JPH10273496A - Lipoteichoic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new compound consisting of a specific lipoteichoic acid having a glycerol part of glycerophosphate part substituted with a glucose partly esterified with alanine and exhibiting an activity to induce tumor necrosis factor(TNF). SOLUTION: This new lipoteichoic acid is expressed by the formula [X is H, Ala (Glc)n (Glc is glucose residue; (n) is a natural number of 5-42) or (Gln)n ; COR is a fatty acid residue]. It can stimulate macrophage, induce the formation and release of a cytokine such as tumor necrosis factor(TNF) and improve the phagocytic action, bactericidal action against cytozoic bacteria, etc. The compound can be produced by culturing a lipoteichoic acid-producing bacterium belonging to the genus Enterococcus such as Enterococcus hirae ATCC9790, recovering the cultured cell, subjecting to a separation treatment taking advantage of hydrophobic interaction and separating and purifying the product by a separation process taking advantage of a cation exchanging action.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to lipoteichoic acid (hereinafter abbreviated as LTA). More specifically, the present invention relates to glycerophosphate in which the glycerol moiety at the 2-position is partially esterified with alanine. Lipoteichoic acid, which is substituted by substituted glucose or oligoglucose.

[0002]

2. Description of the Related Art LTA is available from Streptococcus, Enterococcu.
s, Staphylococcus, Lactobacillus, Bacillus, Lister
It is an amphiphilic bacterial cell surface component widely distributed in Gram-positive cocci and bacilli such as ia and Leuconostoc (with some exceptions). LTA has a structure in which a polymer of sn-glycero-1-phosphate is bonded to the 6-position hydroxyl group of the non-reducing terminal hexose of glycolipid, which is a part of the cytoplasmic membrane component, and protrudes into the cell wall peptidoglycan portion. It is known that a glycosyl group, D-alanine, or the like is often bonded to the 2-position hydroxyl group of the glycerol portion in LTA. It is also known that the structure or substituents of the (glycerophosphoric acid) moiety are generally different,
The chemical structure and physicochemical properties of TA vary widely.

[0003] For example, regarding antigenicity, the poly (glycerophosphate) part itself, its glycosyl substituent and D-
Alanine esters mainly serve as antigenic determinants.
In some Gram-positive bacteria, the glycosyl substituent of lipoteichoic acid is recognized as a group-specific antigen, such as Lactobacillus helveticus group-specific A antigen, L. fe.
rmenti F antigen, Enterococcus faecalis D antigen, Lactococcus lactis N antigen, Streptococcus m
utans serotype-specific antigen a and the like.

[0004] Passive hemagglutination often detects antibodies reactive with LTA in humans, especially in the sera of the late 10s and above. Studies centered on streptococcal and staphylococcal preparations show that LTA exhibits broad immunobiological activity similar to LPS (an amphiphilic endotoxin lipopolysaccharide that forms the outer membrane of Gram-negative bacteria) It has been revealed. That is, LTA stimulates mouse peritoneal stimulating macrophages, and TNF-α, IL-6, I
It is known that it induces the production and release of cytokines such as FN-γ, IL-1, and CSF, and enhances phagocytosis, active oxygen generation, and bactericidal action against intracellular parasites.

[0005] Furthermore, LTA activates the reticulum system by administration to mice and the like, and enhances nonspecific resistance. Also in this connection, in the experimental mouse tumor system, LTA
It shows the growth inhibitory effect on ethA fibrosarcoma, tumor regression / healing effect, and lung metastasis inhibitory effect of L-1 sarcoma. The stimulating effect of LTA on lymphocytes (such as the mitogenic effect) is controversial.

Other functions of LTA include the action of inducing the production of nitric oxide (NO) from vascular smooth muscle cells and the action of activating complement via the classical pathway by directly reacting with the complement component (C1q). And so on. Although the above effect of LTA is weaker than that of LPS, the difference in lethal toxicity and pyrogenicity is large. In addition, LTA is used for human and animal buccal and nasal mucosal epithelial cells, erythrocytes, platelets, polymorphonuclear leukocytes,
It binds to spleen cells, but this effect is due to Streptococcus pyog
Enes and Staphylococcus aureus play a role in the adhesion of the pharynx and nasal mucosa, and may contribute to the development of pathogenicity. On the other hand, host cells with LTA bound to their surface are anti-LT
It is destroyed by the action of antibody A and complement, and suffers from antibody-dependent and cell-mediated cytotoxicity (ADCC). LTA adsorbed on the surface of red blood cells activates complement via another pathway.

[0007] As described above, the chemical structure of LTA, and thus the “physical properties”, vary widely, and the immunobiological activity varies depending on the species, strain, and culture conditions of the fungus, and a sufficiently homogenized purified product. The chemical structure responsible for the above-mentioned immunobiological activity has not yet been elucidated, for example, because of the difficulty in isolating the same.

The chemical structure of LTA derived from species belonging to Enterococcus and Streptococcus has been described so far.
Reported by W. Fischer (see Reference 1 in the cited reference list at the end of the specification). W. Fischer, L
TA is generally a glycolipid anchor as well as a 1,3-linked poly (glycerophosphate) in which position 2 of glycerol is substituted with oligoglucose or D-alanine.
It is reported that it consists of. Also, biological activities of LTA such as cytokine (IL-1, IL-6 and TNF-α) inducing activity and antitumor activity have been reported (see References 2 to 8).

[0009] More recently, the effect of LTA structure on recognition by macrophage capture receptors has been reported (JWGreenberg et al., Infecti).
on and Immunity, Vol. 64, No. 8, p. 3318-3325 (199
6)). However, this document does not describe LTA having glucose or oligoglucose partially esterified with alanine at position 2 of glycerol in the glycerophosphate portion of LTA.

