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
2000-12-18
2003-04-08
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S252300, C435S069100, C435S254110, C435S254200, C435S348000, C435S349000, C435S419000, C536S023100, C536S023500, C530S350000
Reexamination Certificate
active
06544760
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to a new gene that encodes an enzyme inhibitor. In particular, the present invention relates to a novel protein, designated “Zkun11,” and to nucleic acid molecules encoding Zkun11.
BACKGROUND OF THE INVENTION
In animals, proteinases are important in wound healing, extracellular matrix destruction, tissue reorganization, and in cascades leading to blood coagulation, fibrinolysis, and complement activation. Proteinases are released by inflammatory cells for destruction of pathogens or foreign materials, and by normal and cancerous cells as they move through their surroundings.
The activity of proteinases is regulated by inhibitors (Roberts et al.,
Critical Reviews in Eukaryotic Gene Expression
5:385 (1995)). One family of proteinase inhibitors, the Kunitz inhibitors, includes inhibitors of trypsin, chymotrypsin, elastase, kallikrein, plasmin, coagulation factors XIa and IXa, and cathepsin G. These inhibitors thus regulate a variety of physiological processes, including blood coagulation, fibrinolysis, and inflammation.
Proteinase inhibitors regulate the proteolytic activity of target proteinases by occupying the active site and thereby preventing occupation by normal substrates. Although proteinase inhibitors fall into several unrelated structural classes, they all possess an exposed loop (variously termed an “inhibitor loop,” a “reactive core,” a “reactive site,” or a “binding loop”), which is stabilized by intermolecular interactions between residues flanking the loop and the protein core (Bode and Huber,
Eur. J. Biochem.
204:433 (1992)). Interaction between inhibitor and enzyme produces a stable complex, which disassociates very slowly, releasing either uncleaved inhibitor, or a modified inhibitor that is cleaved at the scissile bond of the binding loop.
One class of proteinase inhibitors, the Kunitz inhibitors, are generally basic, low molecular weight proteins comprising one or more inhibitory domains (“Kunitz domains”). The Kunitz domain is a folding domain of approximately 50-60 residues, which forms a central anti-parallel beta sheet and a short C-terminal helix. This characteristic domain comprises six cysteine residues that form three disulfide bonds, resulting in a double-loop structure. Between the N-terminal region and the first beta strand resides the active inhibitory binding loop. This binding loop is disulfide bonded through the P2 Cys residue to the hairpin loop formed between the last two beta strands. Isolated Kunitz domains from a variety of proteinase inhibitors have been shown to have inhibitory activity (see, for example, Petersen et al.,
Eur. J. Biochem.
125:310 (1996); Wagner et al.,
Biochem. Biophys. Res. Comm.
186:1138 (1992); Dennis et al.,
J. Biol. Chem.
270:25411 (1995)).
Proteinase inhibitors comprising one or more Kunitz domains include tissue factor pathway inhibitor (TFPI), tissue factor pathway inhibitor 2 (TFPI-2), amyloid &bgr;-protein precursor (A&bgr;PP), aprotinin, and placental bikunin. TFPI, an extrinsic pathway inhibitor and a natural anticoagulant, contains three tandemly linked Kunitz inhibitor domains. The amino-terminal Kunitz domain inhibits factor VIIa, plasmin, and cathepsin G; the second domain inhibits factor Xa, trypsin, and chymotrypsin; and the third domain has no known activity (Petersen et al.,
Eur. J. Biochem.
125:310 (1996)). TFPI-2 has been shown to be an inhibitor of the amidolytic and proteolytic activities of human factor VIIa-tissue factor complex, factor XIa, plasma kallikrein, and plasmin (Sprecher et al.,
Proc. Nat'l Acad. Sci. USA
91:3353 (1994); Petersen et al.,
Biochem.
35:266 (1996)). The ability of TFPI-2 to inhibit the factor VIIa-tissue factor complex and its relatively high levels of transcription in umbilical vein endothelial cells, placenta and liver suggests a specialized role for this protein in hemostasis (Sprecher et al.,
Proc. Nat'l Acad. Sci. USA
91:3353 (1994)).
