharide composition is fractionated by a polyamine column etc., and the sulfate group binding position and the number of binding of each fraction are analyzed to make it specific to the HGF affinity column. It is possible to determine the oligosaccharides that physically bind, that is, the sugar structures that constitute the sulfated polysaccharides. As a result, the obtained sulfated polysaccharide consisted of 4 units of disaccharide unit structures of hexuronic acid and hexosamine, and among the disaccharide unit structures, 2-O-sulfated hexuronic acid and 6-O, 2 −
In the present invention, the disaccharide unit structure with N-disulfated hexosamine is about 1.5 to 2.5 units among the 4 units, and contains a structural unit portion having an affinity for hepatocyte growth factor. It can be confirmed that it is a sulfated polysaccharide.

The sulfated polysaccharide of the present invention is the above-mentioned HGF.
Can be used in combination with. For example, HGF
The sulfated polysaccharide of the present invention is added in an amount of 0.1 to 10 with respect to 1 mol.
Such a sulfated polysaccharide effectively binds to HGF when added in the range of ⁵ mol. As a result, the binding between heparan sulfate existing on the cell surface and HGF was suppressed, and HG
The decomposition of F is significantly suppressed. Further in vitro
Activity of HGF and pharmacological effect in vivo are improved.

[0013]

EFFECTS OF THE INVENTION Since the sulfated polysaccharide of the present invention has the property of specifically binding to HGF, it can stabilize HGF and improve its proliferative activity. Further, known sulfated polysaccharides are known to suppress the decomposition of heparin-binding growth factors such as HGF, and thus the sulfated polysaccharides of the present invention can be expected to have similar effects,
For example, by adding the sulfated polysaccharide of the present invention to a culture solution for producing a heparin-binding growth factor by recombinant DNA technology, such a heparin-binding growth factor can be efficiently and highly produced from the culture supernatant. .

[0014]

The present invention will be described in more detail with reference to the following examples, but the invention is not limited to the following examples as long as the gist thereof is not exceeded. In the examples below,
HGF was prepared by a gene recombination method in accordance with the method described in Japanese Patent Laid-Open No. 3-285693.
F "was used. Example 1 Size of heparin oligosaccharide capable of binding to HGF Heparin (Seikagaku Corporation) was subjected to nitrite decomposition to perform limited decomposition of heparin. 0.5 mg of the obtained heparin oligosaccharide was tritium-labeled with [ ³ H] NaBH ₄ . This radiolabeled heparin oligosaccharide is converted into S
After addition to an ephadex G-50 (Pharmacia) column (1.2 cm x 127 cm), elution with 0.2 M ammonium acetate was performed, and the peak position of the oligosaccharide was determined from the radioactivity measurement and uronic acid measurement of the eluate. Was fractionated. 2 peaks eluting first
It was assumed that the peak was a sugar-sized oligosaccharide, and that the peaks that were sequentially eluted were the peaks of 4, 6, 8, 10, 12 sugar-sized oligosaccharides. Table 1 shows the size of each fractionated oligosaccharide and its approximate molecular weight.

[0015]

[Table 1] Table 1 ──────────────────── Molecular weight of the number of constituent sugars in heparin oligosaccharides ────────────── ───────── 4 (Hep 4) 1300 6 (Hep 6) 2000 8 (Hep 8) 2600 10 (Hep 10) 3300 12 (Hep 12) 4000 ────────────── ──────

On the other hand, CNBr-activateds
1.2 ml of epharose 4B (Pharmacia)
Coupling buffer (0.4M NaCl in 0.2%).
Recombinant human HGF (1 mg) suspended in 1M NaHCO ₃ (pH 8.3) and contacted with acetylated heparin
/ Ml) solution was added and shaken gently at 4 ° C. overnight.
After thoroughly washing the obtained gel with a coupling buffer,
Suspend in 1M Tris-HCl buffer (pH 8.0) and
Gently shaken overnight at ℃. This gel was packed in a column, washed with 10 mM Tris-hydrochloric acid buffer (pH 7.2) containing 2 M NaCl, and further washed with the buffer used in affinity chromatography to obtain HG.
An F-affinity column was created.