For the purpose of elucidating the chemical structure in LTA essential for the expression of the above-described LTA cytokine-inducing activity, compounds having the basic structure proposed for LTAs have been synthesized (References 9 and 9). 10). However, surprisingly, these synthetic compounds
Neither cytokine-inducing activity nor antitumor activity was shown (see Reference 11). The structure of this synthetic analog is
The structure differed from the proposed structure for A in the following respects: (I) the glycolipid anchor of native LTA contains unsaturated fatty acids, while synthetic LTA contains only saturated acids;
(II) Poly (glycerophosphate) of natural LTA
Is thought to have 9 to 40 repeating units (see Reference 12), while in the case of synthetic LTA it contains only 4 repeating units; (III) natural LTA has a high degree of glycosyl substitution and While having an alanyl substitution, the synthetic poly (glycerophosphate) moiety has no substituent.

That is, the absence of the cytokine-inducing activity of the synthetic compound is considered to be due to one of the following two possibilities: (1) Some or all of the above structural features are those of LTA Essential for biological activity;
(2) Unknown components present in LTA are involved in the activity.

[0012]

SUMMARY OF THE INVENTION The present inventors have recently reported that En
LTA derived from terococcus hirae ATCC 9790,
Separation into a quantitatively low component having IL-6 inducing activity on human whole blood cells and a quantitatively high component having no IL-6 inducing activity on the same cells (occupying 90% or more). (See Reference 13). Thus, in the present study, the present inventors analyzed the chemical structure of the isolated quantitatively high component having no IL-6 inducing activity. That is, one of the objects of the present invention is to elucidate the chemical structure of LTA, thereby elucidating the physical properties of LTA, in particular, biological activity, and more specifically, the relationship between the cytokine-inducing activity and the chemical structure of LTA. is there.

[0013]

Means for Solving the Problems The present inventors have proposed a microorganism En
A fraction containing LTA was obtained from terococcus hirae by a fractionation method, and based on this, the structure of LTA was analyzed. As a result, position 2 of the glycerol portion of the glycerophosphate portion was
Found to be replaced by glucose or oligoglucose partially esterified with alanine,
The present invention has been completed. LTA in which the 2-position of the glycerol moiety of the glycerophosphate moiety is replaced by glucose or oligoglucose partially esterified with alanine is a novel LTA which has not been reported previously.
TA.

That is, according to the present invention, the following structural formula (1):

Embedded image (Wherein, X independently represents H, Ala (Glc) _m or (Glc) _m , wherein m represents a natural number of 1 to 5; n represents a natural number of 5 to 42; and COR Is
(Indicating a fatty acid residue).

In one embodiment of the present invention, at least one X in the definition of X in the structural formula (1) is Al
a (Glc) _m . Note that n represents a natural number of 5 to 42, and the average value of n is about 11.

[0016] The lipoteichoic acid of the present invention is preferably
terococcus genus, more preferably Enterococcus
hirae, particularly preferably Enterococcus hirae A
It is characterized by being derived from TCC9790.

The lipoteichoic acid of the present invention is obtained, for example, by culturing a lipoteichoic acid-producing bacterium, collecting the bacterium, subjecting the bacterium to a separation method utilizing hydrophobic interaction, and then separating the lipoteichoic acid using a cation exchange effect. It can be obtained by applying the law. In the separation method using a cation exchange action performed to obtain lipoteichoic acid of the present invention, a buffer solution containing a salt can be used as an eluate.

In the present specification, Ala means alanine, which may be D-form or L-form, but preferably D-alanine. In the formula represented by Ala (Glc) _m , Ala is bonded to an arbitrary hydroxyl group in an arbitrary glucosyl group by an ester bond, and the position of the alanine bonded to the glucosyl group or oligoglucosyl group is not particularly limited. . However, in the case of an oligoglucosyl group, it is preferable that alanine is bonded to the terminal glucosyl group farthest from the glycerol component. In addition, alanine is 6 of the hydroxyl groups in the glucosyl group.
It is preferably bonded to a hydroxyl group at the position.

In the present specification, Glc indicates a glucosyl group. In the present specification, COR represents a fatty acid residue, where R represents a saturated or unsaturated hydrocarbon group. The number of carbon atoms in the hydrocarbon group is not particularly limited, and any one of a short-chain fatty acid (2 to 4 carbon atoms), a medium-chain fatty acid (5 to 10 carbon atoms), and a long-chain fatty acid (11 or more carbon atoms) It may be a residue. Preferably, the fatty acid residue is a long-chain fatty acid residue, in which case the number of carbon atoms is generally 11 or more, for example, fatty acid residues having 16 and 18 carbon atoms. More specifically, a saturated fatty acid residue having 16 carbon atoms, a fatty acid residue having one unsaturated bond (such as a double bond) having 16 carbon atoms, a saturated fatty acid residue having 18 carbon atoms, or a fatty acid residue having 18 carbon atoms Examples of the fatty acid residue include a fatty acid residue having one unsaturated bond (such as a double bond).

[0020]

DETAILED DESCRIPTION OF THE INVENTION As mentioned hereinabove, lipoteichoic acid can be obtained from Streptococcus, Enterococcus, Staphylococcu.
s, Lactobacillus, Bacillus, Listeria, Leuconostoc
(With some exceptions), such as gram-positive cocci and bacilli. Therefore, it is expected that the lipoteichoic acid having the above structural formula (1) according to the present invention is also produced by any of the above bacteria. That is, the source of the lipoteichoic acid of the present invention is not limited to a specific microorganism, but can be obtained from any microorganism that produces lipoteichoic acid having the structural formula (1). In addition, not only natural LTA produced by microorganisms, but also lipoteichoic acid having the structural formula (1) can be obtained by chemical synthesis in some cases. Such chemically synthesized lipoteichoic acid is also within the scope of the present invention. Inside. The lipoteichoic acid of the present invention can be preferably obtained from a microorganism of the genus Enterococcus, more preferably Enterococcus hirae.
And particularly preferably Enterococc
available from us hirae ATCC 9790.

The method for preparing the lipoteichoic acid from the microorganism producing the lipoteichoic acid of the present invention is not particularly limited, and may be carried out by appropriately combining isolation and purification methods known to those skilled in the art. Can be.

