JPS6245601A - New ganglioside - Google Patents

Abstract

NEW MATERIAL:A ganglioside of the formula (wherein at least one of R1 and R2 is either a saccharide chain having a Gal-GlcNAc unit and a sialic acid residue at a nonreducing terminal or a sialic acid residue and the other is either a glucose chain having a Gal-GlcNAc unit or H, Glc is a glucose residue, Gal is a galactose residue, GlcNAc is an N-acetylglucosamine residue and Cer is ceramide). EXAMPLE:A ganglioside of the formula (wherein R1 is H and R2 is a sialic acid residue). USE:A reagent for medical or biochemical studies. PREPARATION:Fat-globule membranes extracted from butter milk are separated and purified.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to a method that is useful as a pharmaceutical and biochemical research reagent.
It concerns a new ganglioside.

Conventional technology has shown that glycosphingolipids, which exist on the surface of mammalian biological membranes, are composed of hydrophobic ceramide and hydrophilic carbohydrates, among which sialic acid is bound to the end of the carbohydrate chain. The glycolipids that exist are called gangliosides.

There have already been many studies on the role of gangliosides in living organisms, including bacterial toxins (toxins such as cholera, tetanus, populinus, Vibrio parahaemolyticus, staphylococcus, Escherichia coli and Bacillus phosphorus), thyroid stimulating hormone, luteinizing hormone,
In addition, it acts as a receptor molecule for hair gonadal m1m hormone, lymphokines (interferon, macrophage migration inhibitory factor, interleukin, etc.), fibronectin, Sendai virus, influenza virus, serotonin, noradrenaline, histamine, dopamine, fluhiscein, lectin, etc. , mutual identification of cells,
It is thought to play an important role in the physiological functions of cells, such as division, proliferation, differentiation, and immunity.

Gangliosides are not only found in mammalian tissues, but also exist in milk, particularly in high concentrations in fat globule capsules.

As a result of many previous studies on gangliosides in mammalian tissues and milk, several gangliosides were isolated and their molecular structures confirmed, and as a result, the sugar chains in gangliosides were similar to those in other glycolipids. Like a chain,
It has been revealed that there are both linear and branched chains. However, in all gangliosides that have been isolated and confirmed, even those with branched sugar chains, branching occurs between the sugar residue that is 4th or more distant from the ceramide at the end of the molecule and the next sugar residue. and
No gangliosides with branches closer to ceramide have been found.

Structure of the Invention Meanwhile, the present inventors separated and purified a large number of gangliosides from fat globule membranes collected from buttermilk, and as a result of detailed investigation of their structures, discovered a novel ganglioside characterized by a branched sugar chain structure. Based on this discovery, the present invention as detailed below has been completed.

That is, the present invention provides a novel ganglioside whose main feature is that it has a branch between the second sugar residue and the next sugar residue from the ceramide as described below.

(In the formula, R; and R2 represent a sugar chain having a Gal-GlcN Ac unit, a sugar chain having a Gal-GlcN Ac unit and a sialic acid residue at the non-reducing end, a sialic acid residue, or a hydrogen atom. In addition, Glc represents a glucose residue, Gal represents a lactose residue, and G 16N A
c represents N-7 cetylglucosamine residue, and Cer represents ceramide. However, at least one of R3 and R2 is a sugar chain having the above sialic acid residue or a sialic acid residue.

In the above ganglioside of the present invention, R1 and R2
is a sugar chain having a Ga1-GlcNAc unit (one that does not have a sialic acid residue at the non-reducing end), the number of sugar units that the sugar chain has at the chain end or in the chain is not limited, but for example, 7 It is estimated that substances having terminal sugar units such as , amisheptasaccharide, etc. exist in nature. Moreover, the sialic acid residues may be bonded to the end of the sugar chain instead of singly or in the form of a chain in which two or more of them are bonded.

In physiologically active substances with sugar chains such as glycolipids and glycoproteins, the presence or absence of sugar chain branches, the position and number of branch branches are closely related to the physiological effects of the substance, and there are many cases where branching strengthens the physiological effects. This is well known. Therefore, it is fully expected that the novel ganglioside according to the present invention, due to its unique branched structure, will have physiological effects that are more pronounced or qualitatively different than those of previously confirmed gangliosides. Therefore, it is expected that it will be used as a medicine or a diagnostic agent, and it is clear that it can at least serve as a new reagent useful in the study of many biological phenomena.

