Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-04-21
2002-08-20
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S018000, C435S069100, C435S325000, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06436687
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to a eukaryotic DNA sequence and, more particularly, to a eukaryotic cDNA sequence derived from mRNA encoding a sialidase enzyme, the MRNA being expressed in mouse brain cells.
2. Background Description
Sialidases are a family of glycohydrolytic enzymes which cleave sialic acid residues from the oligosaccharide components of glycoproteins and glycolipids. Sialidases have been identified in a number of cellular organelles: the plasma membrane, the lysosomes, and the cytosol (Schengrund, C., Rosenberg, A., and Repman, M. A. (1976) J. Biol. Chem., 79:555; Tulsiani, D.
R. P., and Carubelli, R., (1970) J. Biol. Chem., 245:1821). The sialidase enzyme has alternatively been referred to as “neuraminidase,” and the names are considered synonymous within the relevant art. Saito, M. and Yu, R. K., “Biochemistry and Function of Sialidases” in
Biology of the Sialic Acids
, Plenum Press, N.Y. (1995).
In general, sialidase enzymes catalyze the release of sialic acid from sialo-oligosaccharides, gangliosides, or sialo-glycoprotein. The sialic acid in the oligosaccharide component of glycoproteins is involved in mediation of clearance from the serum and thus affects the physical, chemical and immunogenic properties of the protein molecule. Maintenance of sialic acid residues is thus important to the functional expression of various glycoproteins with pharmaceutic uses as described in U.S. Pat. No. 5,928,915 to Warner et al. It is well known in the art that the oligosaccharide part of various glycoconjugates (especially glycolipids and glycoproteins) have a number of important functions in vivo (Biology of Carbohydrates, vol. 2, Ginsburg et al., Wiley, N.Y., 1984; The Glycoconjugates, vol. I-V, Academic Press, New York. Carbohydrate structures are important for the stability, activity, localization, immunogenicity and degradation of glycoproteins. Carbohydrates are antigenic determinants for blood group antigens and act as receptors for pathogens, proteins, hormones, and toxins, for example. Carbohydrates are also important for oncogenesis, since specific oligosaccharides have been found to be cancer-associated antigenic determinants. The cleavage of carbohydrate moieties from certain precursor glycoproteins, can result in the creation of potent immune stimulators. For example, group-specific component is a precursor of a macrophage activating factor that is normally converted to the active factor by the action of glycosidases of B and T cells. By way of example as an in vitro role for one particular sialidase, U.S. Pat. No. 5,177,002 to Yamamoto discloses a process for the production of a potent macrophage activating factor derived from human vitamin D-binding protein by cleaving Vitamin D protein with a combination of b-galactosidase and sialidase.
A number of bacterial and viral sialidase enzymes have been cloned, their X-ray crystal structures reported, and their biological functions extensively studied. It has been suggested that the
Vibrio cholerae
sialidase may function as a virulence factor for the bacterium, by increasing the binding affinity and penetration of cholera toxin into target cells. The sialidase of influenza virus has been suggested to play a role in the escape of progeny virus from infected cells. Thus, there are numerous sialidase enzymes in nature with a wide variety of physiological significance attributed to the different molecules.
Several mammalian forms of the gene encoding sialidase have been cloned, including rat muscle sialidase, Chinese hamster ovary (CHO) cell sialidase, and sialidases from mouse and human lysosomes. Rat skeletal sialidase has been purported to play a role in the differentiation of skeletal muscle cells. Desialyation of recombinant proteins expressed in CHO cells may result in a reduced clinical effect of such proteins. In addition, mouse and human lysosomal sialidases have been implicated in two metabolic storage disorders, namely sialidosis and galactosialidosis.
Sialidosis is a metabolic disorder that can be inherited as an autosomal recessive trait. It is caused by a deficiency of the enzyme alpha-neuramidinase, and it affects the metabolizing of certain fats and carbohydrates. In this condition an enzyme is missing that results in the accumulation of sialic acid in the nerve cells. The disorder is also associated with excess tissue accumulation and urinary excretion of sialylated oligosaccharides and glycolipids. There are two main clinical variants of the disease: late onset sialidosis type I, characterized by bilateral macular cherry-red spots and myoclonus, and infantile onset type II, which is characterized by skeletal dysplasia, mental retardation and hepatosplenomegaly. Galactosialidosis is distinguished from sialidosis by the additional deficiency in b-galactosidase enzyme activity and concurrent accumulation and excretion of excess sialyloligosaccharides.
Sialidase enzymatic activity has been reported previously in rat and mouse brains. In addition, a sialidase activity has been identified in brain myelin that may play a functional role in developmental changes in myelin gangliosides and membrane adhesion. Saito, M., and Yu, R. K.,
J. Neurochem
. 58, 83-37 (1992); Saito, M., and Yu, R. K.,
J. Neurosci
. 36, 127-132 (1992). It has also been reported previously that two immunologically distinct forms of sialidase can be found in bovine brain, with one type localized to the synaptosomal membrane and one found in both synaptosomal and lysosomal membranes. Miyagi et al. J. Biol. Chem. 244: 5004-5011 (1999). A rat cytosolic sialidase DNA has also been reported. Miyagi et al., J. Biol. Chem. 244, 5004-5011 (1993). Interestingly, the latter genes have been sequenced and the bovine brain sialidase gene sequences do not share significant homology with previously reported bacterial, hamster, rat and human lysosomal and skeletal sialidase DNA sequences.
Treatment of cells with purified preparation of sialidase is known to affect a wide variety of cellular functions. It is thought that the wide variety of changes seen following such treatment is due to either the removal of terminal sialic acid residues from a glycoprotein or alternatively due to exposure of galactose residues. For example, treatment with purified sialidase disrupts receptor function, cell growth and differentiation, cell-cell binding and interactions, immunocyte function, and membrane fluidity, to name but a few Saito, M. and Yu, R. K., “Biochemistry and Function of Sialidases” supra.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to provide an isolated single or double-stranded polynucleotide, typically DNA, having a nucleotide sequence which comprises a nucleotide sequence selected from the group consisting of: (a) the sense sequence of SEQ ID NO:1 from about nucleotide position 1 to about nucleotide position 1699; (b) polynucleotide sequences that, on expression, encode a polypeptide or polypeptides consisting of the amino acid sequences of SEQ ID NO:2; (c) polynucleotide sequences that encode amino acid sequences that are analogous to the amino acid sequence of SEQ ID NO:2, (d) polynucleotide sequences that are the polynucleotide complement of (a), (b) or (c), and (e) polynucleotide sequences that are substantially homologous to the polynucleotides of (a), (b) (c) and (d). It is further an object of the invention to provide contiguous polynucleotide fragments of the sequences in (a), (b), (c) or (d) of at least 16 nucleotides in length. In one particular embodiment, the polynucleotide is a DNA molecule, while in another embodiment the polynucleotide is an RNA molecule.
It is also an object of the invention to provide an expression cassette containing polynucleotide sequences that, on expression, encode a polypeptide or polypeptides consisting of the amino acid sequences of SEQ ID NO:2 (or analogous sequences), or fragments thereof, operably linked to at least one DNA control sequence.
It is also an object of the invention to provide a host cell transform
Fronda Chris
Yu Robert K.
Zeng Guichao
Achutamurthy Ponnathapu
Ramirez Delia M.
Virginia Commonwealth University
Whitham Curtis & Christofferson, P.C.
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