For example, a lipoteichoic acid-producing microorganism is cultured in a suitable medium (generally containing a carbon source, a nitrogen source, a metal salt, an inorganic salt, and the like), and then the bacteria are collected. The culture medium and culture conditions (temperature, time, etc.) can be appropriately adjusted so that desired lipoteichoic acid is produced. Then, the free lipid of the bacteria is removed, extracted with phenol / water, the extract is treated with DNase-RNase, dialyzed, concentrated, and lyophilized to obtain a crude LTA fraction.

Further, the crude LTA fraction is subjected to a separation method utilizing a hydrophobic interaction (such as a batch type fractionation method or chromatography), and then a separation method utilizing an ionic interaction (a batch type separation method). Or chromatography) to obtain a more purified LTA fraction. Further, other means commonly used for isolating or purifying biological substances, such as gel filtration chromatography, can be used in appropriate combination.

In the separation method utilizing hydrophobic interaction, octyl-sepharose, phenyl sepharose,
A carrier having a hydrophobic group, such as polystyrene gel, can be used as the adsorbent. For example, octyl-
In a separation method using hydrophobic interaction using Sepharose, a crude LTA fraction is applied to octyl-Sepharose, and then LTA is eluted with an appropriate eluate. As the eluate, for example, a buffer containing 1-propanol at an appropriate concentration can be used. For example, when eluting with a column chromatography method, 1-propanol having a concentration gradient of 15% to 70% can be used as an eluate, and when performing batch separation, for example, 15%, 40%. A buffer containing 1-propanol at a concentration such as% or 60% can be used as the eluate. The type and concentration of these eluates can be appropriately selected by those skilled in the art in combination with the adsorbent.

In a separation method utilizing ionic interaction, an ion exchange membrane, specifically, QMA-Me
mSep1010 (PerSeptive Biosystems, Framingha
m, MA, USA), DEAE-MemSep1000
(Manufactured by Japan Millipore), QMA-MemSep1000
(Manufactured by Nippon Millipore) or DEAE-Sephac
el, DEAE-Sephadex, QM-Sepha
For example, a rose (all manufactured by Pharmacia) or an ion exchange resin having a strongly basic ion exchange group can be used. For example, QMA-MemSep10
In a separation method using ionic interaction using 10 as an ion exchange membrane, after applying an LTA-containing fraction to the membrane, LTA is eluted with an appropriate eluent.
As an eluate, 1% of an appropriate concentration (for example, 35%) is used.
-A buffer containing propanol can be used, and a suitable concentration or concentration gradient (e.g.
M) (eg, NaCl, KCl, CH ₃ C)
A buffer containing OONH ₄ , HCOONH _4, etc.) can be used.

The LT obtained by the separation method as described above
The structure of the component A can be determined in detail by NMR spectrum, mass spectrum, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), or the like. Further, by appropriately treating the product by hydrolysis, acetylation / deacetylation, alkali treatment, or the like, it can be useful for determination of a chemical structure.

In the following embodiment, Enterococcus hir
Starting from the microorganism represented by ae ATCC 9790,
Bacteria are defatted, extracted with hot phenol / water, and DNase-R
The extract obtained by digestion with Nase was dialyzed, concentrated, and lyophilized to obtain a crude LTA sample. Then, the crude LTA sample was subjected to a batch separation method using octyl-Sepharose to obtain a fraction containing LTA (BOS15, BOS4).
0 and BOS60). This was further applied to an ion exchange membrane to obtain an LTA component (QM-I). By analyzing the structure of the LTA component (QM-I) thus obtained, the present inventors have found an LTA having a novel structure which has not been reported previously.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

[0029]

EXAMPLES Example 1 Culture of Bacterial Cells E. hirae (ATCC 9790) was cultured in batches of 37 in 150 L batches.
The cells were grown for 6 hours with stirring at ℃. This growth medium (1
L) is 10 g glucose, 15 g trypticase, 4 g tryptose, 4 g yeast extract, 2 g K
₂ HPO ₄ , 5 g KH ₂ PO ₄ , 2 g Na ₂ CO ₃ ,
And 2 g of NaCl. Cells are collected by centrifugation and kept in acetone at 4 ° C until use.
Saved in.

NMR spectrum ¹ H- and ¹³ C-NMR spectra were measured using JMN-LA50.
500 each with 0 spectrometers (JEOL, Tokyo)
And 126 MHz. Chemical shifts are represented by δ values, and for ¹ H spectra, tetramethylsilane (δ
0) or water (δ4.65) as internal standard and for ¹³ C spectra tetramethylsilane (δ0) as internal standard or benzene (δ12
8) was used as an external standard.

The mass spectrum FAB-MS was obtained with an SX-102 mass spectrometer (JEOL). Glycerol or m-nitrobenzyl alcohol was used as the matrix. ESI-MS
Is an API III plus mass spectrometer (PE SCI
EX, Thornhill, Ontario, Canada).

Crude LTA by Octyl-Sepharose
Defatting of fractionated bacteria, hot phenol / water extraction, and DN of extract
Aase-RNase digestion was performed as described in the literature (see Reference 13). The digested extract was dialyzed, concentrated and lyophilized to obtain a crude LTA sample. The yield is
2-3% based on dry weight of delipidated cells. The crude LTA (1 g) contains 15% 1-propanol and contains 0.1%.
A solution in 200 ml of 1 M acetate buffer (pH 4.5)
400 ml of octyl-Sepharose (Pharmacia, Sweden, Uppsala) equilibrated with the same buffer. The mixture was left at 4 ° C. for 30 minutes and then filtered. Octyl-Sepharose was again suspended in 250 ml of the same buffer, left at 4 ° C. for 30 minutes, and filtered. This operation was repeated four more times. The recovered octyl-sepharose was treated in the same manner with acetate buffer containing 40% 1-propanol (4 times), and then treated in the same manner with acetate buffer containing 60% 1-propanol (6 times). The obtained filtrates were separately combined, concentrated under reduced pressure to about 100 ml, dialyzed, concentrated again under reduced pressure, and lyophilized to give 3 mL.
Fractions were obtained. These were respectively treated with BOS15 (15% 1-
Batch octyl in buffer containing propanol-
Sepharose eluate), BOS40 and BOS60. The recovery of each fraction was approximately 30, 45, and 10% from crude LTA.