For this column, a buffer solution (0.15 M NaC) containing 4 nmol of radiolabeled heparin oligosaccharide of each size (4 sugar, 6 sugar, 8 sugar, 10 sugar and 12 sugar) was prepared.
1, 0.9 mM CaCl ₂ and 0.2 mg / ml
10 mM Tris-hydrochloric acid buffer solution (pH 7.2) containing chondroitin sulfate (CSA)) was added, and 1 at 4 ° C.
Allowed to react for hours. After this, 0.15 M NaCl, 0.
9 mM CaCl2, and 0.2 mg / ml CSA
Was washed with 10 mM Tris-hydrochloric acid buffer (pH 7.2) containing HMP, and the heparin oligosaccharide bound to the HGF affinity column was added with 1 M containing 2.0 M NaCl.
Elution was performed with 0 mM Tris-HCl buffer (pH 7.2). By measuring the radioactivity of the eluted heparin oligosaccharides, the binding rate between each heparin oligosaccharide and the HGF affinity column was calculated. FIG. 1 shows the binding rate of heparin oligosaccharides to the HGF affinity column. As shown in FIG. 1, it was found that the size of heparin oligosaccharides was 8 or more in order to bind HGF to heparin oligosaccharides.

Example 2 Size of heparan sulfate oligosaccharide capable of binding to HGF Low sulfated heparan sulfate (CLHS) 50 derived from bovine liver
Dissolve 0 mg in 5 ml of water and chondroitinase ABC
A 25 m unit was used to digest contaminating chondroitin sulfate and dermatan sulfate. Further, ethanol precipitation was performed on this fraction. As a result of quantifying this uronic acid, it was found that about 50% of the treated CLHS was present in the chondroitinase ABC resistant fraction. This fraction was applied to a DEAE-Sephacryl column, and then 0.2M NaCl, 0.02M Tris-HCl buffer (pH 7.2) to 1.0M NaCl, 0.02 was added.
Elution was performed with a linear concentration gradient of M Tris-hydrochloric acid buffer (pH 7.2), and CLHSI, CLHS II and C were used.
Fractionation into LHS III fractions. The obtained low-sulfate fraction, CLHS I fraction, was compared to hepatylinase I fraction.
Was added and the enzyme reaction was carried out at 37 ° C. for 1 hour, and then the reaction was stopped by boiling for 3 minutes. 0.5 mg of the obtained heparan sulfate oligosaccharide was tritium-labeled with [ ³ H] NaBH ₄ . The radiolabeled heparan sulfate oligosaccharide was loaded onto a Sephadex G-50 column (1.2 cm x
127M) and eluted with 0.2M ammonium acetate, and its approximate molecular weight (800, 1300, 17)
00, 2100, 2600, 3000). The relationship between the molecular weight and the elution position is that oligosaccharides (ΔDi-6S (unsaturated GAG disaccharide kit (for HPLC), Seikagaku Corporation), which are known molecular weight standards, radioisotope-labeled 4 of chondroitin , 6 and 8 sugars (all obtained by digesting chondroitin with hyaluronidase, separating and purifying by gel filtration, and reducing with [ ³ H] NaBH ₄ ), were eluted under the same conditions, and pre-determined ( Biochem. J., 285 , 805-813 (1.
992)). The approximate molecular weight of the obtained fraction,
The size of each oligosaccharide was assumed from the molecular weight of the disaccharide unit structure consisting of iduronic acid and 2-N-sulfated glucosamine. The fractionation pattern of each oligosaccharide is shown in FIG. 2, and the size of each fractionated oligosaccharide (the number of constituent sugars assumed above) and its approximate molecular weight are shown in Table 2.

[0019]

[Table 2] Table 2 ───────────────────── Heparan sulphate oligosaccharides Approximate molecular weight of the constituent sugars ──────────── ────────── 4 (CLHS 4) 800 6 (CLHS 6) 1300 8 (CLHS 8) 1700 10 (CLHS10) 2100 12 (CLHS12) 2600 14 (CLHS14) 3000 ─────── ──────────────

