Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1998-10-21
2001-01-30
Stole, Einar (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007100, C435S007400, C435S007210, C435S252300, C435S254110, C435S320100, C435S325000, C435S455000, C435S471000, C536S023100, C536S023500
Reexamination Certificate
active
06180334
ABSTRACT:
BACKGROUND OF THE INVENTION
Mechanisms of proteoglycan breakdown in connective tissue are complex and involve multiple agents and pathways. Aggrecan is the large aggregating chondroitin sulfate proteoglycan of cartilage. See, for example, Doege, et al.
J. Biol. Chem.
266:894 (1991); GenBank/EMBL Accession Number M55172 (human aggrecan). In studies investigating the catabolism of aggrecan, experimental systems used have included monolayer cultures of primary chondrocytes from established chondrocyte cell lines (Hughes C E, et al.,
Biochem. J.
(1995) 305:799-804; Lark M W et al.,
J. Biol. Chem.
(1995) 270(6):2550-2556) and explant cultures using cartilage from a variety of anatomical sites and animal species (Flannery C R, et al.
J. Biol. Chem.
(1992) 267:1008-1014; Sandy J D, et al.
Biochim Biophys Acta
(1978) 543:536-44; Tyler J A,
Biochem. J.
1985; 225:493-507). The addition of cytokines such as IL-1 and TNF have been extensively used as agents which promote the degradation of the extracellular matrix (Hughes C E et al., supra (1995) Morales T I, et al.
Arch Biochem Biophys
(1992) 293(1):79-84; Fosang A J, et al.
Matrix
(1991) 11:17-24).
In particular, these two cytokines have been shown to target the catabolism of aggrecan.
Several studies have now lead to a number of important discoveries which have defined specific cleavage sites along the protein core of aggrecan (Ilic M Z, et al.
Arch Biochem Biophys
(1992) 294(1):115-22; Loulakis P, et al. Putative site(s) of enzymic cleavage.
Biochem J
(1992) 284:589-593; Sandy J D, et al.
J. Biol. Chem.
(1991) 266:8683-8685). In total there appear to be at least seven cleavage sites, and amino acid sequence analysis of cartilage proteoglycan breakdown products have defined two major sites of proteolytic cleavage in aggrecan which occur within the interglobular domain (IGD) between amino acid residues Asn
341
-Phe
342
(“AF”) and Glu
373
-Ala
374
(“EA”) (human sequence enumeration). Doege K J, et al.
J. Biol. Chem.
(1991) 266:894-902. The AF cleavage site generates a C-terminal catabolic fragment (ending with the C-terminal sequence DIPEN) consisting of the 50-60 kDa G1 domain that remains in the tissue complexed to hyaluronate (Flannery C R, et al., 1992). In recent studies Fosang et al. (
Trans. Orthop. Res. Soc.
(1995)20:4) have also identified N-terminal fragments (beginning with the N-terminal sequence FFG) from this cleavage site in synovial fluids from patients diagnosed with a variety of different arthritides (joint disease). Similarly, Witt et al (
Trans. Orthop. Res. Soc.
1995; 20:122) have found these glycosaminoglycan-containing N-terminal fragments in media samples from control and IL-1 stimulated porcine explant cultures.
The EA cleavage site produces glycosaminoglycan-containing N-terminal fragments (ARG . . . ) that appear as the major aggrecan degradation products isolated from synovial fluid of patients with arthritis (Lohmander LS, et al.
Arth. Rheum.
(1993) 36:1214-1222). These N-terminal fragments are also found in media from cartilage explant cultures treated with IL-1 or retinoic acid (Hughes C E, et al., 1995; Sandy J D, et al., 1991). A recent study (Lark M W, et al., 1995) identified the C-terminal fragment (ending with the C-terminal sequence EGE) in rat chondrosarcoma cells treated with retinoic acid.
The proteolytic activity responsible for this Glu
373
-Ala
374
cleavage has not been identified but it appears to have specificity for Glu-Xaa peptide bonds where Xaa is Ala, Gly or Leu. This activity has been termed “aggrecanase.” Fosang A J, et al.