The main octyl-sepharose fraction (BOS4
Further separation of 0) A portion (50 mg) of BOS40 was dissolved in 6 ml of 0.01 M acetate buffer (pH 4.5) containing 35% 1-propanol and ion exchange membrane QMA equilibrated with the same buffer.
-Applied to MemSep 1010 (PerSeptive Biosystems, Mass., USA). This film is 100m
Washed with 1 liter of the same buffer at a flow rate of 3.3 ml / min. The eluate was concentrated under reduced pressure to about 40 ml with a rotary evaporator. The material bound to the membrane was then eluted with 100 ml of buffer containing 35% 1-propanol and 1 M NaCl. The eluate was concentrated under reduced pressure to about 40 ml with a rotary evaporator. Both eluates were dialyzed separately, concentrated and lyophilized to give two fractions, namely QM-
A and QM-I were obtained. The same operation was repeated several times.
The recovery of these two fractions based on BOS40 was 2-3% and 80-90%, respectively.

Analytical Procedure The contents of phosphorus, glycerol, glucose, fatty acids and alanine were analyzed as described in the literature (see Reference 13). TLC was performed on silica gel plates (Merck Ke
iselgel 60 F ₂₅₄ Art. 5715). The following solvent system was used:
A, chloroform / methanol / water (65/25/4,
B, chloroform / methanol / ethyl acetate (100/4/6, volume ratio). Spots on the plate were visualized by using iodine vapor, water spray, or anisaldehyde-sulfuric acid reagent.

The HPLC was performed on an LC-6AC liquid chromatograph (Shimadzu Corporation) equipped with a RID-6A refractive index detector and a C-R7A chromatopack. Gel permeation chromatography was performed using a 0.01 M phosphate buffer (pH 7.0) containing 0.5 M NaCl on an Asahi Pack GFA-30 column (500 mm × 7.5 mm ID, Asahi Kasei).
0) was used as eluent at a flow rate of 0.8 ml / min. Several heparin standards (molecular weight: 1.8 × 1
^{0 4, 9.8 × 10 3,} 4.4 × 10 3, 2.4 × 10
³ and 1.2 × 10 ³ ) and dextran standards (1.7 × 10 ⁴ , 1.15 × 10 ⁴ and 9.4 ×).
Using 10 ³ ), a calibration curve for molecular weight measurement was prepared.

Phos in high anionic main component (QM-I)
Measurement of Homonoester QM-I (1.5 mg) was added to 1.5 ml of alkaline phosphatase solution (derived from Escherichia coli, Wako Pure Chemical Industries, Ltd., 5 units / m in 0.04 M ammonium carbonate)
1). This mixture was incubated at 37 ° C. for 24 hours. Then, the amount of free phosphoric acid in the reaction mixture was measured as described above.

Hydrolysis of QM-I Hydrolysis of QM-I with 47% hydrofluoric acid and phase separation of the reaction product were carried out according to the operation of Fischer (see Reference 1). Briefly, 10 mg of QM-I
Was hydrolyzed with 50 μl of hydrofluoric acid at 4 ° C. for 24 hours. The reaction mixture was dried over sodium hydroxide under reduced pressure. The residue was mixed with chloroform, methanol and water (2/1
/ 3, volume ratio). The product of the aqueous phase is combined with alkaline phosphatase 3
Treated in 0.04 M ammonium carbonate at 7 ° C. for 24 hours. After lyophilization, the mixture was completely acetylated with acetic anhydride / pyridine (1/1, by volume) at room temperature for 24 hours. After distilling off under reduced pressure, the completely acetylated product was extracted with chloroform.
Separated using.

Induction of IL-6 and measurement of its level Human peripheral whole blood cell culture (see references 14 and 15)
IL-6 induction in E. coli and measurement of its level using ELISA was performed as described in the literature (see Reference 13).

Deacylation of QM-I Deacylation of QM-I was performed according to the procedure of Kochanowski et al. (See Reference 16). That is, the fatty acids released after hydrolysis with 0.2 M sodium hydroxide at 37 ° C. for 2 hours were removed, and desalted by dialysis.

The present inventors have made sure that the test materials and test equipment are not contaminated with foreign endotoxins. For example,
Purchase from Otsuka (Tokyo) or Toray pure LV-308
Endotoxin-free water prepared by combining (Toray, Tokyo) and GSL-200 (Advantech, Tokyo) was used throughout the study.

Results In our earlier studies, the fractionation of crude LTA was performed by gel filtration chromatography followed by hydrophobic interaction chromatography on octyl-Sepharose with a primary gradient of 1-propanol. , Three fractions, namely a nucleic acid-containing fraction (NA), LTA-1 (recovery rate: 30 to 50%),
And LTA-2 (recovery: 7-14%). However, this method was not practical for large-scale fractionation due to flow rate limitations. Thus, in the present study, a rapid and efficient separation was realized using the batch type fractionation method on octyl-Sepharose. The 1-propanol concentration chosen for this elution corresponds to the concentration at the center of each of the three fractional peaks in our primary gradient chromatography. 3 fractions, BOS
15, BOS40 and BOS60 are 15
Eluted with 0.1 M acetate buffer containing%, 40%, and 60% 1-propanol. Judging from their recoveries (approximately 30, 45, and 10% based on crude LTA) and fatty acid content, BOS15, BOS40, and BOS60 were NA, LT, and LT in the previous study, respectively.
It was estimated to correspond to A-1 and LTA-2. BO
The IL-6 inducing activity of S40 and BOS60 also supported this assumption. BOS40 and BOS60 induced IL-6 to the same extent as the LTA-2 inducing activity when tested on human peripheral whole blood cells in the same manner as described previously (see Reference 13). (FIG. 1). Due to its high yield, BOS40 was used for further fractionation in this study.

