Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1999-02-10
2001-03-06
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S193000
Reexamination Certificate
active
06197535
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is relates to a method for purifying a &ggr;-glutamyl carboxylase (also referred to herein as &ggr;-carboxylase) from Conus snails and to a method for assaying the activity of this &ggr;-carboxylase.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by number and are listed numerically in the appended List of References.
The vitamin K-dependent &ggr;-carboxylation of glutamate residues was originally discovered as a novel post-translational modification in the blood coagulation cascade (1); some of the key clotting factors such as prothrombin must be &ggr;-carboxylated in order for proper blood clotting to occur. Somewhat later, this post-translational modification was also found in certain bone proteins (2). This modification was restricted to these rather specialized mammalian systems until a very unusual peptide, conantokin-G, was described from the venom of the predatory marine snail,
Conus geographus
(3). Conantokin-G is a 17-amino acid peptide that inhibits the N-methyl-D-aspartate receptor (4). Unlike most Conus peptides, which are multiply disulfide-bonded, conantokin-G has no disulfide cross-links but has five residues of &ggr;-carboxyglutamate residues; this remains the highest density of &ggr;-carboxyglutamate found in any functional gene product characterized to date.
Most of the biologically active components of the Conus venom are multiply disulfide bonded peptides (the conotoxins). These have been shown to be initially translated as prepropeptide precursors, which are then post-translationally processed to yield the mature disulfide-crosslinked conotoxin. Conantokin-G differs strikingly from most conotoxins not only in having &ggr;-carboxyglutamate residues, but also because it has no disulfide crosslinks. We report below an analysis of a cDNA clone encoding the conantokin-G precursor. Furthermore, we establish the probable function of one region of the precursor that is excised during the maturation of the functional conantokin-G peptide.
The presence of &ggr;-carboxyglutamate in a non-mammalian system was initially controversial because vitamin K-dependent carboxylation of glutamate residues had primarily been thought to be a highly specialized mammalian innovation. However, we have found that conantokin-G is only one member of a family of peptides; a variety of other conantokins have been found including conantokin-T and conantokin-R from two other fish-hunting cone snails (5, 6). All three peptides have a high content of &ggr;-carboxyglutamate (4-5 residues). &ggr;-Glutamyl carboxylase has been purified from mammalian sources (7, 8) and has been expressed both in mammalian and insect cell lines (9, 10). Recently it was shown that, as is the case in the mammalian system, the carboxylation reaction in Conus venom ducts absolutely requires vitamin K, and the net carboxylation increases greatly in the presence of high concentrations of ammonium sulfate. In these respects, the mammalian and the Conus &ggr;-carboxylation venom systems are very similar (11).
The propeptides of vitamin K-dependent blood coagulation proteins share extensive sequence similarity. This sequence is believed to interact with the carboxylase and constitutes the &ggr;-carboxylation recognition sequence (&ggr;-CRS). In this report, we analyze the conantokin-G precursor sequence for potential &ggr;-CRS sequences. The results described below identify a sequence present in the −1 to −20 region of the conantokin-G prepropeptide which, when covalently linked to the N-terminal of the substrate enzyme, stimulates carboxylation by the Conus enzyme.
SUMMARY OF THE INVENTION
The present invention is relates to a method for purifying a &ggr;-carboxylase from Conus snails and to a method for assaying the activity of this &ggr;-carboxylase.
Conantokin-G isolated from the marine snail
Conus geographus
is a 17-amino acid &ggr;-carboxyglutamate (Gla)-containing peptide that inhibits the N-methyl-D-aspartate receptor. We describe the cloning and sequence of conantokin-G cDNA and the possible role of the propeptide sequence. The cDNA encodes a 100-amino acid peptide. The N-terminal 80 amino acids constitute the prepro-sequence, and the mature peptide is derived from the remaining C-terminal residues after proteolysis, C-terminal amidation and a unique post-translational modification, &ggr;-carboxylation of glutamate residues to Gla. Mature conantokin-G peptide containing Glu residues (E.Con-G) in place of Gla is a poor substrate for the vitamin K-dependent &ggr;-glutamyl carboxylase (apparent K
m
=3.4 mM). Using peptides corresponding to different segments of the propeptide, we investigated a potential role for the propeptide sequences in &ggr;-carboxylation. Propeptide segment −20 to −1 covalently linked to E.Con-G or the synthetic pentapeptide FLEEL increased their apparent affinities two orders of magnitude. These substrates are not efficiently carboxylated by the bovine microsomal &ggr;-glutamyl carboxylase, suggesting differences in specificities between the Conus and the mammalian enzyme. However, the role of propeptide in enhancing the efficiency of carboxylation is maintained. It has been found that the the Conus carboxylase properly carboxylates conantokin-G. That is, Glu
2
of conantokin-G is not carboxylated by the enzyme whereas Glu
3
, Glu
4
, Glu
7
, Glu
10
and Glu
14
are carboxylated when the enzyme is added to the propeptide.
The −20 to −1 amino acid sequence of the prepropeptide is highly conserved among the conantokins. This region, or the entire prepropeptide, therefore can be used to prepare an affinity column to purify the carboxylase which post-translationally modifies Glu to &ggr;-carboxyglutamate. Once the carboxylase is purified, it can be sequenced and cloned. In addition, the prepropeptide can be used to assay activity of the carboxylase from Conus.
REFERENCES:
patent: 5830998 (1998-11-01), Maccecchini
patent: 5854217 (1998-12-01), Maccecchini
patent: 9820026 (1998-05-01), None
patent: 9831705 (1998-07-01), None
Gray, W.R. and Olivera, B.M. (1988). “Peptide Toxins from VenomousConusSnails.”Ann. Rev. Biochem.57:665-700.
Bandyopadhyay, P.K. et al. (1998). “Conantokin-G Precursor and Its Role in &ggr;Carboxylation by a Vitamin K-dependent Carboxylase from aConusSnail.”J. Biol. Chem.273:5447-5450.
Czerwiec et al., “Vitamin K-dependent Carboxylase: Comparison of the Bovine Gamma-Carboxylase with the Gamma-Carboxylase from the Marine Cone Snail”, Blood, (1996) vol. 88, No. 10 Suppl. 1 Part 1-2, p. 523A, Abstract 2079, Dec. 1996.
Stanley et al., “Identification of a Vitamin K-dependent Carboxylase in the Venom Duct of a Conus Snail”, FEBS Letters, vol. 407, No. 1, pp. 85-88, Apr. 1997.
Bush et al., “Hydrophobic Amino Acids Define the Carboxylation Recognition Site in the Precursor of the Gamma-Carboxyglutamic-Acid-Containing Conotoxin Epsilon-TxIX from the Marine Cone Snail Conus Textile”, Biochemistry, vol. 38, No. 44, pp. 14660-14666, SciSearch Abst AN 1999:888719, Nov. 1999.
Bandyopadhyay Pradip K.
Olivera Baldomero M.
Walker Craig S.
Rothwell Figg Ernst & Manbeck p.c.
University of Utah Research Foundation
Weber Jon P.
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