Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-11-22
2001-05-01
Stucker, Jeffrey (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C424S094100, C424S094610, C424S520000, C435S183000, C530S300000, C530S350000, C536S023500
Reexamination Certificate
active
06225454
ABSTRACT:
TECHNICAL FIELD
The present invention provides a novel sialidase and DNA coding for it. More precisely, the present invention provides sialidase that localizes in plasma membrane, and specifically hydrolyzes gangliosides, and DNA coding for it.
The sialidase of the present invention and the DNA coding for it are expected to be utilized as a reagent used for saccharide chain studies and a medicament used for gene diagnosis and gene therapy.
BACKGROUND ART
Sialidase is a glycohydrolytic enzyme present in living bodies, which eliminates a sialic acid residue from a non-reducing terminal of saccharide chains of glycoproteins or glycolipids. It has been known that, when sialic acid is removed from saccharide chain molecules, not only the degradation of these molecules begins, but also molecular conformation and many of important cell functions such as recognition mechanism by receptors, cell adhesion, and immunomechanism may be changed. It has also become clear that sialidase exhibits rapid activity change in connection with proliferation and canceration of cells, and it is involved in the metastatic ability of cancer cells. However, there is little knowledge about how sialic acid is eliminated in vivo. This is because the studies of mammalian sialidases on the molecular level are behindhand, and hence there are many unknown points concerning their structures and expression mechanism.
Because mammalian sialidase exhibits only low activity, and is extremely unstable, isolation and purification of the enzyme have been difficult. Sialidase has been often considered for a long time to be one of the mere lysosomal enzymes involved in the dissimilation and degradation. Under such a situation, we isolated and purified the enzyme by using mainly rat tissues as the source of the enzyme, and found that there were four types of sialidases which differ from sialidases of bacteria, viruses, protozoa and the like in their natures (Miyagi, T. and Tsuiki, S.,
Eur. J. Biochem.
141, 75-81, 1984; Miyagi, T. et al.,
J. Biochem.
107, 787-793, 1990; Miyagi, T. and Tsuiki, S.,
J. Biol. Chem.
260, 6710-6716, 1985). These enzymes each localize in lysosomal matrix, lysosome membrane, plasma membrane (cell surface membrane), and cytoplasm within a cell, respectively, and they are different from each other not only in enzymological characteristics such as substrate specificity, but also in immunological properties. Among those sialidases, the sialidase localized in cytoplasm can be obtained as a homogenous purified product from rat skeletal muscles. Its cDNA cloning has been succeeded for the first time in the world as for animal sialidases, and its primary structure has been determined (Miyagi T. et al.,
J. Biol. Chem.,
268, 26435-26440, 1993). Its genomic structure analysis has also been done, and as for its function, it has been elucidated that the enzyme participates in the differentiation and the growth of skeletal muscle cells by using the cDNA as a probe. These studies can be considered a part of pioneer researches in sialidase studies in the world.
By the previous studies, it has become clear that there is possibility that the sialidase localized in plasma membrane exhibits activity elevation upon proliferation and canceration of cells, and it is also deeply concerned with the differentiation of nerve cells and the signal transduction of cells. To date, however, it has not been understood at all about the structure of this enzyme, the mechanism causing the activity change and the like. In order to answer these questions, what many researchers in this field have long been desired is cloning of its cDNA. For example, if the mechanism of cancerous change due to this enzyme could be elucidated, it would be possible to utilize the results in diagnosis and therapy of cancers. Further, because gangliosides exist in surface membranes of many cells and participate in important cell functions such as cell adhesion and informational communication, and they are also main cerebral components, the sialidase utilizing them as a specific substrate is estimated to be involved in certain important cranial nerve functions.
SUMMARY OF THE INVENTION
The present invention has been accomplished in view of the aforementioned present condition. An object of the present invention is to provide the sialidase localized in plasma membrane and DNA that codes for it.
The inventors of the present invention earnestly conducted studies in order to achieve the aforementioned object, and as a result, succeeded in isolating the sialidase localized in plasma membrane and cloning of cDNA coding for it. Furthermore, they found that the aforementioned sialidase was unique in that it substantially specifically hydrolyzed gangliosides (glycolipids containing sialic acid), which are substrates that similarly localize mainly in plasma membrane, and it was completely different from other mammalian sialidases and microbial sialidases in enzymatic substrate specificity. Thus, they accomplished the present invention.
That is, the present invention provides a protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or
(B) a protein which has the amino acid sequence including substitution, deletion, insertion, or transition of one or several amino acid residues in SEQ ID NO: 2 or SEQ ID NO: 4, and exhibits activity to eliminate a sialic acid residue from a non-reducing terminal of ganglioside.
The present invention also provides DNA coding for the protein defined in the above (A) or (B). Specifically, such DNA may be DNA which has the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
A sialidase which has the aforementioned characteristics will be referred to as the “sialidase of the present invention” hereinafter, and DNA coding for it will be referred to as the “DNA of the present invention” hereinafter.
Hereafter, the present invention will be explained in detail.
<1> Sialidase of the Present Invention
The sialidase of the present invention is a protein which has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Moreover, the sialidase of the present invention include a protein which has the amino acid sequence including substitution, deletion, insertion, or transition of one or several amino acid residues in SEQ ID NO: 2 or SEQ ID NO: 4, so long as it exhibits activity for eliminating a sialic acid residue from a non-reducing terminal of ganglioside.
Among the aforementioned sialidases, the sialidase that has the amino acid sequence of SEQ ID NO: 2 has the following physicochemical properties.
(1) Activity
It eliminates sialic acid residues from a non-reducing terminal of ganglioside.
(2) Substrate Specificity
It acts on gangliosides, but not act on glycoproteins and oligosaccharides. Specifically, it acts on GD3-ganglioside, GD1a-ganglioside, GM3-ganglioside, and synthetic gangliosides (GSC-17(&agr;2-3) and GSC-61(&agr;2-6)), but it does not substantially act on GM2-ganglioside, GM1-ganglioside, orosomucoid, fetuin, glycophorin, ovine submaxillary gland mucin, and bovine submaxillary gland mucin. It weakly acts on &agr;2-3 sialyllactose, and 4-MUNeuAc (4-methylumbelliferyl N-acetylneuraminic acid).
(3) Optimum pH
4.7 to 5.0
(4) Molecular Weight
About 65,000 as determined by sucrose density gradient centrifugation,
About 52,000 as determined by SDS-polyacrylamide gel electrophoresis under reducing condition
(5) Inhibition and Activation
A surface active agent is required for the activity. For example, it is highly active in the presence of 0.1 to 0.2% of Triton X-100.
It is strongly inhibited by heavy metal ions such as Cu
2+
, and 4-hydroxy mercury benzoate.
It is stabilized by dithiothreitol, Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetylneuraminic acid), and glycerol. However, it is weakly inhibited by Neu5Ac2en.
Among the sialidases of the present invention, the sialidase which has the aforementioned characteristics is an enzyme derived from bovine, whereas the sialidase which has the amino acid sequence of SEQ ID NO: 4 in one derived from human. These
Miyagi Taeko
Wada Tadashi
Yoshikawa Yuko
Kilpatrick & Stockton LLP
Miyagi Ken
Stucker Jeffrey
Winkler Ulrike
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