Furthermore, a buffer solution (0.15M) containing 4 nmol of oligosaccharides of each size labeled with radioisotope was added to the HGF affinity column prepared in Example 1.
NaCl, 0.9 mM CaCl ₂ and 0.2 mg /
10 mM Tris-hydrochloric acid buffer solution (pH 7.2) containing ml CSA was added and reacted at 4 ° C. for 1 hour. After this, 0.15 M NaCl, 0.9 mM Ca
10 m containing Cl ₂ and 0.2 mg / ml CSA
Wash with M Tris-HCl buffer (pH 7.2),
Further, the heparan sulfate oligosaccharide bound to the HGF affinity column was eluted with 10 mM Tris-hydrochloric acid buffer solution (pH 7.2) containing 2.0 M NaCl. By measuring the radioactivity of the eluted heparan sulfate oligosaccharide, the binding rate between each heparan sulfate oligosaccharide and the HGF affinity column was calculated. FIG. 3 shows the binding rate of heparan sulfate oligosaccharides to the HGF affinity column. As shown in FIG. 3, it was found that the size of heparan sulfate oligosaccharides was 8 or more, and more preferably 10 or more, in order to bind HGF to heparan sulfate oligosaccharides.

Example 3 Structures of heparan sulfate oligosaccharide capable of binding to HGF and heparan sulfate oligosaccharide not capable of binding to HGF The CLHS I-derived heparan sulfate oligosaccharide having a size of 10 sugars prepared in Example 2 (CLHS10) or dodecasaccharide (CLHS12) was added to the HGF affinity column by the method described in Example 1 and reacted at 4 ° C. for 1 hour, and the fraction bound to this column and The unbound fraction was separated. That is, 0.15
M NaCl, 0.9 mM CaCl2, and 0.2
Washing was performed with 10 mM Tris-hydrochloric acid buffer (pH 7.2) containing mg / ml CSA (unbound fraction), and further elution was performed with 10 mM Tris-hydrochloric acid buffer (pH 7.2) containing 2.0 M NaCl. (Bound fraction).

CL for HGF affinity column
The adsorption fraction and non-adsorption fraction of HS10 are MonoQ
Add to column (Pharmacia) and add 0M NaCl,
Elution was performed with a linear concentration gradient from 0.05 M Tris-hydrochloric acid buffer (pH 7.2) to 1.0 M NaCl, 0.05 M Tris-hydrochloric acid buffer (pH 7.2). Then, elution was further performed with 2M NaCl and 0.05M Tris-hydrochloric acid buffer (pH 7.2).

The results are shown in FIG. C is shown in FIG.
[Fig. 4] Fig. 4 shows the chromatography when the fraction adsorbed to the HGF affinity column of LHS10 was added.
The B-III fraction shown in (A) was collected. On the other hand, FIG.
FIG. 4 (B) shows the chromatography when the HGF affinity column non-adsorbed fraction of CLHS10 was added, and the UB-I, UB-II and UB-III fractions shown in FIG. Recovered.

Next, the obtained B-III, UB-I and U
The B-II and UB-III fractions were enzymatically digested with heparitinase I, II, and III to decompose into unsaturated disaccharides, and the unsaturated disaccharide composition of each fraction was analyzed by a polyamine column (new chemistry). Experimental Course 3, Carbohydrate II, p49-62
(Tokyo Kagaku Doujinsha)). The notation of unsaturated disaccharide is shown in FIG.

The unsaturated disaccharide composition analyzed is shown in FIG. 6 in%. From the sulfation degree of B-III (the number of sulfate groups present in the disaccharide unit structure), it was found that this oligosaccharide was composed of eight constituent sugars. Here, a method for obtaining the number of constituent sugars is shown below. The degree of sulfation of CLHS (10) -B-III in FIG. 6, that is, the value of SO4 / Δdimer, 2.41 is B-
It is the average value of the degree of sulfation of unsaturated disaccharides obtained by enzymatically digesting III. Since the value of the degree of sulfation obtained is an average value, in practice, from theory, 60% of unsaturated disaccharides having a degree of sulfation of 2 and 40% of unsaturated disaccharides having a degree of sulfation of 3 are present. ing. The disaccharide unit structure having a degree of sulfation of 2 before the enzymatic digestion is 2-O-sulfated iduronic acid and 2-N-sulfated glucosamine or iduronic acid and 6-O, 2-N-disulfated glucosamine. And the disaccharide unit structure with a sulfation degree of 3 is 2
-O-sulfated iduronic acid and 6-O, 2-N-disulfated glucosamine, each having a molecular weight of 529 and 62.
It is 5. Therefore, the value obtained by multiplying the above-mentioned existence ratio by each molecular weight is 567, which is the molecular weight of the disaccharide unit structure having the sulfation degree of 2.41. From Table 2 above, it can be seen that CLHS10 has an approximate molecular weight of 2100, so this is divided by 567, and CLHS10 has 3.7 disaccharide unit structures. Since there are 3.7 disaccharide unit structures, CLHS10 is a 7.4 saccharide.
In fact, it is considered to be a 6-8 sugar, and it was found that the size of the oligosaccharide needs to be 8 or more in order to bind HGF to heparan sulfate oligosaccharide.
0 was composed of eight constituent sugars. Therefore, () in FIG.
The number of units of various unsaturated disaccharide units having different sulfation positions and degrees of sulfation with respect to the constituent sugars (4 units when the repeating structure of the disaccharide is one unit) is shown in the table.

