Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-11-19
2002-05-07
Schwartzman, Robert A. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C435S252300, C435S320100, C435S325000, C536S023500
Reexamination Certificate
active
06383780
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a newly identified human aminopeptidase. The invention also relates to polynucleotides encoding the aminopeptidase. The invention further relates to methods using the aminopeptidase polypeptides and polynucleotides as a target for diagnosis and treatment in aminopeptidase-related disorders. The invention further relates to drug-screening methods using the aminopeptidase polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the aminopeptidase polypeptides and polynucleotides. The invention further relates to procedures for producing the aminopeptidase polypeptides and polynucleotides.
BACKGROUND OF THE INVENTION
Proteases may function in carcinogenesis by inactivating or activating regulators of the cell cycle, differentiation, programmed cell death, or other processes affecting cancer development and/or progression. Consistent with the model involving protease activity and tumor progression, certain protease inhibitors have been shown to be effective inhibitors of carcinogenesis both in vitro and in vivo.
Aminopeptidases
Aminopeptidases (APs) are a group of widely distributed exopeptidases that catalyse the hydrolysis of amino acid residues from the amino-terminus of polypeptides and proteins. The enzymes are found in plant and animal tissue, in eukaryotes and prokaryotes, and in secreted and soluble forms. Biological functions of aminopeptidases include protein maturation, terminal degradation of proteins, hormone level regulation, and cell-cycle control.
The enzymes are implicated in a host of conditions and disorders including aging, cancers, cataracts, cystic fibrosis and leukemias. In eukaryotes, APs are associated with removal of the initiator methionine. In prokaryotes the methionine is removed by methionine aminopeptidase subsequent to removal of the N-formyl group from the initiator N-formyl methionine, facilitating subsequent modifications such as N-acetylation and N-myristoylation. In
E. coli
AP-A (pepA), the xerB gene product is required for stabilization of unstable plasmid multimers.
APs are also involved in the metabolism of secreted regulatory molecules, such as hormones and neurotransmitters, and modulation of cell-cell interactions. In mammalian cells and tissues, the enzymes are apparently required for terminal stages of protein degradation, and EGF-induced cell-cycle control; and may have a role in protein turnover and selective elimination of obsolete or defective proteins. Furthermore, the enzymes are implicated in the supply of amino acids and energy during starvation and/or differentiation, and degradation of transported exogenous peptides to amino acids for nutrition. As leukotriene A4 hydrolase may be an aminopeptidase, APs may further have a role in inflammation. Industrial uses of the enzymes include modification of amino termini in recombinantly expressed proteins. See A. Taylor (1993)
TIBS
18: 1993:167-172.
A variety of aminopeptidases have been identified from a wide variety of tissues and organisms, including zinc aminopeptidase and aminopeptidase M from kidney; arginine aminopeptidase from liver; aminopeptidase N
b
from muscle; leucine aminopeptidase (LAP) from lens and kidney; aminopeptidase A (xerB gene product) from
E. coli
; yscl APE1/LAP4 and aminopeptidase A (pep4 gene product) from S. cerevisiae; LAP from Aeromonas; dipeptidase from mouse ascites; methionine aminopeptidase from Salmonella,
E. coli, S. cerevisiae
and hog liver; and D-amino acid aminopeptidase from Ochrobactrum anthropi SCRC C1-38.
Of these aminopeptidases, the structure of bovine lens leucine aminopeptidase (blLAP) is well-characterized and consists of a homohexamer synthesized as a large precursor, each monomer containing two thirds of the protein in a major lobe and one third in a minor lobe. The minor lobe contains the N-terminal 150 residues. All putative active site residues, presumably also the inhibitor bestatin-binding site, are found in the C-terminal lobe and include Ala-333, Asn-330, Leu360, Asp332, Asp255, Glu-334, Lys250, Asp273, Met-454, Ala-451, Gly362, Thr-359, Met270, Lys262, Gly362 and Ile-421.