Further fractionation of BOS40 was carried out by ion-exchange membrane chromatography on QMA-MemSep1010 in the same manner as described above (see Reference 13) except that the stepwise elution method was employed. 2 fractions, QM
-A and QM-I were obtained from the eluate with 0.01M acetate buffer containing 35% 1-propanol and the same buffer containing both 35% 1-propanol and 1M NaCl, respectively. IL-6-inducing activity was QM-
A, but not QM-I (FIG. 2).
This was in good agreement with the results of the previous study by the present inventors (see Reference 13).

QM-I eluted as a broad but single peak by gel permeation HPLC of its deacyl derivative.
Its molecular weight was estimated to be in the (standard heparin calibrated by the use of) 2 to 8 × 10 ³ or (with calibration by the use of standard dextran) 7-14 range of × 10 ^3. QM-I is also hydrophobic and anionically homogeneous, as determined from the elution profile in the corresponding chromatography. Therefore, QM-I
Can be regarded as a homogeneous high molecular weight sugar conjugate having the above molecular weight range. The chemical composition is summarized in Table I.

[0044]

Table I Chemical composition of QM-1 The molar ratio was calculated by assuming that 2 moles of fatty acids were present. Weight% (molar ratio) phosphate 11.5 (12.0) glycerol 18.7 (14.0) glucose 42.4 (17.3) fatty acid 7.6 (2.0) C16: 0 (0.7) C16: 1 (0.1) C18: 0 (0.2) C18: 1 (0.9) Alanine 2.3 (2.0)

The direct alkaline phosphatase digestion of QM-I did not detect free free phosphate, thus excluding the possible presence of the phosphomonoester. Thus, it was concluded that the phosphorus in this molecule was present in the form of a phosphodiester. The molar ratio of phosphorus to glycerol is approximately 1, indicating the presence of the poly (glycerophosphate) structure shown for LTA. To cleave phosphodiester bonds, which are thought to be abundant in this molecule, QM
-I digested by hydrofluoric acid (HF) hydrolysis,
The product was converted into chloroform / methanol / water (2/1 /
(3, volume ratio). T with solvent system A
LC analysis showed that 1% of the HF hydrolyzate was in the organic phase.
A major product of the species and some minor products were detected (FIG. 3). They were isolated by preparative TLC. Those F
AB-MS and ¹ H-NMR spectra revealed that this major product had a Glc ₂ acyl ₂ Gro structure and that minor products were lyso derivatives, diacylglycerols, and fatty acids.

One-dimensional ¹ H-NMR data of glycerol and glucose residues in Glc ₂ acyl ₂ Gro is shown in Table 1.
Summarized in II. The proton coupling constants at the 1st to 4th positions of both glucose residues showed a pyranosyl ring structure (see Reference 17). Chemical shifts of the anomeric protons (δ 4.98 and 4.95) and coupling constants ( ³
At 3.5 and 3.8 Hz as J _1.2 ), the α-anomeric configuration was confirmed (see Reference 18). For glycerol residues, the downfield shift of H-1 and H-2 compared to H-3 indicated that fatty acids were located at these 1 and 2 positions. QM- shown in FIG.
In the NOESY spectrum of I, between Glc ^nr H-1 and Glc ^r H-2, and Glc ^r H-1 and GroH-
An NOE between residues of 3 was observed (Glc ^nr represents non-reducing glucose, Glc ^r represents reducing glucose). Upon complete acetylation, no downfield shift of Glc ^r H-2 was observed in the ¹ H-NMR data (data not shown). From these results, this glycolipid structure is represented by Glc ^nr (α1-2) Glc ^r (α1
-3) It was determined to be acyl ₂ Gro. These NMR data were in good agreement with the data for the synthetic equivalents listed in Table II (see Reference 9). These results also demonstrate that the stereochemistry of this glycerol residue is the same as present in normal sugar glycerolipids.

[0047]

TABLE 2 Glycolipid Glc ₂ obtained by HF hydrolysis of QM-I
acyl ¹ for ₂ Gro and synthetic equivalents H-
NMR data. This spectrum, CDCl ₃ -CD ₃
Measured at 303K in OD (3: 1). Attribution ¹ H-
And ¹³ C-NMR one-dimensional spectrum method and DQF-C
The measurement was performed by a two-dimensional method of OSY, TOCSY and HMQC. Glc ^r and Glc ^nr represent reducing and non-reducing glucose residues, respectively. Data on synthetic glycolipids were obtained from reference (9). Glc ₂ acyl ₂ Gro Synthetic glycolipid proton Chemical shift ( ³ J _H , _H ) Chemical shift ( ³ J _H , _H ) δ (Hz) δ (Hz) Glycerol Gro H-1 4.20 (6.6, 12.2) 4.20 (6.4, 12.1) 4.43 (3.4, 12.2) 4.43 (3.0, 12.1) Gro H-2 5.23 5.23 Gro H-3 3.64 (5.1, 10.8) 3.64 (5.3, 10.6) 3.83 (5.3, 10.9) 3.83 (5.8, 10.6) Glucose Glc ^r H-1 4.98 (3.5) 4.98 (3.6) Glc ^r H-2 3.59 (3.5, 9.8) 3.58 (3.6, 9.6) Glc ^r H-3 3.78 (8.9, 9.8) 3.78 Glc ^r H-4 3.45 (8.9, 9.7) 3.44 (9.2, 9.9) Glc ^r H-5 3.57 3.57 Glc ^r H-6 3.78 3.7-3.8 Glc ^nr H-1 4.95 (3.8) 4.95 (3.8) Glc ^nr H-2 3.45 (3.9, 9.6) 3.44 ( 3.8, 9.5) Glc ^nr H-3 3.70 (9.0, 9.6) 3.69 (9.2, 9.5) Glc ^nr H-4 3.33 (9.1, 9.8) 3.34 (9.2, 9.5) Glc ^nr H-5 3.88 3.88 Glc ^nr H-6 3.69 (6.1, 12.2) 3.70 (6.2, 12.4) 3.86 3.86

The fatty acid ester in this glycolipid is mainly in a molar ratio of 0.63 / 0.23 / 0.10 / 1.00,
It consisted of hexadecanoate, hexadecenoate, octadecanoate, and octadecenoate. Cation mode F of this glycolipid moiety
AB-MS analysis showed the molecular ion peak shown in FIG. This molecular weight heterogeneity is explained by the substitution of different fatty acid combinations at positions 1 and 2 of glycerol. For example, the peak at m / z 942 corresponds to a glycolipid having one hexadecanoic acid and one octadecenoic acid, while the peak at m / z 968 contains two octadecenoic acids.

