Core 2 &bgr;-1,6-N-acetylglycosaminyltransferase gene

Details Core 2 &bgr;-1,6-N-acetylglycosaminyltransferase gene Core 2 &bgr;-1,6-N-acetylglycosaminyltransferase gene

2001-02-28

2003-07-15

Prouty, Rebecca E. (Department: 1657)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S015000, C435S320100, C435S252300, C435S325000, C435S069100, C536S023200

Reexamination Certificate

active

06593119

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to novel core 2 &bgr;-1,6-N-acetylglycosaminyltransferase nucleic acid molecules, polypeptides encoded by such nucleic acid molecules, and uses of the nucleic acid molecules and polypeptides.
BACKGROUND OF THE INVENTION
The enzyme UDP-GlcNAc:Gal[&bgr;] 1,3GalNAc-R (GlcNAc to GalNAc) [&bgr;] 1,6-N-acetylglucosaminyltransferase (i.e. core 2 &bgr;-1,6-N-acetylglycosaminyltransferase) converts core 1 (i.e. Gal[&bgr;]1,3GalNAc[&agr;]-O) to core 2 structures (i.e. Gal[&bgr;]1,3[GlcNAc[&bgr;]1,6]GalNAc[&agr;]-O in the O-linked glycan biosynthesis pathway (Williams and Schachter, 1980 J. Biol. Chem 255:11247, 1980 and Schachter H. and Brockhausen, I, In: Allen,, H. J. and Kisailus, E. C. (eds) Glycoconjugates. Composition, Structure, and Function. Marcel Dekker, New York, pp 263-332). Core 2 GlcNAc-T activity is important in the extension of O-linked sugars with poly(N-acetyllactosamine) (i.e. repeating Gal [&bgr;] 1-4GlcNAc [&bgr;] 1-3). These structures have been associated with malignant transformation (Yousefi et al, 1991) and proliferative activation of lymphocytes (Higgins et al, 1991), they affect cellular adhesion (Zhu and Laine, 1985; Laferte and Dennis, 1988), and they may act as ligands for mammalian lectins (Merkle and Cummings, 1988)
Synthesis of branched, complex core 2-based O-linked structures has been found to be controlled by the relative levels of core 2 GlcNAc-T and [&agr;]-2,3 sialyl-T (Whitehouse et al, 1997) which compete for the same core 1 acceptor substrate. Therefore, core 2 is a key enzyme in the modulation of cell—cell interactions through glycosylation of target molecules. For example, glycosylation of PSGL-1 modulated by core 2 GlcNAc-T has been found to be a critical step for binding to P-selectin (Kumar et al, 1996; Li et al, 1996).
Expression of Core 2 GlcNAc-T in diabetic heart has also been associated with a stress-response and myocardial hypertrophy (Nishio Y. et al, J. Clin Invest 1995 October; 96(4): 1759-67). Diabetes and hyperglycemia induces core 2 GlcNAc-T gene expression specifically in cardiac myocytes of rats.
GalNAc&agr;R prevents core 2 synthesis by blocking one enzyme earlier in the O-linked pathway, and it reduces invasion and metastasis. A somatic mutation that prevents UDP-Gal transport into the Golgi blocking O- and N-linked extensions including core 2 structures causes a more severe attenuation of metastasis than a block in either pathway alone, suggesting both O-linked core 2 and N-linked branched oligosaccharides contribute to the malignant phenotype. Most recently, it was demonstrated that an increased expression of core 2 GlcNAc-T in colorectal cancer cells is closely correlated with the progression of the disease (Shimodaira K., at al 97, Cancer Res.).
The identification of new core 2 GlcNAc-transferases and nucleic acids encoding the enzymes satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of disorders mediated by the enzymes including cancer and inflammatory disorders.
The citation of any reference herein is not an admission that such reference is available as prior art to the instant invention.
SUMMARY OF THE INVENTION
The present inventors have identified novel core 2 &bgr;-1,6-N-acetylglycosaminyltransferase nucleic acid molecules, and polypeptides encoded by such nucleic acid molecules. The nucleic acid molecules are herein designated “core2c GlcNAc-T” or “core2c GlcNAc-T”, and the polypeptides are herein designated “Core 2c”, “Core 2c GlcNAc-T”, or “Core 2c GlcNAc-T Polypeptide”. The core 2c GlcNAc-T nucleic acid molecules were found to be primarily expressed in the placenta, kidney, liver, and thymus.