Many aminopeptidases are metalloenzymes, requiring divalent cations, with specificities for Zn
2
+ or Co
2
+. However, particular sites of certain aminopeptidases can readily utilize Mn
2
+ and Mg
2
+. Residues used to ligand Zn
2
+ include the His Glu and Asp Glu Lys configurations. In addition to bestatin, boronic and phosphonic acids, &agr;-methylleucine and isoamylthioamide are identified as competitive inhibitors for most aminopeptidases. See A. Taylor (1993)
TIBS
18: 1993:167-172; Burley et al. (1992)
J. Mol. Biol
. 224:113-140; Taylor et al. (1993)
Biochemistry
32:784-790.
Aminopeptidases from various organisms and various tissues within an organism have high degrees of primary sequence homology, as indicated by immunological assays. Some enzymes also exhibit very similar kinetic profiles. Direct amino acid sequence comparison of blLAP and aminopeptidase-A from
E. coli
shows 18, 44 and 35% identity for the amino- and carboxy-terminals, and the entire protein, respectively. The comparison shows 46, 66, and 60% identity for the respective regions. See Burley et al. (1992)
J. Mol. Biol
. 224:113-140.
Bovine lens leucine aminopeptidase (blLAP), bovine kidney LAP, human lens and liver LAPs, hog lens, kidney and intestine LAPs, proline-AP,
E. coli
AP-A, AP-I and the
S. typhimurium pepA
gene product have been categorized as belonging to the family of zinc aminopeptidases which utilize the residues Asp Glu Lys for zinc binding and the active site amino acid configuration described above for bovine LAP for substrate binding. This family, possibly also including Aeromonas LAP, is suggested to be distinguished from zinc proteases which utilize His His Glu in zinc binding and Arg in substrate binding. The Saccharomyces methionine-AP is characterized to contain two zinc finger like motifs in the amino-terminus and shares little homology with blLAP. See A. Taylor (1993)
TIBS
18:167-171; Watt et al. (1989)
J. Biol. Chem
. 264:5480-5487.
Leucine aminopeptidase expression is regulated at the transcriptional level, evidenced by enhancement of both activity and mRNA upon removal of serum in in vitro aged and/or transforming lens epithelial cells. Furthermore, LAP mRNA and protein are induced by interferon y in human ACHN renal carcinoma, A549 lung carcinoma, HS153 fibroblasts and A375 melanoma. Regulation by development and growth is also implicated. The
E. coli
pepN gene is transcriptionally regulated upon anaerobiosis and phosphate starvation. Membrane bound AP-N (CD 13) is expressed in a lineage-restricted manner by subsets of normal and malignant cells, apparently through regulation by physically distinct promoters. Expression of the yeast yscI product APE1 is dependent upon the levels of yscA and PEP4 gene products. Synthesis of APE1 is sensitive to media glucose levels, and the activity of yeast aminopeptidase is sensitive to substitution of ammonia rather than peptone as the source of nitrogen. See Harris et al. (1992)
J. Biol. Chem
. 267:6865-6869; Jones et al. (1982)
Genetics
102:665-677.
Aminopeptidase B
Aminopeptidase B is an exopeptidase that removes arginine and/or lysine from various amino terminal peptide substrates. This enzyme is structurally related to leukotriene A4 hydrolase. The activity of aminopeptidase B is dependent upon Zn
2
+. With respect to primary structure, the enzyme isolated from rat testis exhibits an amino terminal potential signal peptide and a Zn
2
+ binding consensus sequence (HEXXHX
18
E). In view of the fact that the enzyme contains this consensus sequence, the enzyme can be classified as a M1 family metallopeptidase.
In the M1 family, the aminopeptidase is most closely related to the leukotriene A
4
hydrolase. Accordingly, in addition to the aminopeptidase activity, the enzyme contains leukotriene A
4
epo
Kapeller-Libermann Rosana
MacBeth Kyle J.
White David
Alston & Bird LLP
Millennium Pharmaceuticals Inc.
Schnizer Richard
Schwartzman Robert A.
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