The product extracted from the HF hydrolyzate of QM-I into the aqueous phase was analyzed by ESI-MS. In the positive ion mode, m / z 277.2, 439.3, 60
1.2, 763.4, 925.4 and m / z 326.
Two sets of ion peaks of 2, 488.3, 650.4, and 812.4 were observed (FIG. 6). The former set is Glc _n G
ro (n = 1 to 5), the latter set being the protonated form of AlaGlc _n Gro (n
= 1 to 4). Negative ion mode ESI-MS showed the presence of compounds still retaining phosphate groups, so the mixture was further treated with alkaline phosphatase to remove residual phosphate. ESI-MS analysis of the resulting dephosphorylated product (positive ion mode)
Showed only the molecular ion peak of the sodium adduct of Glc _n Gro (n = 1 to 5), and the alanine substituent was no longer detected. Since the phosphatase treatment was performed under mildly alkaline conditions, the disappearance of the alanine residues suggests that they were linked to Glc _n Gro via an ester bond. When the dephosphorylated product was completely acetylated, five compounds were isolated by preparative TLC using solvent system B (analytical TLC).
7 is shown in FIG. 7).

These isolated compounds were analyzed by ¹ H-NMR
Based on the spectra, fully acetylated Gro,
Glc ^I (α1-2) Gro, Glc ^II (α1-2) G
lc ^I (α1-2) Gro, Glc ^III (α1-2) G
^{lc II (α1-2) Glc I (} α1-2) Gro, and ^{Glc IV (α1-2) Glc III (} α1-2) Glc II
(Α1-2) Glc ^I (α1-2) Gro was identified (glucosyl residues are numbered from those linked to glycerol toward the non-reducing end). E above
Despite being detected in SI-MS, Glc ₅ G
The presence of ro was not confirmed by TLC due to its low content.

The ¹ H-NMR data of these products are shown in Table II.
Put together in I. The coupling constants of the anomeric and other ring protons indicated that all glucose residues were in the α-glucopyranosyl configuration. GroH-2
Was not observed in the complete acetylation of Glc _n Gro, but this was due to Glc (α1-
2) Indicates Gro binding. Fully acetylated Gl
No downfield shift was observed for any of Glc ^I H-2 and Glc ^II H-2 of c ₃ Gro. This corresponds to Glc (α1-2) in Glc _n Gro.
This demonstrates Glc binding. The position of this interresidue bond was further confirmed by HMBC. Thus, the possible inter-residue relationships in fully acetylated Glc ₂ Gro are that between Glc ^II C-1 and Glc ^I H-2, Glc ^II
Between H-1 and Glc ^I C-2, Glc ^I C-1 and Gro
H2 and between Glc ^I H-1 and GroC-2.

[0052]

Table 3 Fully acetylated Gro, GlcGro, Glc ₂
Gro, Glc ₃ Gro, ¹ about Glc ₄ Gro
1 H-NMR data. The spectra were measured at 303K in CDCl _3. Assignment was performed by ¹ H- and ¹³ C-NMR one-dimensional spectroscopy, DQF-COSY, and HMQC. Glucosyl residues are numbered from those linked to glycerol toward the non-reducing end. Proton chemical shift δ (coupling constant, Hz) Gro GlcGro Glc ₂ Gro Gro H-1 4.16 (5.7, 11.9) 4.09 4.18 4.29 (4.1, 11.9) 4.27 4.28 Gro H-2 5.24 4.06 4.09 Gro H-3 4.16 (5.7 , 11.9) 4.09 4.21 4.29 (4.1, 11.9) 4.27 4.27 Glc ^I H-1 5.31 (3.9) 5.21 (3.7) Glc ^I H-2 4.84 (3.8, 10.3) 3.75 (3.6, 9.9) Glc ^I H-3 5.47 ( 9.6, 10.1) 5.45 (9.4, 9.8) Glc ^I H-4 5.06 (9.8, 9.9) 4.98 (9.2, 10.3) Glc ^I H-5 4.20 4.21 Glc ^I H-6 4.17 4.05 4.23 (4.6, 12.4) 4.29 Glc ^II H-1 5.16 (3.5) Glc ^II H-2 4.87 (3.5, 10.4) Glc ^II H-3 5.37 (9.5, 10.2) Glc ^II H-4 5.07 (9.5, 10.2) Glc ^II H-5 4.07 Glc ^II H- 6 4.16

[0053]

[Table 4] Table III (continued) Proton chemical shift δ (coupling constant, Hz) Glc ₃ Gro Glc ₄ Gro Gro H-1 4.18 4.09 4.38 4.43 Gro H-2 4.26 4.18 Gro H-3 4.17 4.24 4.33 4.30 Glc ^I H-1 5.33 (3.9) 5.34 (3.9) Glc ^I H-2 3.73 (3.9, 10.1) 3.93 (3.7, 9.9) Glc ^I H-3 5.45 (9.6, 9.6) 5.49 (9.4, 9.6) Glc ^I H-4 5.00 (9.6, 9.9) 5.04 (9.1, 9.4) Glc ^I H-5 4.26 4.27 Glc ^I H-6 4.06 4.11 Glc ^II H-1 5.04 (3.2) 5.05 (3.0) Glc ^II H-2 3.76 (3.2, 10.3) 3.70 (3.2, 10.3) Glc ^II H-3 5.40 (9.6, 9.9) 5.33 Glc ^II H-4 4.99 (9.6, 10.1) 5.00 (9.6, 9.9) Glc ^II H-5 4.16 4.04 Glc ^II H-6 4.07 4.18 Glc ^III H-1 5.10 (3.7) 5.20 (3.4) Glc ^III H-2 4.94 (3.7, 10.3) 3.76 (3.4, 9.9) Glc ^III H-3 5.32 (9.4, 10.3) 5.35 Glc ^III H-4 5.08 (9.4, 10.1) 5.01 (9.6, 10.0) Glc ^III H-5 4.11 4.08 Glc ^III H-6 4.18 4.17 Glc ^IV H-1 5.12 (3.9) Glc ^IV H-2 4.92 (3.6, 10.2) Glc ^IV H-3 5.31 Glc ^IV H-4 5.10 (9.6, 10.1) Glc ^IV H-5 4.00 Glc ^IV H-6 4.19