Broadly stated the present invention contemplates an isolated Core 2c GlcNAc-T nucleic acid molecule encoding a polypeptide of the invention, including mRNAs, DNAs, cDNAs, genomic DNAs, PNAs, as well as antisense analogs and biologically, diagnostically, prophylactically, clinically or therapeutically useful variants or fragments thereof, and compositions comprising same.
The invention also contemplates an isolated Core 2c GlcNAc-T polypeptide encoded by a nucleic acid molecule of the invention a truncation, an analog, an allelic or species variation thereof, or a homolog of a polypeptide of the invention or a truncation thereof. (Truncations, analogs, allelic or species variations, and homologs are collectively referred to herein as “Core 2c GlcNAc-T Related Polypeptides”). The polypeptide comprises cytosolic, transmembrane, and catalytic regions.
The nucleic acid molecules of the invention permit identification of untranslated nucleic acid sequences or regulatory sequences that specifically promote expression of genes operatively linked to the promoter regions. Identification and use of such promoter sequences are particularly desirable in instances, such as gene transfer or gene therapy, which may specifically require heterologous gene expression in a limited environment. The invention therefore contemplates a nucleic acid molecule comprising a non-coding sequence such as a 5′ and/or 3″ sequence.
The nucleic acid molecules which encode for the mature core 2c GlcNAc-T polypeptide (may include only the coding sequence for the mature polypeptide (SEQ ID NO. 1, 7 or 10); the coding sequence for the mature polypeptide and additional coding sequences (e.g. leader or secretory sequences, proprotein sequences); the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5′ and/or 3′ of the coding sequence of the mature polypeptide (e.g. SEQ ID NO. 9).
Therefore, the term “nucleic acid molecule encoding a polypeptide” encompasses a nucleic acid molecule which includes only coding sequence for the polypeptide as well as a nucleic acid molecule which includes additional coding and/or non-coding sequences.
The nucleic acid molecules of the invention may be inserted into an appropriate vector, and the vector may contain the necessary elements for the transcription and translation of an inserted coding sequence. Accordingly, vectors may be constructed which comprise a nucleic acid molecule of the invention, and where appropriate one or more transcription and translation elements linked to the nucleic acid molecule.
Vectors are contemplated within the scope of the invention which comprise regulatory sequences of the invention, as well as chimeric gene constructs wherein a regulatory sequence of the invention is operably linked to a heterologous nucleic acid, and a transcription termination signal.
A vector can be used to transform host cells to express a Core 2c GlcNAc-T Polypeptide or Core 2c GlcNAc-T Related Polypeptide, or a heterologous polypeptide (i.e. a polypeptide not naturally expressed in the host cell). Therefore, the invention further provides host cells containing a vector of the invention. The invention also contemplates transgenic non-human mammals whose germ cells and somatic cells contain a vector comprising a nucleic acid molecule of the invention in particular one that encodes an analog of Core 2c GlcNAc-T, or a truncation of Core 2c GlcNAc-T.
The polypeptides of the invention may be obtained as an isolate from natural cell sources, but they are preferably produced by recombinant procedures. In one aspect the invention provides a method for preparing a Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide utilizing the purified and isolated nucleic acid molecules of the invention. In an embodiment a method for preparing a Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide is provided comprising:
(a) transferring a vector of the invention comprising a nucleic acid sequence encoding a Core 2c GlcNAc-T Polypeptide, or Core 2c GlcNAc-T Related Polypeptide, into a host cell;
(b) selecting transformed host cells from untransformed host cells;
(

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