Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-12-18
2003-07-01
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S069100, C435S325000, C435S183000, C435S006120, C435S252300, C435S091200, C536S023100, C536S023200, C536S024300, C536S024310, C536S024330, C514S04400A
Reexamination Certificate
active
06586217
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains to compositions related to proteins which function in controlling development and differentiation of mammalian cells, e.g., cells of a mammalian immune system. In particular, it provides proteins which exhibit an enzymatic activity of phosphorylating a selenium compound to produce a selenophosphate, a first step in the production of a selenocysteine, which is incorporated into various proteins.
BACKGROUND OF THE INVENTION
Selenocysteine was identified as the selenium moiety in a number of prokaryotic and eukaryotic proteins. See, e.g., Stadtman, (1991)
J. Biol. Chem.
266:1625-16260; and Berry, et al (1993)
Biochem Soc. Trans.
21:827-832. The presence of selenocysteine in proteins was shown to be responsible for a substantial increase in their enzymatic activity relatively to their cysteine-containing counterparts. See, e.g., Axley et al. (1990)
J. Biol. Chem.
265:18213-18218; Axley, et al. (1991)
J Biol. Chem.
266:13731-13736; Axley, et al. (1991)
Proc. Nat'l Acad. Sci. USA
88:8450-8454; and Berry, et al. (1991)
Nature
349:438-440. Selenocysteine is located in the active site of three different groups of bacterial enzymes: glycine reductase, see, e.g., Garcia, et al. (1992)
J. Bacteriol.
174:7080-9, formate dehydrogenases, see, e.g., Zinoni, et al. (1987)
Proc. Natl. Acad. Sci. USA
84:3156-3160; and Berg, et al. (1991)
J. Biol. Chem.
266:22380-22385, and hydrogenases, see, e.g., Stadtman (1990)
Ann. Rev. Biochem.
59:111-127. A number of selenocysteine containing proteins have also been described in mammalian organisms. These include the Cytoplasmic, see, e.g., Flohé, et al. (1979)
FEBS Letts
32:132-134; Plasma, see, e.g., Takahashi, et al. (1987)
Arch. Biochem. Biophys
256:677-686; and Maddipati, et al. (1987).
J. Biol. Chem
262:17396-17403; Phospholipid Hydroperoxide Glutathione Peroxidases, see, e.g., Ursini, et al. (1985)
Biochim. Biophys. Acta
839:62-70; and Schuckelt, et al. (1991)
Free Radical Res. Commun.
14:343-361; Type I iodothyronine deiodinase, see, e.g., Behne, et al. (1990)
Biochem. Byophis. Res. Commun.
173:1143-1149; and Berry, et al. (1991)
Nature
349:438-440; Selenoprotein P. see, e.g., Read, et al. (1990)
J. Biol. Chem
265:17899-17905; and Hill, et al. (1991)
J. Biol. Chem.
265:10060-10063; and Selenoprotein of the Sperm Mitochondial Capsule, see, e.g., Kleene, et al. (1990)
Dev. Biol.
137:395-402; and Karimpour, et al. (1992)
DNA and Cell Biology
11:693-699. With notable exceptions, these proteins catalyze oxidation-reduction reactions and some act as peroxide scavengers, see, e.g., Stadtman (1990).
Ann. Rev. Biochem.
59:111-127.
As in bacteria, see, e.g., Zinoni, et al. (1990)
Proc. Natl Acad. Sci. USA
87:4660-4664; and Heider, et al. (1992)
EMBO J.
11:3759-3766, the incorporation of selenocysteine in eukaryotic proteins is directed by UGA codons, commonly read as stop codons by the translational machinery. See, e.g., Bock, et al. (1991)
TIBS
16:463-467; and Berry and Larsen (1993)
Biochem. Soc. Trans.
21:827-832. The co-translational insertion of selenocysteine in the polypeptide chain is made possible by the existence of a specific secondary structure in selenoprotein-encoding mRNAs, see, e.g., Zinoni, et al. (1990)
Proc. Natl Acad. Sci. USA
87:4660-4664; Heider, et al. (1992)
EMBO J.
11:3759-3766; and Berry, et al (1993)
EMBO J.
12:3315-3322. Stem-loops have been described in bacterial selenocysteine-encoding mRNAs, which are located inside the translating region, immediately after the UGA codon, and are required for the incorporation of selenocysteine into bacterial proteins. See, e.g., Zinoni, et al. (1990)
Proc. Natl Acad. Sci. USA
87:4660-4664; and Heider, et al. (1992)
EMBO J.
