Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2003-10-09
2004-11-30
Monshipouri, Maryam (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S320100, C435S006120, C435S325000, C435S219000, C536S023200
Reexamination Certificate
active
06825025
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of enzyme proteins that are related to the metalloprotease enzyme subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
BACKGROUND OF THE INVENTION
Many human enzymes serve as targets for the action of pharmaceutically active compounds. Several classes of human enzymes that serve as such targets include helicase, steroid esterase and sulfatase, convertase, synthase, dehydrogenase, monoxygenase, transferase, kinase, glutanase, decarboxylase, isomerase and reductase. It is thefore important in developing new pharmaceutical compounds to identify target enzyme proteins that can be put into high-throughput screening formats. The present invention advances the state of the art by providing novel human drug target enzymes related to the metalloprotease subfamily.
Endothelin-Converting Enzymes
The novel human protein, and encoding gene, provided by the present invention is related to the family of metalloprotease enzymes (also referred to as the peptidase family M13, zinc metalloprotease family, and the neprilysin family) in general and shows a high degree of similarity to the endothelin-converting enzyme subfamily of metalloproteases. Furthermore, the protein of the present invention may be a novel isoform of the gene provided in Genbank gi7662200 (see the amino acid sequence alignment in FIG.
2
).
Endothelin-coverting enzymes (ECE) are membrane-bound metalloproteases that catalyze the proteolytic activation of endothelins, which are potent vasoactive peptides. Endothelins are produced from biologically inactive intermediates known as big endothelins by ECE-catalyzed proteolytic processing. ECE function in secretory pathways as well as on the cell surface. ECE-1 and ECE-2 have been characterized. ECE-2 is structurally related to ECE-1, neural endopeptidase 24.11, and human Kell blood group protein. ECE-1 and ECE-2 are both inhibited by phosphoramidon. ECE-1 is most active at neutral pH, whereas an acidic pH is optimum for ECE-2. It is though that ECE-2 converts endogenously synthesized big endothelin-1 to mature endothelin-1 at the acidic environement of the trans-Golgi network (Emoto et al.,
J Biol Chem
1995 Jun. 23;270(25): 15262-8).
Metalloproteases
The metalloproteases may be one of the older classes of proteinases and are found in bacteria, fungi as well as in higher organisms. They differ widely in their sequences and their structures but the great majority of enzymes contain a zinc atom which is catalytically active. In some cases, zinc may be replaced by another metal such as cobalt or nickel without loss of the activity. Bacterial thermolysin has been well characterized and its crystallographic structure indicates that zinc is bound by two histidines and one glutamic acid. Many enzymes contain the sequence HEXXH, which provides two histidine ligands for the zinc whereas the third ligand is either a glutamic acid (thermolysin, neprilysin, alanyl aminopeptidase) or a histidine (astacin). Other families exhibit a distinct mode of binding of the Zn atom. The catalytic mechanism leads to the formation of a non covalent tetrahedral intermediate after the attack of a zinc-bound water molecule on the carbonyl group of the scissile bond. This intermediate is further decomposed by transfer of the glutamic acid proton to the leaving group.
Metalloproteases contain a catalytic zinc metal center which participates in the hydrolysis of the peptide backbone (reviewed in Power and Harper, in Protease Inhibitors, A. J. Barrett and G. Salversen (eds.) Elsevier, Amsterdam, 1986, p. 219). The active zinc center differentiates some of these proteases from calpains and trypsins whose activities are dependent upon the presence of calcium. Examples of metalloproteases include carboxypeptidase A, carboxypeptidase B, and thermolysin.
Metalloproteases have been isolated from a number of procaryotic and eucaryotic sources, e.g.
