Process for producing asialo GM1

Details Process for producing asialo GM1 Process for producing asialo GM1

1991-06-28

1994-01-04

Rollins, John W.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435 74, 435101, 424 946, 424 9461, 536 41, 536 53, 536 553, 536124, C12P 1926, C12P 1944, C07H 500

Patent

active

052759393

DESCRIPTION:

BRIEF SUMMARY
Technical Field

This invention relates to a process for producing asialo G.sub.M1.
2. Prior Art
"Ganglioside" is a general name of the group of sialic acid-containing sphingoglycolipids and is known to exist in the brain of higher animals, especially in the nervous system. In the living body, the gangliosides not only take part in nervous function and recognition, differentiation, proliferation, canceration, senescence, etc. of cells, but also, from a cell sociological view point, are responsible for receptor function of cytotoxins such as cholera toxin and botulinum toxin, peptide hormones such as thyroid hormone, interferons and so on. The gangliosides are also supposed to contribute to negative charges on the surface of cells. In fact, it is reported that the gangliosides extracted from bovine brain promote regeneration of damaged peripheral nerve and useful for treatment of alcoholic polyneuritis, diabetic neuropathy, etc (Iyaku to Yakugaku, 13(6), pp1407-1412 (1985); Practice, 4(4), pp 452-456 (1987); Iyaku Journal 22(7), pp 55-62 (1986)).
As described above, gangliosides are important substances having various functions in the living body. Therefore, it is desired to establish a process for collecting each of various gangliosides in a pure form. In order to prepare a specific ganglioside, conventionally attempted are methods using various kinds of chromatography. However, since the starting material, i.e., ganglioside components as extracted from bovine brain or the like is a ganglioside mixture containing various gangliosides each in trace amounts, it is difficult to obtain the desired products even by conducting cumbersome procedures, and any proper purification methods have not been found yet. Further, although an attempt is made to synthesize gangliosides by chemical technique, its process is complicated and side reactions are likely to occur, entailing difficulty in removing by-products.
On the other hand, neuraminidase is an enzyme which has been considered not to act directly on the gangliosides on its own. As a matter of fact, it is only reported that asialoganglioside G.sub.Al is formed from ganglioside G.sub.M1 in the presence of detergents such as cholates (Masaki Saito, Taisha, 16, p761 (1977)). Moreover, it has been unknown at all that any isozyme of the neuraminidase exists.


DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a process for collecting a specific ganglioside, i.e., asialo G.sub.M1 from gangliosides.
Another object of the present invention is to provide a process for producing asialo G.sub.M1 in high purity and in good yield.
Namely, the present invention provides a process for producing asialo G.sub.M1 characterized in that neuraminidase isozyme L is allowed to act on gangliosides to obtain asialo G.sub.M1.
The present inventors have conducted intensive research and consequently found that the neuraminidase derived from the bacteria of the genus Arthrobacter has a novel isozyme and that, when said neuraminidase isozyme L is allowed to act on gangliosides, asialo G.sub.M1 can be selectively obtained with high purity and in good yield. According to the process of the present invention, by-products are not produced, and therefore asialo G.sub.M1 obtained can be readily purified. Further, the present invention has the advantage that asialo G.sub.M1 can be prepared using said enzyme in the absence of any detergent which was used in the conventional process.
The neuraminidase isozyme L (hereinafter referred to as "Isozyme L") to be used in the present invention has the following physicochemical properties :
The molecular weight was determined according to gel filtration chromatography and SDS-PAGE electrophoresis as described below.


Gel filtration chromatograph

Gel filtration method was done using Sephadex G150 (product of Pharmacia). As the eluent, 50 mM phosphate buffer (pH 6.8) was used.


SDS-PAGE electrophoresis

SDS-polyacrylamide gel electrophoresis was conducted according to the method of U. K. Laemmli (Nature, 227, 680 (1970)).
(3) Optim

REFERENCES:
patent: 4071408 (1978-01-01), Flashner et al.
patent: 5116752 (1992-05-01), Sugimori et al.
Sugano et al.; FEBS Lett. 89(2):321-325 (1978).
Saito et al.; J. Biol. Chem. 254(16):7845-7854 (1979).
Suzuki et al.; Biochim. Biophys. Acta 619:632-639 (1980).
Macher et al.; J. Biol. Chem. 256(4):1968-1974 (1981).
Sander-Weiner et al.; Chemical Abstracts 98:49369q (1983).
Saito et al.; Anal. Biochem. 148:54-58 (1985).
Wieraszko et al.; Chemical Abstracts 104:204605b (1986); 107:151908f (1987).
Sonnino et al.; Chemical Abstracts 108:204912q (1988).
Genetics, vol. 114, No. 1, (1986), pp. 247-258, published in 1986 and authored by P. B. Samollow et al.
Chemical Abstracts, vol. 103, No. 11, (Sep. 16, 1985) p. 320, Abstract No. 84523d, published in U.S.A. and authored by Saito Megumi et al.
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Journal of Biological Chemistry, vol. 254, No. 16 (1979) pp. 7845-7854, 1979 by M. Saito et al.
Journal of Biochemistry, vol. 82, No. 5 (1977), pp. 1425-1433, 1977, by Y. Uchida et al.

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