In order to elucidate the mode of attachment between the glycolipid and the Glc _n Gro moiety, the deacylated QM-I N
The MR spectrum was examined. FIG. 8 shows the ¹³ C-NMR spectrum. Δ6 with a coupling constant of about 5 Hz
The signals at 7.38 and 67.52 are assigned to GroC-1 and GroC-3. The coupling constants corresponding to the expected values for the downfield shift and ² J _c , _p indicate the phosphodiester linkages at the 1 and 3 positions of glycerol, so that the 1,3-phosphodiester linked poly (glycero The presence of a (phosphate) chain. ¹ H and ¹³ C-for the glycolipid region
The NMR data is summarized in Table IV. Their attribution is DEPT
(135 °), by TOCSY, NOESY, HMQC, and HMBC spectra and by reference to the reported data (see reference 16). Observed Glc ^nr C-6 (about 5 Hz) and Glc ^nr C-5
(Approximately 7 Hz), the coupling constants are ² J _c and _p , respectively.
And ³ J _c , _p , consistent with expected values. This clearly indicates a phosphodiester linkage at position 6 of the non-reducing glucose. From these results, it was revealed that this poly (glycerophosphate) chain was bonded to the 6th position of glucose on the non-reducing side of the glycolipid moiety.

[0055]

Table IV ¹ H- and ¹³ C-NMR data for glycerol and glucose residues in the glycolipid region within deacylated QM-I. The spectra were measured at 303K in D ₂ O. Assignments were made by ¹ H- and ¹³ C-NMR one-dimensional spectroscopy and DQF-COSY, TOCSY, NOESY, H
This was performed by referring to the MQC and HMBC, and the data in the literature (16). Glc ^r represents a reducing glucose residue, and Glc ^nr represents a non-reducing glucose residue. Position Chemical shift ^{(δ) 1 H 13 C Gro} 1 3.65 64.62 Gro 2 71.52 Gro 3 3.81 70.00 Glc r 1 5.11 97.15 Glc r 2 3.63 76.74 Glc r 3 3.76 72.55 Glc r 4 3.42 70.69 Glc r 5 3.54 72.55 Glc r 6 3.67 61.88 3.76 Glc ^nr 1 5.04 97.40 Glc ^nr 2 3.56 72.33 Glc ^nr 3 3.73 73.80 Glc ^nr 4 3.50 70.69 Glc ^nr 5 3.97 72.01 Glc ^nr 6 4.06 65.25

Taking all data into account, QM-I, the major component in BOS40 from E. hirae ATCC 9790, but IM in experiments using human peripheral whole blood cells
The chemical structure of the component having no L-6 inducing activity was determined to be as shown in FIG.

Example 2 TNF-Inducing Activity of QM-I QM-I was added to a THP-1 (human monocyte cell) culture system, and the TNF-inducing activity was examined in the presence and absence of serum. THP-1 was cultured in a medium (RPMI 1640), and QM-I or LPS was added at various concentrations to a TPH-1 culture solution having a cell number of 1 × 10 ⁶ / ml. This culture was prepared in the presence and absence of 1% fetal calf serum.
After further culturing at 37 ° C. for 24 hours in a CO ₂ incubator, the amount of TNF-α present in the culture supernatant was measured by ELISA. The results are shown in Table V.

[0058]

Table V Additives in the presence of serum QM-I LPS additive concentration (μg / ml) 0 0.1 1 10 0 0.01 0.1 1 TNF-α (pg / ml) 20 20 158 2262 20 144 768 1754 No serum Lower additive QM-I LPS additive concentration (μg / ml) 0 0.1 1 10 0 0.01 0.1 1 TNF-α (pg / ml) 20 20 1423 3326 20 20 20 20

The TNF-inducing activity of QM-I is suppressed in the presence of serum but becomes stronger in the absence of serum, contrary to the case of LPS.

Reference list: 1. Fischer, W. (1990) (Kates, M., ed.), Pp. 123.
-234, Plenum Press, New York. 2. Yamamoto, A., Usami, H., Nagamuta, M., Sugaw
ara, Y., Hamada, S., Yamamoto, T., Kato, K., Kokegu
chi, S. and Kotani, S. (1985), Br. J. Cancer 51, 7
39-742. 3. Yamamoto, A., Nagamuta, M., Usami, H., Sugaw
ara, Y., Watanabe, N., Niitsu, Y. and Urushizaki,
I. (1985), Immunol. Lett. 11, 83-88. 4. Usami, H., Yamamoto, A., Sugawara, Y., Hamad.
a, S., Yamamoto, T., Kato, K., Kokeguchi, S., Takad
a, H. and Kotani, S. (1987), Br. J. Cancer56, 797-
799.

5. Usami, H., Yamamoto, A., Yamashi
ta, W., Sugawara, Y., Hamada, S., Yamamoto, T., Ka
to, K., Kokeguchi, S., Ohokuni, H. and Kotani, S.
(1988), Br. J. Cancer 57, 70-73. 6. Tsutsui, O., Kokeguchi, S., Matsumura, T. an
d Kato, K. (1991), FEMS Microobiol. Immunol., 76, 2
11-218. 7. Bhakdi, S., Klonisch, T., Nuber, P. and Fisc
her, W. (1991), Intfect. Immun. 59, 4614-4620. 8. Kotani, S. (1992), Adv. Exp. Med. Biol. 319,
145-164.

