22012, human carboxypeptidase

Details 22012, human carboxypeptidase 22012, human carboxypeptidase

1999-06-30

2002-04-09

Caputa, Anthony C. (Department: 1642)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S069100, C530S350000, C536S001001, C536S018700, C536S022100, C536S023100, C536S023200

Reexamination Certificate

active

06369210

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a newly identified human carboxypeptidase. The invention also relates to polynucleotides encoding the carboxypeptidase. The invention further relates to methods using the carboxypeptidase polypeptides and polynucleotides as a target for diagnosis and treatment in carboxypeptidase-related disorders. The invention further relates to drug-screening methods using the carboxypeptidase polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the carboxypeptidase polypeptides and polynucleotides. The invention further relates to procedures for producing the carboxypeptidase polypeptides and polynucleotides.
BACKGROUND OF THE INVENTION
Proteolytic enzymes are involved in many cellular processes. The carboxypeptidase family of enzymes catalyze, the cleavage of C-terminal amino acids of peptides and proteins, altering their biological activity. Lysosomal carboxypeptidase enzymes are highly concentrated in lysosomes, but may also be active extracellularly after their release from lysosomes in soluble form or bound to transmembrane or other membrane-associated proteins. Carboxypeptidases may cleave peptides in a sequence-specific manner. For example, prolylcarboxypeptidases cleave only peptides linked to proline residues (for example, des-Arg9-bradykinin, angiotensin II). There is also evidence that these enzymes are involved in terminating signal transduction by inactivating peptide ligands after receptor endocytosis.
In contrast to endoproteases which cleave internal peptide bonds of proteins and polypeptides, carboxypeptidases (CPs) catalyze the cleavage of only the C-terminal peptide bond, releasing one amino acid at a time. The two main groups of CPs include serine CPs and metallo-CPs, the serine CPs containing a signature trio of Ser, Asp, His in the active site. This trio is also contained in prolylendopeptidase serine proteases. Serine CPs include polycarboxypeptidase (PRCP) also referred to as angiotensinase C; and deamidase, also referred to as cathepsin A and lysosomal protective protein. See Skidgel et al. (1998)
Immunological Reviews
161:129-141.
Metallo-CPs contain a signature glutamic acid as the primary catalytic residue and require zinc-binding for activity. Metallo-CPs can be grouped by substrate specificity into CPA and CPB types; the CPA type preferentially cleaving C-terminal hydrophobic residues, and the CPB type cleaving only peptides with C-terminal basic Arg or Lys residues. See R. A. Skidgel (1993) In: Hooper N M, ed.
Zinc Metalloproteases in Health and Disease
, London: Taylor & Francis, Ltd., p. 241-283.
CPM is a B type carboxypeptidase which is anchored on cell membranes via gylcosylphosphatidylinositol (GPI) association with its mildly hydrophobic stretch of 15 C-terminal amino acids. As in many other proteins sharing this anchoring mechanism, CPM is released from the membrane by bacterial phosphatidylinositol-specific phospholipase C. Human CPM is a glycoprotein of 426 amino acid residues with 43% identity to human intracellular secretory granular CP (CPE), 41% with the active 50 kDa subunit of human plasma CPN, and 15% with bovine pancreatic CPA or CPB. The active sites of these CPs contain conserved amino acid residues corresponding to the zinc binding residues His
66
Glu
69
and His
173
, substrate binding residues Arg
137
and Tyr
242
, and the catalytic Glu
264
, as designated for CPM. Sequence homologies around these conserved residues is high, with an identity between CPs M, E and N of approximately 70-90%. See Tan et al. (1989)
J. Biol. Chem
. 264:13165-13170; Deddish et al. (1990)
J. Biol. Chem
. 265:15083-15089; R. A. Skidgel (1993) In: Hooper N M, ed.
Zinc Metalloproteases in Health and Disease
, London: Taylor & Francis, Ltd., p. 241-283. CPM has been mapped to the chromosomal location of chromosome 12q13-q15 which is associated with a variety of solid tumors.