11:3759-3766. On the other hand, stem-loops, structurally different from their bacterial homologues, have been identified in the 3′ untranslated region of eukaryotic mRNAs encoding selenocysteine-containing proteins. These mRNA structures, designated SECIS elements, see, e.g., Berry, et al (1993).
EMBO J.
12:3315-3322, are required for the UGA directed incorporation of selenocysteine into eukaryotic proteins. Specific tRNAs, designated tRNA(ser)sec, complementary to the UGA codons, were described both in bacteria, see, e.g., Leinfelder, et al. (1990)
Proc. Natl. Acad. Sci. USA
87:543-547; and Heider, et al. (1989)
Nucleic Acids Res.
17:2529-2540; and eukaryotic organisms, see, e.g., Lee, et al. (1990)
Mol. Cel. Biol.
10:1940-1949; and Hatfield, et al. (1992)
Biochem. Biophys. Res. Commun.
184:254-259. Furthermore, a specific elongation factor necessary for the incorporation of selenocysteine into proteins, designated SELB, was isolated in bacterial organisms, see, e.g., Forchhammer, et al. (1989)
Nature
342:453-456. This elongation factor was shown to bind to the loop region of the hairpin structure required for selenocysteine incorporation. In the presence of selenocysteine-tRNA, SELB was shown to interact with selenocysteine-tRNA and GTP to form a ternary complex that recognizes the ribosome bound mRNA forming a selenocysteinyl-tRNA-SELB-GTP-mRNA complex that can tether protein factors to the translational complex during protein synthesis, see, e.g., Baron, et al. (1993)
Proc. Natl. Acad. Sci. USA
90:4181-4185; and Ringquist, et al. (1994)
Genes and Development
8:376-385.
Enzymatic reactions required for the synthesis of selenocysteine have been described in bacterial organisms. First, monoselenophosphate (MSP) is synthesized from selenide and ATP in an enzymatic reaction catalyzed by SPS. See, e.g., Veres, et al. (1994)
J. Biol. Chem.
269:10597-10603. MSP is a highly reactive compound, see, e.g., Glass, et al. (1993)
Biochemistry
32:12555-9; and Stadtman (1994)
Biofactors
4:181-185, used as an active selenium donor in the synthesis of selenocysteine from seryl-tRNAs. See Leinfelder, et al. (1990)
Proc. Natl. Acad. Sci. USA
87:543-547. Additionally, MSP is required for an entirely different process: the posttranscriptional modification of 2-thiouridine tRNAS in which sulfur is replaced with selenium forming 2-selenouridine tRNAs, see, e.g., Leinfelder, et al. (1990)
Proc. Natl. Acad. Sci. USA
87:543-547; Veres, et al. (1992)
Proc. Natl. Acad. Sci. USA
89:2975-2979; and Veres and Stadtman (1994)
Proc. Natl. Acad. Sci. USA
91:8092-8096.
However, the isolation of a mammalian counterpart enzyme has eluded biochemists for years. As such, the elucidation of whether similar mechanisms of regulation and expression occur in mammals has not been determined. Moreover, in the absence of ready sources of selenophosphate and selenocysteine-tRNAS, further discovery of what other proteins useful in various developmental and regulatory pathways has remained a mystery. The present invention solves these and many other needs.
SUMMARY OF THE INVENTION
The present invention is based, in part, upon the discovery of mammalian selenocysteine-containing enzymes. It embraces means to use the enzymes to screen for chemical modulators of the selenophosphate synthetase (SPS), e.g., mutations (muteins) of the natural sequences, fusion proteins, and other structural or functional analogs. Isolation of the proteins allows also for production of antibodies at high efficiency. The invention also embraces isolated genes and fragments thereof encoding proteins of the invention. Various uses of these different protein or nucleic acid compositions are also provided.
The present invention provides:
1. A sustantially pure or isolated protein comprising a fragment exhibiting sequence homology to a corresponding portion of a mammalian selonophosphate synthetase wherein:
a) said homology is at least about 90% identity and said portion is at least about 9 amino acids;
b) said homology is at least about 80% identity and said portion is at least about 17 amino acids; or
a) said homology is at least about 700% identity and said portion is at least about 25 amino acids;
2. The protein of claim
1
, wherein:
a) said mammalian SPS is a rodent or primate protein;
b) sa
Bazan J. Fernando
Guimarães M. Jorge
Zlotnik Albert
Ching Edwin P.
Hutson Richard
Prouty Rebecca E.
Schering Corporation
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