9. Fukase, K., Matsumoto, T., Ito,
N., Yoshimura, T., Kotani, S. and Kusumoto, S. (199
2), Bull. Chem. Soc. Jpn. 65, 2643-2654. 10. Fukase, K., Yoshimura, T., Kotani, S. and Ku
sumoto, S. (1994), Bull. Chem. Soc. Jpn. 67, 473-48.
2. 11. Takada, H., Kawabata, Y., Arakaki, R., Kusum
oto, S., Fukase, K., Suda, Y., Yoshimura, T., Kokeg
uchi, S., Kato, K., Komuro, T., Tanaka, N., Saito,
M., Yoshida, T., Sato, M. and Kotani, S. (1995),
Infect. Immun. 63, 57-65. 12. Leopold, K. and Fischer, W. (1991), Eur. J.
Biochem. 196, 475-482.

13. Suda, Y., Tochio, H., Kawano,
K., Takada, H., Yoshida, T., Kotani, S. and Kusumo
to, S. (1995), FEMS Immunol. Med. Microbiol. 12, 9
7-112. 14. Hirano, T., Taga, T., Nakano, N., Yasukawa,
K., Kashiwamura, S., Shimizu, K., Nakajima, K., Pyu
n, KH and Kishimoto, T. (1985), Proc. Natl. Aca
d. Sci. USA 82,5490-5494. 15. Matsuda, T., Hirano, T. and Kishimoto T. (19
88), Eur. J. Immunol. 18, 951-956. 16. Kochanowski, B., Fischer, W., Iida-Tanaka,
N. and Ishizuka, I. (1993), Eur. J. Biochem. 214,
747-755.

17. Altona, C. and Haasnoot, CAG
(1980), Org. Magn. Reson. 13, 417-429. 18. Dabrowski, J., Hanfland, P. and Egge H. (198
2), Methods Enzymol. 83, 69-86. 19. Keller, R., Fischer, W., Keist, R. and Basse.
tti, S. (1992), Infect. Immun. 60, 3664-3672.

[0065]

The lipoteichoic acid of the present invention is a substance having a novel structure which has not been reported before. Moreover, the lipoteichoic acid of the present invention has TNF-inducing activity.

[Brief description of the drawings]

FIG. 1 shows IL-6 of BOS40 and BOS60.
Shows inducing activity. Sample doses are BOS40 and BO
100 and 10 μg / ml for S60;
1000, 100, and 10 pg / ml for PS
Met. Data are the mean of two experiments ± standard deviation.
The blood donor was JY.

FIG. 2 shows QM- from BOS40 and BOS60.
A and QM-I show IL-6-inducing activity. Sample doses are 10 and 1 μg for QM-A and QM-I
/ Ml for LPS and 1000, 100, and 10 pg / ml for LPS. Data are the mean of two experiments ±
Standard deviation. The blood donor was MH.

FIG. 3 shows a TLC analysis of the product extracted into the organic phase from the HF hydrolyzate of QM-I. TLC is a silica gel plate (Merck Keiselgel 60 F ₂₅₄ Art. 5
715) in solvent system A; chloroform / methanol / water (6
(5/25/4, volume ratio). The product was visualized with anisaldehyde-sulfuric acid reagent and recorded with an image scanner (ScanJet IIcx, Hewlett Packard).

FIG. 4 shows NOES of Glc ₂ acyl ₂ Gro.
2 shows a part of the Y spectrum. The spectrum was measured at 303K in chloroform / methanol (3: 1).
The mixing time was 650 minutes.

FIG. 5 shows FAB- of Glc ₂ acyl ₂ Gro.
2 shows MS analysis. m-Nitrobenzyl alcohol was used as the matrix.

FIG. 6 shows an ESI-MS analysis of the product extracted into the aqueous phase from the HF hydrolyzate of QM-I. 0.
1 mg / ml solution (20% 2-propanol in water)
Was continuously injected at 0.3 ml / h.

FIG. 7 shows a TLC analysis of the product extracted into the aqueous phase from the HF hydrolyzate of QM-I. TLC is a silica gel plate (Merck Keiselgel 60 F ₂₅₄ Art. 571).
5) Using solvent system B: chloroform / methanol / ethyl acetate (100/4/6, volume ratio). The product was visualized with anisaldehyde-sulfuric acid reagent and recorded with an image scanner (ScanJet IIcx, Hewlett Packard). Fully acetylated Gro not visualized by this method was detected with iodine vapor (data not shown).

FIG. 8 shows a ¹³ C-NMR spectrum of deacylated QM-I. The spectrum in D ₂ O 303
Measured in K. Attribution is DEPT (135 °), HMQ
C, HMBC, DQF-COSY, TOCSY, and N
Performed by OESY. Glc ^nr means non-reducing glucose in the glycolipid region.

FIG. 9 shows the structure of QM-I from BOS40. Some of the oligoglucosyl substituents on glycerol can be further substituted with alanine.

Claims (7)

[Claims] 1. The following structural formula (1): (Wherein, X independently represents H, Ala (Glc) _m or (Glc) _m , wherein m represents a natural number of 1 to 5; n represents a natural number of 5 to 42; and COR Is
(Indicating a fatty acid residue). 2. At least one X is Ala (Glc)
The lipoteichoic acid according to claim 1, wherein _m is _m . 3. The lipoteichoic acid according to claim 1, which is derived from the genus Enterococcus. 4. The lipoteichoic acid according to claim 1, which is derived from Enterococcus hirae. 5. An Enterococcus hirae ATCC 9790
The lipoteichoic acid according to claim 1, wherein the lipoteichoic acid is derived. 6. A method of culturing a lipoteichoic acid-producing bacterium, collecting the bacterium, subjecting the bacterium to a separation method utilizing hydrophobic interaction, and then subjecting the lipoteichoic acid-producing bacterium to a separation method utilizing a cation exchange action. The lipoteichoic acid according to any one of claims 1 to 5, wherein the lipoteichoic acid can be used. 7. The lipoteichoic acid according to claim 6, wherein a buffer containing a salt is used as an eluate in a separation method utilizing a cation exchange action.