Specific examples of its uses are listed below.

■ Usefulness as a cancer antigen Monoclonal antibodies against milk fat globule membranes have already been produced, and it has been suggested that some fat globule membrane components may serve as antigens for certain cancers. The ganglioside according to the present invention is isolated from the fat globule membrane of milk and is therefore derived from mammary gland cells, and is not found at all in other cell tissues, so it is thought that it is specifically produced by mammary gland cells. Therefore, moaclonal antibodies against gangliosides with such strong specificity are likely to be effective in the diagnosis of metastatic cancer and the like and in the treatment by missile therapy. In addition, some monoclonal antibodies that have already been developed for breast cancer diagnosis may react with the gangliosides of the present invention, and those that are reactive may be linked to the expression of cancer-related sugar chains in cancer cells and antigens. It is useful as a reagent for analyzing sexual changes.

■Usefulness for newborns Gangliosides show remarkable changes as cells differentiate and proliferate, and are thought to play an important role in mutual cell recognition. In fact, it has been reported that the induction of differentiation of multipotent myeloblastoid cells and the proliferation and process formation of adrenal gland-derived neuroblastoma are promoted by the addition of gangliosides.

Since the ganglioside according to the present invention is originally pore-specific, it is likely to have important effects such as growth of the neonatal intestinal tract and involvement in nervous system tissue, and applications that utilize such effects are expected. Ru.

■Usefulness as a myxovirus inactivating substance Gangliosides have recently attracted attention as receptors for myxoviruses. To date, various types of gangliosides with receptor functions have been identified, among which the paragloboside structure (Galβ1→4
GlcNAcβ1-3Galβ1→4Gldβ1-Ce
r), those in which the bond between galactose and sialic acid at the terminal is an α2-6 bond, and branched types.
It is said to have strong receptor functions. The ganglioside of the present invention isolated from milk has all of these characteristics and is likely to act as a potent myxovirus receptor. Therefore, if this ganglioside acts before the virus is adsorbed to cells, it is possible to prevent viral infection and is considered to be effective in preventing influenza and other diseases.

Among the gangliosides according to the present invention, those in which R7 in the above general formula is a hydrogen atom and R2 is a sialic acid residue, and those in which R1 and R7 are both sialic acid residues, can be prepared from milk according to the method of the example below. It can be separated from the fat globule membrane. Using these gangliosides as starting materials, any glycosyltransferase such as sialyltransferase, 7fusyltrans7erase, galactosyltransferase, N-7 cetylgalactosaminyltransferase, and N-a7tylglucosaminyltransferase is used. Many of the gangliosides belonging to the present invention can be easily produced by modifying the sugar chain moiety through an enzymatic reaction.

Example The operation of adding about three times the amount of A7Tone to buttermilk Balagu, stirring thoroughly, and then filtering to remove glyceride was repeated three times, and the residue was mixed with chloroform-methanol-water (20-:10:1). ) mixture and chloroform-methanol (1:1) mixture in 10 times the volume,
Extract 11 lipids at 45°C.

After adjusting the chloroform concentration and methanol concentration of the extract to 2:1, 115 volumes of a 0.74% aqueous potassium chloride solution were added to the extract to perform 7-fold distribution. Take the upper layer, concentrate it, dissolve it in water, dialyze it, concentrate it, add chloroform-methanol (1:1), and then add it to a DEA E-Sephalometric column (acetic acid form). . Elution is 0.05
M, 0.15M #, J: Perform a gradient using 0.45M ammonium acetate-methanol, and collect the monosialoganglioside fraction.

The obtained bunglioside fraction was further adsorbed on an Iatrobeads column and treated with taloloform-methanol-water (
Elute with a gradient method using a 55:45:, 1) mixture and a chloroform-methanol-water (10:80:4) mixture. By repeating this operation, i) a new ganglioside which shows a single band in thin layer chromatography and which appears at a different position from any of the known gangliosides (close to each spot of the known gangliosides GD1a and GDlb) can be obtained; Collect both portions (hereinafter referred to as “Ryobu A”) that are thought to contain them.

Fraction A obtained as described above was analyzed as follows to confirm its structure.