Aprotinin (bovine pancreatic trypsin inhibitor) is a broad spectrum Kunitz-type serine proteinase inhibitor that has been shown to prevent activation of the clotting cascade. Aprotinin is a moderate inhibitor of plasma kallikrein and plamin, and blockage of fibrinolysis and extracorporeal coagulation have been detected in patients given aprotinin during open heart surgery (Davis and Whittington,
Drugs
49:954 (1995); Dietrich et al.,
Thorac. Cardiovasc. Surg.
37:92 (1989)). Aprotinin has also been used in the treatment of septic shock, adult respiratory distress syndrome, acute pancreatitis, hemorrhagic shock, and other conditions (Westaby,
Ann. Thorac. Surg.
55:1033 (1993); Wachtfogel et al.,
J. Thorac. Cardiovasc. Surg.
106:1 (1993)). The clinical utility of aprotinin is believed to arise from its inhibitory activity towards plasma kallikrein or plasmin (Dennis et al.,
J. Biol. Chem.
270:25411 (1995)). Placental bikunin is a serine proteinase inhibitor containing two Kunitz domains (Delaria et al.,
J. Biol. Chem.
272:12209 (1997)). Individual Kunitz domains of bikunin have been expressed and shown to be potent inhibitors of trypsin, chymotrypsin, plasmin, factor XIa, and tissue and plasma kallikrein (Delaria et al.,
J. Biol. Chem.
272:12209 (1997)).
Known Kunitz-type inhibitors lack specificity and may have low potency. Lack of specificity can result in undesirable side effects, such as nephrotoxicity that occurs after repeated injections of high doses of aprotinin. These limitations may be overcome by preparing isolated Kunitz domains, which may have fewer side effects than traditional anticoagulants. Hence, there is a need in the art for additional Kunitz-type proteinase inhibitors.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a novel Kunitz inhibitor protein, designated “Zkun11.” The present invention also provides Zkun11 variant polypeptides and Zkun11 fusion proteins, as well as nucleic acid molecules encoding such polypeptides and proteins, and methods for using these nucleic acid molecules and amino acid sequences.
DESCRIPTION OF THE INVENTION
1. Overview
The present invention provides nucleic acid molecules that encode a new human Kunitz inhibitor protein, designated as “Zkun11.” An illustrative nucleotide sequence that encodes Zkun11 is provided by SEQ ID NO:1. The encoded polypeptide has the following amino acid sequence: CNYPLKKGTC NSYLTRFYYN TLTFLCEPFV FSGCGGNRNN FKQKYFCEKM C (SEQ ID NO:2).
Sequence analysis revealed that Zkun11 can be represented by the following Kunitz domain sequence: C-X(8)-C-X(15)-C-X(7)-C-X(12)-C-X(3)-C (SEQ ID NO:5), where “X” is any amino acid residue and the values in parentheses indicate the number of such variable residues. Referring to SEQ ID NO:2, disulfide bonds are predicted to be formed by paired cysteine residues Cys1 to Cys51, Cys10 to Cys34, and Cys26 to Cys47. Amino acid substitutions can be made within the Zkun11 sequence so long as the conserved cysteine residues are retained and the higher order structure is not disrupted.
Example 1 illustrates that the Zkun11 gene is expressed differentially. For example, the Zkun11 gene is expressed in prostate tissue, but is not expressed in a variety of tissues including adrenal gland, bladder, colon, kidney, liver, ovary, pituitary, placenta, rectum, spleen, stomach, and testis. Thus, prostate tissue can be distinguished from other tissues identified herein, using nucleic acid molecules that encode Zkun11, as well as Zkun11 polypeptides, and antibodies (or antibody fragments) to Zkun11 polypeptides.
As detailed below, the present invention provides isolated polypeptides having an amino acid sequence that is at least 70%, at least 80%, or at least 90% identical to the amino acid sequence of SEQ ID NO:2, wherein such isolated polypeptides specifically bind with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO:2. Typically, such isolated polypeptides comprise an amino acid sequence with the structure: C-X(8)-C-X(15)-C-X (7)-C-X(12)-C-X(3)-C (SEQ ID NO:5), where “X” is any amino acid resi
Jones Phillip B. C.
Parker Gary E.
Prouty Rebecca E.
Ramirez Delia
ZymoGenetics Inc.
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