B-III fraction capable of adsorbing to HGF, and H
Comparing the unsaturated disaccharide composition of the UB-I, UB-II and UB-III fractions which are non-adsorbed fractions to GF,
It was found that the ratio of ΔDi-triS and ΔDi-di (N, 6) S was high in the unsaturated disaccharide composition of B-III which was the HGF adsorbed fraction. Particularly, in the B-III fraction capable of binding to HGF, it was found that almost 2 units of 4 units of oligosaccharides were occupied by ΔDi-triS. From these results, 2 units of IdoA (2S)
-GlcNS (6S) was found to be important for HGF binding (ΔDi-triS, IdoA (2S) -G
It is an unsaturated disaccharide obtained by enzymatically digesting lcNS (6S)).

Similarly, heparan sulfate oligosaccharide (CLHS12), which is derived from CLHS I and consists of 12 sugars, is converted into H
It was applied to a GF affinity column and the bound and unbound fractions were collected. Furthermore, HG of CLHS12
The adsorbed fraction and the non-adsorbed fraction with respect to the F affinity column were each added to a MonoQ column (Pharmacia), and the mixture was diluted with 0M NaCl, 0.05M Tris-hydrochloric acid buffer (pH 7.2) to 1.0M NaCl, 0. 05M
Elution was performed with a linear concentration gradient of Tris-hydrochloric acid buffer (pH 7.2), and 2.0 M NaCl, 0.05 M was added.
Elution was performed with Tris-hydrochloric acid buffer (pH 7.2).

The results are shown in FIG. CLH is shown in FIG.
[Fig. 7] Fig. 7 (A) shows the chromatography when the SGF HGF affinity column adsorption fraction was added.
The BI fraction shown in was collected. On the other hand, in FIG.
FIG. 7 shows the chromatography when the non-adsorbed fraction of LGF12 on the HGF affinity column was added, and FIG.
The UB-I and UB-II fractions shown in (B) were collected.

Next, the obtained B-I, UB-I and U
Heparitinase I, II, II against B-II fraction
It was enzymatically digested with I to decompose it into unsaturated disaccharides, and the unsaturated disaccharide composition of each fraction was analyzed by a polyamine column. The unsaturated disaccharide composition analyzed is shown in FIG. From the sulfation degree of BI,
It was found that this oligosaccharide consists of 8 to 10 constituent sugars. In () of FIG. 8, the number of units of various unsaturated disaccharide units having different sulfation positions and degrees of sulfation with respect to the constituent sugars (4 units when the repeating structure of the disaccharide is 1 unit) is shown. Indicated.

As a result, it was found that in the unsaturated disaccharide composition of the HGF adsorbed fraction, BI, almost 2 units of 4 units of oligosaccharides were occupied by ΔDi-triS. From these results, 2 units of IdoA (2S)
-GlcNS (6S) was confirmed to be important for HGF binding (ΔDi-triS, IdoA (2S)
-An unsaturated disaccharide obtained by enzymatically digesting GlcNS (6S)).

Example 4 Heparan sulfate oligosaccharide-releasing activity of HGF bound to rat proteoglycan Digoxigenin-HGF 20 was added to wells coated with heparan sulfate proteoglycan prepared from rat liver.
ng / well with 0.2 mg / ml CSA and 0.9
After binding with a blocking buffer containing mM CaCl ₂ , it was replaced with PBS containing 1 ng to 10 μg / ml heparan sulfate oligosaccharide, and HG bound to the plate
F was released. Free activity was 50% of HGF bound to plate-coated heparan sulfate proteoglycan.
It was determined as the released concentration. The results are shown in Table 3.

