Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1999-10-22
2003-04-29
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C536S023200, C536S023100, C435S193000, C435S252300, C435S320100
Reexamination Certificate
active
06555371
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a sialyltransferase and to a DNA encoding the same. More particularly, the present invention relates to an enzyme which synthesizes ganglioside G
M3
by transferring sialic acid to a galactose residue of lactosylceramide and to a DNA encoding the enzyme.
Human myelogenous leukemia cell line EL-60, which is a cell line that has acquired the ability of infinite proliferation as a result of tumorigenic transformation, is used generally and widely as a model for leukemia cells (Collins, S. J. Gallo, R. C., and Gallagher, R. E., Nature (London), 270, 347-349 (1977); Collins, S. J., Blood, 70, 1223 (1987)). The cell line does not differentiate even after continued cultivation and continues to proliferate while it remains as undifferentiated cells. However, when cultivation is continued with addition of phorbol ester, which is widely used as a differentiation inducer, the cell line stops the proliferation of cells and takes an appearance similar to that of monocytes or macrophages. This indicates that. differentiation has been induced. It has been reported that during this process, the amount of G
M3
, which is a kind of ganglioside, increases considerably (Nojiri, E., Takaku, F., Tetsuka, T., and Saito, M., Blood, 64, 534-541(1984)), and when the ganglioside G
M3
is added exogenously, the cell line shows the same change as that observed with the addition of phorbol ester, i.e., the cells undergo monocytic differentiation (Saito, M., Terui, Y., and Nojiri, H., Biochem. Biophys. Res. Commun., 132, 223-231 (1985)). Also, it has been proved that in this differentiation process, G
M3
itself has an activity of inducing differentiation (Nojiri, H., Takaku, F., Miura, Y., and Saito, M., Proc. Natl. Acad. Sci. U.S.A., 83, 782-786 (1986)), and that chemically synthesized G
M3
also induces differentiation (Sugimoto, M. and Ogawa, T., Glycoconj. J., 2, 5-9 (1985); Saito, M., Nojiri, H., Ogino, H., Yuo, A., Ogura, H., Itoh, M., Tomita, K., Ogawa, T., Nagai, Y., and Kitagawa, S., FEBS Lett., 271, 85-88 (1990)).
On the other hand, it has been elucidated that sialic acid-containing glycolipids, in particular ganglioside, bear important functions in various biological phenomena and not only its functions but also its biosynthesis are being clarified. In vertebrates, many gangliosides (ganglio-series gangliosides) have a common precursor, G
M3
, which has the simplest structure among major gangliosides and the G
M3
synthesis affords a basis for the biosynthesis of gangliosides which have major functions.
As described above, ganglioside G
M3
itself participates in the proliferation/differentiation of cells and tissues and it is suggested that the ganglioside G
M3
is a precursor for a group of higher gangliosides having various functions in vertebrates.
G
M3
has been considered to be synthesized from lactosylceramide by transfer of sialic acid to the galactose residue in lactosylceramide by CMP-sialic acid:lactosylceramide sialyltransferase (CMP-NeuAc: Gal&bgr;1-4Glc&bgr;1-1′Cer&agr;2,3-sialyltransferase; SAT-I). However, neither the transferase from mouse and human has been isolated nor the genes thereof have been identified.
Enzymes which transfer sialic acid through an &agr;2-3 ketoside bond are described in, for example, Wienstein et al., J. Biol. Chem., 257, 13835 (1982); Gillespie et al., Glycoconj., 7, 469 (1990); Gillespie, W., Kelm, S. and Paulson, J C., J. Biol. Chem., 267, p21001-21010 (1992); Lee, Y C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hashimoto, T. and Tsuji, S., J. Biol. Chem., 269, p10028-10033 (1994); Kim. Y J., Kim, K S., Kim, S H., Kim, C H., K o, J H., Choe, I S., Tsuji, S. and Lee, Y C., Biochem. Biophys. Res. Commun., 228, p324-327 (1996); and JP-A 5-336963. However, none of the enzymes is known to be involved in the synthesis of G
M3
or shows an enzyme activity of transferring sialic acid to lactosylceramide through an &agr;2-3 ketoside bond. Sandhoff, K. et al. presume that &agr;2-3 sialyltransferase (SAT4) is identical with the enzyme which synthesizes G
M3
(J. Biol. Chem., 268, 5341 (1993)). However, this is a presumption based on an indirect method, which fails to support that the enzymes are identical to each other as a substance.