■ Analysis of sugar-binding sites by partial menalization and alcitol acetation Fraction A was analyzed by Hakomori's method (J, Biochem, 55.2).
05) to completely methylate the free hydroxyl groups, followed by hydrolysis, followed by Sterner's method (Arch,
B iochem, B 1ophys, 155
.

464) with sodium borohydride to obtain partially methylated alditol, which is then acetylated after adding acetic anhydride to obtain partially methylated alditol acetate. Of these, partially methylated alditol acetate of neutral sugar is 3% 0V-275on chromos
orb W (100-200 mesh award Applie
Gask t7? at 170'C by d 5science Lab, ) Perform Togelahui. On the other hand, partially methylated alditol acetate of amine sugar is ○V-17on
celite 545 (10o-200 mesh yA
pplied 5science Lab, ) by 19
Gas chromatography is performed at 0'C. Thereafter, each peak component is identified by gas chromatography-Maku spectrometer.

The retention time of gas chromatography and comparison with the mass spectrogram in Biochemistry Handbook ■, pages 641-650 indicate that components a to e separated by gas chromatography have the following sugar residues. (The numbers in parentheses are their relative molar ratios).

a Galβ], -(0,9) b-4Glcβ1-(1,0) c-6Galβ1-(0,9) e →4GIcNAcβ1→ (1,7) ■ Complete analysis of sialic acid residues by methylation analysis method The methylated gangliosides of fraction A are subjected to metatolysis with 0.3 M hydrochloric acid and methanol at 80° C. for 18 hours, and after removing the liberated fatty acid methyl ester with hexane, the methanol layer is dried and trimethylsilylated. The obtained trimethylsilylated product was heated at 2% OV -101 on Chr.
Perform 1.200°c'r, v chromatography using ice. As a control, fully methylated N-acetylneuraminic acid and fully methylated N-glycolylphilaminic acid are also subjected to gas chromatography under the same conditions. In these gas chromatographies, the retention time of trimethylsilylate from fraction A was 5
The retention time of fully methylated N-7 cetyl filaminic acid was 5.13 minutes (5.13 minutes).
The retention time of glycolylneuraminic acid was 8.06 minutes). As a result, it was confirmed that the sialic acid in the ganglioside of Ryobu A was of the N-7 cetyl firamic acid type.

■ The gangliosides in fraction A are sequentially treated with β-galactosiguse 2, filaminiguse 3, and N-acetyl-β-D-glufusaminiguse, and the products at each treatment stage are examined by thin layer chromatography. .

As a result of the above enzyme treatment and because →3Galβ1 can be detected by the complete methylation method of this product, the final product is paragloboside (Galβ1→4GleNAcβ1→
It was found that 3Galβ1−=4Glcβ1→Cer).

In addition, N-7 cetylneuraminic acid is was found to bind to the 3 side chain of

From the above analysis results, it was concluded that the ganglioside of fraction A is a novel ganglioside having the following structure.

(However, R3 represents an N-7 cetyl/iraminic acid residue.) Furthermore, as a result of conducting the same analysis as above on another ganglioside fraction collected in the separation process in which the above-mentioned fraction A was collected, it was found that the above-mentioned new ganglioside The existence of a new ganglioside in which one N-acetylneuraminic acid residue is further bonded to the end of the sugar chain and a new ganglioside in which two N-acetylneuraminic acid residues are bonded to the end of the sugar chain was confirmed. Ta.

Effects of the Invention The ganglioside according to the present invention is a ganglioside with a structure completely unknown in the past, in which a branch occurs between the second sugar residue and the third sugar residue from Cerami V. Therefore, it is expected that the present invention will enable the provision of new reagents useful for biochemical research, and will also enable the development of many pharmaceuticals and diagnostic agents.

Claims (3)

[Claims] (1) General formula ▲ Numerical formulas, chemical formulas, tables, etc. chain, a sialic acid residue, or a hydrogen atom.Glc also represents a glucose residue,
Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, and Cer represents a ceramide. However, at least one of R_1 and R_2 is a sugar chain having the above sialic acid residue or a sialic acid residue. ) A novel ganglioside. (2) The ganglioside according to claim 1, wherein R_1 is a hydrogen atom and R_2 is a sialic acid residue. (3) The ganglioside according to claim 1, wherein R_1 and R_2 are both sialic acid residues.