[0032]

[Table 3] Table 3 ────────────────────────── Heparan sulfate oligosaccharide free activity (ng / ml) ─────── ─────────────────── CLHS 8 3360 CLHS10 350 CLHS12 100 CLHS12-B 3.5 CLHS12-UB 4230 ────────────── ───────── Heparin 1.0 ───────────────────────────

As is clear from the above results, heparan sulfate oligosaccharides had a high size-dependent release activity.
In addition, non-binding heparan sulfate dodecasaccharide fraction (CLHS12
-UB) has a free activity of 4230 ng / ml, whereas the binding heparan sulfate dodecasaccharide fraction (CLHS12-).
The free activity of B) is 3.5 ng / ml, and the activity is 1000
The opening was more than doubled. Moreover, the activity of CLHS12-B is about 1/3 of that of heparin, indicating that it has a strong activity.

Therefore, the HGF-binding heparan sulfate is HG for the heparan sulfate proteoglycan existing on the cell surface.
It is expected to strongly inhibit the binding of F and suppress its degradation, and as a result, to improve the pharmacological action of HGF.

[Brief description of drawings]

FIG. 1 is a drawing showing the binding rate of heparin oligosaccharides to HGF affinity columns.

Figure 2: Sephadex G with heparan sulfate oligosaccharide
It is a figure showing the result of having added to -50 column and fractionated by the oligosaccharide size.

FIG. 3: Heparan sulfate oligosaccharides from CLHS I and H
It is a figure showing the binding rate with a GF affinity column.

FIG. 4 is a drawing showing chromatography when a fraction adsorbed / non-adsorbed on an HGF affinity column of CLHS10 was added to a MonoQ column. In the figure, (A) is H
GF affinity column adsorption fraction, (B) HGF
The non-adsorbed fractions on the affinity column are shown.

FIG. 5 shows the relationship between the structures of various unsaturated disaccharides produced by enzymatically digesting sulfated polysaccharides and their abbreviations.

FIG. 6 shows CLHS10 and its fractions obtained by HGF affinity column, B-III, UB-I and UB-.
2 is a table showing the composition of unsaturated disaccharides of II and UB-III fractions.

FIG. 7 is a diagram showing chromatography when a fraction adsorbed / non-adsorbed on an HGF affinity column of CLHS12 was added to a MonoQ column. In the figure, (A) is H
GF affinity column adsorption fraction, (B) HGF
The non-adsorbed fractions on the affinity column are shown.

FIG. 8: CLHS12, BI, UB-I and UB-
It is a table | surface which showed the unsaturated disaccharide composition of II fraction.

Claims (7)

[Claims] 1. A sulfated polysaccharide containing a structural unit moiety of at least 8 sugars having an affinity for hepatocyte growth factor. 2. The structural unit portion comprises hexuronic acid and hexosamine or an N-acetyl-substituted product thereof,
The sulfated polysaccharide according to claim 1, which contains 4 or more disaccharide unit structures containing a sulfate group. 3. Hexuronic acid is L-iduronic acid or D
-Glucuronic acid, and hexosamine is D-glucosamine or D-galactosamine or its N-acetyl-substituted form, and the sulfated polysaccharide according to claim 2. 4. A partially decomposed or limited decomposed product of heparin or heparan sulfate.
The sulfated polysaccharide according to item 3. 5. A disaccharide unit structure consisting of 4 units of hexuronic acid and hexosamine.
The disaccharide unit structure of O-sulfated hexuronic acid and 6-O, 2-N-disulfated hexosamine has about 1.5 of the 4 units.
~ 2.5 units, a sulfated polysaccharide containing a structural unit moiety having an affinity for hepatocyte growth factor. 6. Heparin or heparan sulfate, or a mixture of a partial decomposition product or a limited decomposition product thereof is brought into contact with an affinity carrier bound with hepatocyte growth factor as a ligand to have an affinity for hepatocyte growth factor. A method for producing a sulfated polysaccharide, which comprises fractionating a fraction into a fraction having no affinity for the factor. 7. The method is characterized in that contact with an affinity carrier is performed in the presence of chondroitin sulfate, and after fractionation, chondroitin sulfate is separated and removed from the fraction having an affinity for hepatocyte growth factor, if necessary. Item 6. The method according to Item 6.