Bacillus subtilis
(McConn et al., 1964, J. Biol. Chem. 239:3706);
Bacillus megaterium
; Serratia (Miyata et al., 1971, Agr. Biol. Chem. 35:460);
Clostridium bifermentans
(MacFarlane et al., 1992, App. Environ. Microbiol. 58:1195-1200),
Legionella pneumophila
(Moffat et al., 1994, Infection and Immunity 62:751-3). In particular, acidic metalloproteases have been isolated from broad-banded copperhead venoms (Johnson and Ownby, 1993, Int. J. Biochem. 25:267-278), rattlesnake venoms (Chlou et al., 1992, Biochem. Biophys. Res. Commun. 187:389-396) and articular cartilage (Treadwell et al., 1986, Arch. Biochem. Biophys. 251:715-723). Neutral metalloproteases, specifically those having optimal activity at neutral pH have, for example, been isolated from
Aspergillus sojae
(Sekine, 1973, Agric. Biol. Chem. 37:1945-1952). Neutral metalloproteases obtained from
Aspergillus
have been classified into two groups, npI and npII (Sekine, 1972, Agric. Biol. Chem. 36:207-216). So far, success in obtaining amino acid sequence information from these fungal neutral metalloproteases has been limited. An npII metalloprotease isolated from
Aspergillus oryzae
has been cloned based on amino acid sequence presented in the literature (Tatsumi et al., 1991, Mol. Gen. Genet. 228:97-103). However, to date, no npI fungal metalloprotease has been cloned or sequenced.
Alkaline metalloproteases
, for example, have been isolated from
Pseudomonas aeruginosa
(Baumann et al., 1993, EMBO J 12:3357-3364) and the insect pathogen
Xenorhabdus luminescens
(Schmidt et al., 1998, Appl. Environ. Microbiol. 54:2793-2797).
Metalloproteases have been devided into several distinct families based primarily on activity and sturcture: 1) water nucleophile; water bound by single zinc ion ligated to two His (within the motif HEXXH) and Glu, His or Asp; 2) water nucleophile; water bound by single zinc ion ligated to His, Glu (within the motif HXXE) and His; 3) water nucleophile; water bound by single zinc ion ligated to His, Asp and His; 4) Water nucleophile; water bound by single zinc ion ligated to two His (within the motif HXXEH) and Glu and 5) water nucleophile; water bound by two zinc ions ligated by Lys, Asp, Asp, Asp, Glu.
Examples of members of the metalloproteinase family include, but are not limited to, membrane alanyl aminopeptidase (
Homo sapiens
), germinal peptidyl-dipeptidase A (
Homo sapiens
), thimet oligopeptidase (
Rattus norvegicus
), oligopeptidase F (
Lactococcus lactis
), mycolysin (
Streptomyces cacaoi
), immune inhibitor A (
Bacillus thuringiensis
), snapalysin (
Streptomyces lividans
), leishmanolysin (
Leishmania major
), microbial collagenase (
Vibrio alginolyticus
), microbial collagenase, class I (
Clostridium perfringens
), collagenase 1 (
Homo sapiens
), serralysin (
Serratia marcescens
), fragilysin (
Bacteroides fragilis
), gametolysin (
Chlamydomonas reinhardtii
), astacin (
Astacus fluviatilis
), adamalysin (
Crotalus adamanteus
), ADAM 10 (
Bos taurus
), neprilysin (
Homo sapiens
), carboxypeptidase A (
Homo sapiens
), carboxypeptidase E (
Bos taurus
), gamma-D-glutamyl-(L)-meso-diaminopimelate peptidase I (
Bacillus sphaericus
), vanY D-Ala-D-Ala carboxypeptidase (
Enterococcus faecium
), endolysin (bacteriophage A118), pitrilysin (
Escherichia coli
), mitochondrial processing peptidase (
Saccharomyces cerevisiae
), leucyl aminopeptidase (
Bos taurus
), aminopeptidase I (
Saccharomyces cerevisiae
), membrane dipeptidase (
Homo sapiens
), glutamate carboxypeptidase (
Pseudomonas
sp.), Gly-X carboxypeptidase (
Saccharomyces cerevisiae
), O-sialoglycoprotein endopeptidase (
Pasteurella haemolytica
), beta-lytic metalloendopeptidase (
Achromobacter lyticus
), methionyl aminopeptidase I (
Escherichia coli
), X-Pro aminopeptidase (
Escherichia coli
), X-His dipeptidase (
Escherichia coli
), IgA1-specific metalloendopeptidase (
Streptococcus sanguis
), tentoxilysin (
Clo
Beasley Ellen M.
Di Francesco Valentina
Wei Ming-Hui
Yan Chunhua
Applera Corporation
Genomics Celera
Karjala Justin D.
Monshipouri Maryam
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