Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-08-21
2004-08-31
Prouty, Rebecca (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S320100, C435S325000, C435S252300, C536S023200
Reexamination Certificate
active
06783966
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the production of a polypeptide using a recombinant DNA, or to a tool useful for diagnosis or treatment of diseases, or more specifically to &agr;1,4-galactosyltransferase and DNA encoding thereof, a recombination vector containing the DNA, and a transformed cell transfected with the DNA or by the recombination vector, and to a method for producing Gb3/CD77 or globo-series glycolipids by using the transformed cell.
2. Description of the Related Art
Glycosphingolipids are amphipathic molecules(ref.1) that are synthesized by sequential actions of glycosyltransferases(ref.2). Addition of one of three different sugars onto lactosylceramide (which may be termed “LacCer” hereinafter) results in the synthesis of either one of three major glycolipid series, i.e., ganglioside-series (&agr;2,3-sialic acid), lacto
eolacto-series (&bgr;1,4-N-acetylglucosamine) and globo-series (&agr;1,4-galactose). Although a number of genes coding for enzymes responsible for the synthesis of the carbohydrate moiety of glycosphingolipids have been recently isolated(ref.3), no glycosyltransferase genes specific for the synthesis of globo-series glycolipids have been isolated to date.
Globotriaosylceramide (hereinafter sometimes referred to as “Gb3”) is synthesized by &agr;1,4-galactosyltransferase (&agr;1,4 Gal-T) from LacCer(ref.4). This glycolipid has been characterized on red blood cells as the P
k
antigen of the P blood group system(ref.5). Since Wiels et al(ref.6) reported that Gb3 was a Burkitt's lymphoma associated antigen, the expression and biological significance of Gb3 have been vigorously studied(ref.7, 8 and 9). Since Gb3 was clustered as CD77, this antigen will be referred to as Gb3/CD77.
Gb3/CD77 was reported to be expressed in high amounts on Burkitt's lymphoma cells. However, it is now considered to be a differentiation antigen expressed on B cells, and can also be found in some malignant tumors of B cell lineage(ref.7). Among normal leukocytes, it is only expressed on a subset of tonsillar B cells in the germinal centers (GC)(ref.9). Interestingly, GC B lymphocytes expressing Gb3/CD77 undergo rapid and spontaneous apoptosis when isolated and cultured in vitro(ref.11). Furthermore, Burkitt's lymphoma cells with Gb3/CD77 antigen were also easily induced to enter apoptosis upon culture at low serum concentration or cross-linking by anti-immunoglobulin M antibodies(ref.12).
Gb3/CD77 has been recognized as a receptor for verotoxins (VTs), the Shiga-like toxin from
E. coli
O157 strain that can trigger serious cytotoxic effects(ref.13 and 14). VT B-subunit specifically binds to Gb3/CD77, then A subunit is incorporated into cells, resulting in the degradation of 28 S ribosomal RNA and cell death(ref.15). However, only B-subunit is also able to induce apoptosis when cross-linked(ref.16). These results indicate that Gb3/CD77 is a critical molecule in mediating apoptosis signals, although the precise mechanisms remain to be investigated.
As stated above, it was revealed that Gb3/CD77, a globo-series glycolipid, is syntheized by addition of &agr;1,4-galactose to LacCer(ref.4), but the glycosyltransferase specific for this synethetic reaction has not been isolated yet. An object of the present invention is to isolate a Gb3/CD77 synthase, that is, &agr;1,4-galactosyltransferase, and DNA encoding thereof, and to provide the use thereof.
The present inventors have studied hard to achieve the above objects, and succeeded in isolating DNA encoding &agr;1,4-galactosyltransferase, revealing its nucleotide sequence, and confirming that the DNA is responsible for the expression of active &agr;1,4-galactosyltransferase. Thus, they achieved the present invention.
SUMMARY OF THE INVENTION
The present invention provides a polypeptide of (a) or (b) below (hereinafter sometimes referred to as “the polypeptide of the present invention”):
(a) a polypeptide consisting of an amino acid sequence represented by the amino acid Nos. 46-353 in SEQ ID NO: 2; or
(b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a galactose residue from a galactose donor to C4 position of galactose residue of lactosylceramide or galactosylceramide which serves as an acceptor.
The polypeptide of the present invention also includes a polypeptide of(a′) or (b′) below:
(a′) a polypeptide consisting of an amino acid sequence represented by the amino acid Nos. 20-353 in SEQ ID NO:2; or
(b′) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a′) and which has an enzymatic activity to transfer a galactose residue from a galactose donor to C4 position of galactose residue of lactosylceramide or galactosylceramide which serves as an acceptor.
The polypeptide of the present invention also includes a polypeptide of (a″) or (b″) below:
(a″) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO:2; or
(b″) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a″) and which has an enzymatic activity to transfer a galactose residue from a galactose donor to C4 position of galactose residue of lactosylceramide or galactosylceramide which serves as an acceptor.
The present invention also provides a DNA encoding the polypeptides according to any one of above polypeptides(hereinafter sometimes referred to as “the DNA of the present invention”). The DNA of the present invention includes a DNA represented by (a) or (b) below:
(a) a DNA comprising a nucleotide sequence represented by nucleotide Nos. 269 to 1192 in SEQ ID NO:1; or
(b) a DNA hybridizable with a DNA comprising a nucleotide sequence represented by SEQ ID NO:1, a nucleotide sequence complimentary to SEQ ID NO:1, or a part of those sequences, under a stringent condition.
The DNA of the present invention also includes a DNA encoding a polypeptide having an enzymatic activity to transfer a galactose residue from a galactose donor to C4 position of galactose residue of lactosylceramide or galactosylceramide which serves as an acceptor.
The present invention still further provides a recombination vector containing the DNA of the present invention.
The present invention still further provides a transformed cell obtained by transfecting a host cell with the DNA of the present invention or the recombination vector.
The present invention still further provides a method for producing the polypeptide of the present invention, comprising the steps of:
producing the polypeptide of the present invention by culturing the transformed cell above in a medium; and
recovering said polypeptide from the medium and/or a cell extract of the cultured transformed cell.
10. The present invention still further provides a method for producing Gb/CD77 comprising the steps of:
exposing the polypeptide according to any one of claims 1 to 3, or a cultured product of the transformed cell according to claim 8, to lactosylceramide, to cause thereby enzymatic reaction; and recovering Gb3/CD77.
The present invention still further provides a method for producing a glycolipid as represented by the following formula (1) comprising the steps of:
exposing the polypeptide of the present invention, or a cultured product of the transformed cell above, to galactosylceramide, to cause thereby enzymatic reaction; and
recovering the glycolipid represented by the following formula (1):
Gal&agr;1→4Gal-Cer (1)
wherein Gal represents a galactose residue, Cer represents a ceramide residue and &agr;1→4 represents an &agr;1-4 glycosidic linkage.
In the present invention, the enzyme having an activity to transfer a galactose residue from a galactose do
Fukumoto Satoshi
Furukawa Keiko
Furukawa Koichi
Kojima Yoshinao
Okajima Tetsuya
Prouty Rebecca
Seikagaku Corporation
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