The optimal pH range of CPM is in the neutral range of 6.5-7.5. As no endogenous inhibitors are known for CPM, the enzyme is considered to be constitutively active. Synthetic inhibitors including Arg analogs DL-2mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA) and guanidinoethylmercaptosuccinic acid (GEMSA) inhibit CPM. See R. A. Skidgel (1991) In: Conn P M, ed.
Methods in Neurosciences: Peptide Technology
Vol. 6, Orlando: Academic Press, p. 373-385; Plummer et al. (1981)
Biochem. Biophys. Res. Comm
. 98: 448-254.
As with other B type regulatory CPs, CPM cleaves only C-terminal Arg or Lys residues; however, CPM has a preference for the C-terminal Arg. The penultimate amino acid also affects the rate of hydrolysis. Naturally occurring peptide substrates of CPM include bradykinin, Arg
6
- and Lys
6
enkephalins, dynorphin A
1-13
and epidermal growth factor (EGF). See Sidgel et al. (1989)
J. Biol. Chem
. 264:2236-2241; McGwire et al. (1995)
J. Biol. Chem
. 270:17154-17158.
CPM is primarily found on the plasma membrane, with highest levels found in lung and placenta. It is also present in kidney, blood vessels, intestine, brain and peripheral nerves. See R. A. Skidgel (1988)
Trends Pharm. Sci
. 9:299-304; Skidgel et al. (1984)
Biochem. Pharmacol
. 33: 3471-3478; Skidgel et al. (1991)
FASEB J
. 5: 1578; Nagae et al. (1992)
J. Neurochem
. 59:2201-2212; Nagae et al. (1993)
Am. J. Respir. Cell Mol. Biol
. 9:221-229. Expression of CPM is responsive to differentiation of monocytes and lymphocytes. See de Saint-Vis et al. (1995)
Blood
86:1098-1105; Rehli et al. (1995)
J. Biol. Chem
. 270:15644-15649.
CPM participates in the control of peptide hormone activity at the cell surface and degradation of extracellular proteins and peptides. It catalyzes the second step in prohormone processing and removes C-terminal Arg or Lys residues from peptides released from prohormones. CPM functions as a soluble enzyme after its release from the plasma membrane and may function in the plasma membrane form to control peptide receptor activities. CPM can regulate receptor specificity of kinins by cleaving the C-terminal ARG
9
, for example, from bradykinin. The intact bradykinin binds the B2 receptor. The cleaved bradykinin (des-ARG
9
-bradykinin). Des-ARG
9
-bradykinin also binds the B1 receptors: stimulates IL-1 and tumor necrosis factor release from macrophages. Regulation of the B1 receptor is associated with injury or inflammation. CPM may also be involved with other inflammatory mediators, such as anaphylatoxin C5a which mediates histamine release. In addition, CPM may metabolize growth factors containing terminal Arg or Lys, such as EGF, EGF-like peptides, nerve growth factor (NGF) amphiregulin, hepatocyte growth factor, erythropoietin, and macrophage-stimulating protein. In the lung, varying levels of CPM are associated with pneumocystic or bacterial pneumonia or lung cancer, and in the placenta, CPM may protect the fetus from maternally derived peptides. See R. A. Skidgel (1992)
J. Cardiovasc. Pharmacol
. 20(Suppl. 9):S4-S9; Bhoola et al. (1992)
Pharmacol. Rev
. 44:1-80; R. A. Skidgel (1993) In: Hooper N M, ed.
Zinc Metalloproteases in Health and Disease
, London: Taylor & Francis, Ltd., p. 241-283; Dragovic et al. (1995)
Am. J. Respir. Crit. Care Med
. 152:760-764; Nagae et al. (1992)
J. Neurochem
. 59:2201-2212; MacFadden et al. (1988)
FASEB J
. 2:1179 (Abstract).
Another B-type regulatory CP metalloprotein is CPD, a membrane-bound glycoprotein. Human CPD is a protein of 1,377 amino acids with 75% identity with duck GP180 and 90% identity with rat CPD. Human CPD contains two hydrophobic regions located at the C- and N-termini. A 55-60 residue cytoplasmic domain is highly conserved among duck, human and rat sequences and may be significant in intracellular sorting, protein-protein interactions or endocytosis. CPD contains three tandem CP homology domains numbered sequentially from the N- to the C-terminus, and thereby may contain more than one active site. See Tan et al. (1997)
Biochem. J
. 327:81-87; Skidgel et

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