Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-08-21
2001-08-07
Wang, Andrew (Department: 1635)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
Reexamination Certificate
active
06271353
ABSTRACT:
TECHNICAL FIELD
This invention relates to a gene encoding adseverin, which is a Ca
2+
-dependent actin filament-severing protein and has a function of regulating exocytosis, a recombinant vector containing this gene, a recombinant transformed by this vector, a process for producing adseverin by using the above-mentioned gene and a recombinant adseverin protein obtained by this process. The present invention also relates to an oligonucleotide hybridizable specifically with a base sequence encoding the adseverin protein, a method for regulating the formation of adseverin which comprises administering an oligonucleotide hybridizable specifically with a base sequence encoding the adseverin protein to an animal, and an antibody capable of recognizing the adseverin protein.
BACKGROUND ART
In many secretory cells in the resting state, secretion products such as neurotransmitters and hormones are stored in the form of granules or vesicles. When the cells receive adequate signals, these substances are released from the cells by exocytosis. In the process of exocytosis, the granules and vesicles migrate toward plasma membrane. Then they come into contact with the plasma membrane followed by fusion therewith, thus opening the membrane.
This exocytosis is tightly controlled by the concentration of intracellular free calcium [Ca
2+
]
i
(Knight et al., Ann. N.Y. Acad. Sci. 493:504-523, 1987). Namely, it is considered that in resting cells where [Ca
2+
]
i
is low, exocytosis is blocked at several steps depending on [Ca
2+
]
i
(Burgoyne, Biochem. Biophys. Acta 779:201-216, 1984). A number of secretory cells including chromaffin cells which are adrenal medulla secretory cells have a microfilament network composed of actin filaments under the plasma membrane which is supposed to serve as a barrier against the migration of granules and vesicles toward the plasma membrane (Cheek et al., FEBS Lett. 207:110-114, 1986; Lelkes et al., FBES Lett. 208:357-363, 1986). Prior to the release of the secretion products by exocytosis, this network is disassembled due to the increase in [Ca
2+
]
i
by Ca
2+
-dependent mechanisms (Vitale et al., J. Cell Biol. 113:1057-1067, 1991).
Actin is a globular protein with a molecular weight of 42 kD which is commonly distributed in eukaryocytes. It is a cytoskeleton protein closely relating to the contraction of muscle cells, etc. Actin monomers are polymerized to form filaments. Under the physiological ionic strength, actin undergoes polymerization in vitro at a ratio of about 100% so as to give filaments. In actual cells, however, various actin-regulating proteins contribute to the reversible conversion of filaments (gel) and monomers (sol) and changes occur depending on extracellular stimuli.
In bovine chromaffin cells, gelsolin, which seemingly relates directly to this process, was identified (Yin et al., Nature 281:583-586, 1979). Gelsolin shows a Ca
2+
-dependent actin filament severing activity in vitro and exerts barbed end capping and nucleating activities on actin filaments. Recently, adseverin (a protein of 74 kDa), which is similar to gelsolin in activity but different from it, was isolated from bovine adrenal medulla by Prof. Nonomura et al., Department of Pharmacology, Faculty of Medicine, University of Tokyo (Maekawa et al., J. Biol. Chem. 265:10940-10942).
Gelsolin is relatively widely distributed in various tissues and blood plasma (Stossel et al., Annu. Rev. Cell Biol. 1:353-402, 1985), while the distribution of adseverin is restricted mainly to the tissues with secretory functions (Sakurai et al., Neuroscience 38:743-756, 1990). This difference in tissue distribution of these proteins suggests that adseverin more closely relates to the secretory process (i.e., control of the release of neurotransmitters, endocrine substances or physiologically active substances) than gelsolin does. Accordingly, it is highly interesting to reveal the structure and function of adseverin to thereby clarify the role and regulatory mechanisms of actin filaments in exocytosis.
In former days, it was generally regarded that this process was regulated by fused proteins, etc. [Nishizaki, “Kaiko Hoshutsu Gesho ni okeru Saiboshitsu Tanpakushitsu no Yakuwari (Roles of Cytoplasmic Proteins in Exocytosis)”, Saibo Kogaku (Cell Technology), 13:353-360, 1994]. However, Nonomura et al. newly point out in their hypothesis that this process finally depends on an interaction between actin and myosin. This hypothesis further provides an epoch-making idea that the regulation by the actin-severing protein takes place in non-muscular cells on the actin side, differing from the regulation on the myosin side by myosin light chain kinase [Mochida, “Miosin Keisa Kinaze Shinkei Dentatsu Busshitsu Hoshutsu to sono Chosetsu ni okeru Miosin Keisa Kinaze no Yakuwari (Role of Myosin Light Chain Kinase in Release of Myosin Light Chain Kinase Neutrotransmitter and Regulation thereof)”, Saibo Kogaku (Cell Technology), 13:381-388, 1994].
It is thought that actin is liberated from broken cells and induces or enhances platelet agglutination in the blood so as to trigger thrombus development (Scarborough et al., Biochem. Biophys. Res. Commun. 100:1314-1319, 1981). On the other hand, adseverin has a gelsolin-like activity (i.e., an actin filament-severing activity) in vivo as described above. These facts indicate that adseverin might be applicable to drugs relating to thrombus (for example, thrombosis inhibitors).
It is furthermore expected that the release of, for example, a physiologically active substance might be regulated at the gene level by administering the antisense DNA sequence constructed on the basis of the base sequence encoding adseverin. Since adseverin might closely relate to the multiplication of vascular smooth muscles, it is considered that the administration of the antisense DNA would regulate the function of adseverin to thereby inhibit the multiplication of the smooth muscles. Accordingly, it is expected that the administration of the antisense DNA of adseverin might be usable in the inhibition of angiostenosis in blood vessel transplantation in bypass operation, etc. or in the inhibition of restenosis after percutaneous transluminal coronary angioplasty (PTCA).
To use the actin-regulating protein adseverin in the medicinal purposes as described above, it is necessary to produce adseverin in a large amount and in a uniform state. However, it is difficult to obtain uniform adseverin in a large amount by the conventional method wherein adseverin is isolated from an animal tissue per se or the culture supernatant of adseverin-producing cells. It is therefore required to clarify the base sequence of the gene encoding adseverin so as to produce adseverin in a large amount by using gene recombination techniques.
An object of the present invention is to identify the base sequence of the gene encoding adseverin. Another object of the present invention is to produce adseverin in a large amount by using gene recombination techniques with the use of a recombinant vector containing the above-mentioned sequence and to construct a screening system, etc. by using the same, thus developing novel drugs. Another object of the present invention is to produce the antisense DNA on the basis of the base sequence of the gene encoding adseverin and use it as a drug for inhibiting the formation of adseverin. Another object of the present invention is to provide an antibody capable of recognizing the adseverin protein.
The present inventors isolated and purified adseverin from bovine adrenal medulla and clarified its properties (Sakurai et al., Neuroscience 38:743-756, 1990; Sakurai et al., J. Biol. Chem. 226:4581-4584, 1991; Sakurai et al., J. Bio. Chem. 266:15979-15983, 1991).
Further, a hydrolyzed fragment of this protein was obtained and, based on the partial information of its amino acid sequence, oligonucleotide primers were synthesized. On the other hand, cDNA was prepared by reverse transcription from mRNA pr
Nakamura Seiji
Nezu Juni-ichi
Sakurai Takashi
Birch Stewart Kolasch & Birch, LLP.
Chugai Seiyaku Kabushiki Kaisha
Wang Andrew
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