In spite of various attempts which have been made in order to elucidate and control its biosynthesis according as the clarification of importance of ganglioside G
M3
proceeds, the above-mentioned sialyltransferase, which relates closely to the synthesis of G
M3
, has not been isolated yet from mouse and human because of difficulty in preparing the enzyme protein and, hence, neither its gene expression control mechanism has been clarified yet nor its proteo-chemical or enzymological analysis has been performed successfully.
SUMMARY OF THE INVENTION
As a result of intensive investigation with view to elucidating the control mechanism of cell differentiation by carrying forward studies on gene expression control mechanism of and proteo-chemical and enzymological analyses of the above-mentioned sialyltransferase, the present inventors have-been successful in isolating cDNA having a nucleotide sequence encoding the sialyltransferase which participates in the above-mentioned G
M3
synthesis from mouse and human, by using an expression cloning method and based on the nucleotide-sequence, they have clarified the structure of the above-mentioned sialyltransferase. As a result, it revealed that the enzyme is low in homology with the known sialyltransferase and is believed to be a new enzyme, differing from the &agr;2-8 sialyltransferase, with which the identity was presumed by Sandhoff, K. supra.
Accordingly, the present invention provides a sialyltransferase having the following properties and a DNA having a nucleotide sequence encoding it.
(1) Activity:
The sialyltransferase transfers sialic acid from a sialic acid donor selectively to a 3-hydroxyl group of a galactose residue contained in lactosylceramide as a sialic acid acceptor to produce ganglioside G
M3
.
(2) Optimal Reaction pH;
6.0 to 7.0.
(3) Activation:
The activity increases at least 1.5 times with 10 mm of Mn
2+
as compared with the case in the absence thereof.
Also, the present invention provides a sialyltransferase having the above-mentioned activity and having a C-terminal amino acid sequence shown by SEQ ID NO: 5 and a DNA encoding it as well as a sialyltransferase having the above-mentioned activity and having an amino acid sequence shown by SEQ ID NO: 6 or 11 and/or 12 and a DNA encoding it.
The sialic acid donor is preferably cytidine 5-monophosphsate-sialic acid (CMP-sialic acid).
The above-mentioned enzymes and DNAs are preferably those derived from a mammal, most preferably those derived from human.
The present invention also provides a sialyltransferase comprising the polypeptide (a) or the polypeptide (b) below and a DNA encoding it
(a) A polypeptide having an amino sequence shown by SEQ ID NO: 2 or 8.
(b) A polypeptide having an amino acid sequence (a) above, which has therein substitution, deletion, insertion or rearrangement of one or a few amino acid residues, said sialyltransferase having an enzyme activity of transferring sialic acid from a sialic acid donor selectively to the 3-hydroxyl group of galactose residue contained in lactosylceramide as a sialic acid acceptor to produce ganglioside G
M3
.
Specific examples of the DNA of the present invention include a DNA having a nucleotide sequence encoding all the amino acid sequence shown by SEQ ID NO: 2 or 8, or a DNA having partial sequences thereof, for example, DNA having a nucleotide sequence shown by SEQ ID NO: 1 or 7.
Further, the present invention provides a polypeptide comprising all or part of the polypeptide of sialyltransferase encoded by the nucleotide sequence of the above-mentioned DNA. From the polypeptide, a transmembrane domain may be deleted.
In addition, the present invention provides a recombinant vector comprising the DNA of the present invention; a transformant into which the DNA of the present invention is in
Knobbe Martens Olson & Bear LLP
Prouty Rebecca E.
Seikagaku Kogyo Kabushiki Kaisha
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