J. Biol. Chem.
(1992) 267:19470-19474). As used in this specification, “aggrecanase” means a polypeptide (or polypeptides) that will catalyze cleavage of such Glu-Xaa peptide bonds in aggrecan. Although the sites of cleavage within the molecule have been well characterized, many of the agents responsible for generating the large number of different proteoglycan degradation products are still unidentified. It is believed that more than one enzyme is responsible for degradation of proteoglycans. Culture systems have been manipulated with a variety of agents that enhance proteoglycan catabolism, in an effort to discover the agent(s) responsible for its breakdown. However, these studies have been unsuccessful in defining any specific agent responsible for the degradation of aggrecan. Flannery C R, et al.
Trans. Orthop. Res. Soc.
(1993). Nonetheless, experimental data using purified aggrecan and modified aggrecan as a substrate for purified enzyme preparations has yielded some information on the specific cleavage sites of the enzymes involved in proteoglycan degradation. However, this in vitro work has not definitively ascertained the mechanism or identity of agents involved in the degradation of aggrecan in cartilage. Fosang A J, et al. (1992); Fosang A J, et al.
J. Biol. Chem.
(1991) 266:15579-15582; Fosang A J, et al.,
Biochem. J.
(1994) 304:347-351.
In previous studies (Hughes C E, et al., supra, (1995); Hughes C E, et al.
J. Biol. Chem.
(1992) 267:16011-16014) a number of monoclonal antibodies have been developed that are specific for the products (proteoglycan aggregate catabolites) that have been cleaved by specific proteinases. These antibodies have proved useful as tools in studying the mechanisms of breakdown of proteoglycan in cartilage explant culture systems, by allowing monitoring of specific cleavage products.
SUMMARY OF THE INVENTION
In one embodiment, the invention provides an isolated recombinant polypeptide substrate for aggrecanase. In another embodiment, the invention provides a recombinant substrate for aggrecanase that comprises the following components, beginning with the N-terminus and ending with the C-terminus:
a) the signal sequence of CD5;
b) the FLAG-epitope;
c) the interglobular domain of human aggrecan;
d) the hinge region of human IgG1;
e) the CH2 region of human IgG1; and
f) the CH3 region of human IgG1.
In another embodiment, the invention relates to a recombinant substrate for aggrecanase that comprises the amino acid sequence as set forth in
FIG. 6
(SEQ. ID NO. 3). In yet another embodiment, the invention relates to a recombinant substrate that comprises a portion of the amino acid sequence as set forth in
FIG. 6
(SEQ. ID NO. 3).
In a further embodiment, the invention relates to a recombinant substrate for aggrecanase that comprises the amino acid sequence as set forth in
FIG. 6
(SEQ. ID NO. 3) and wherein amino acid 34 is mutated to Ala. In yet a further embodiment, the invention relates to a recombinant substrate for aggrecanase that comprises a portion of the amino acid sequence as set forth in
FIG. 6
(SEQ. ID NO. 3) and wherein amino acid 34 is mutated to Ala.
In another embodiment, the invention relates to an isolated DNA sequence encoding a recombinant substrate for aggrecanase. In a further embodiment, one such DNA sequence comprises the nucleotide sequence of nucleotides 2350 to 4114 of
FIG. 7
(SEQ. ID NO. 4). In another embodiment, another such DNA sequence comprises a portion of the nucleotide sequence of nucleotides 2350 to 4114 of
FIG. 7
(SEQ. ID NO. 4). In yet a further embodiment, the invention relates to a DNA sequence encoding a substrate for aggrecanase, wherein said DNA sequence hybridizes under stringent conditions with the nucleotide sequence of nucleotides 2350 to 4114 of
FIG. 7
(SEQ. ID NO. 4).
The invention also relates to a vector comprising a DNA sequence encoding a recombinant substrate for aggrecanase. In another embodiment, the invention relates to a host cell comprising such a vector. In a further embodiment, the invention relates to a vector comprising the nucleotide sequence set forth in
FIG. 7
(SEQ ID NO:4).
In still another embodiment, the invention relates to a cell culture system (or method) for monitoring aggrecanase activity in a sample comprising:
(a) mixing freshly isolated chondrocyte cells and a recombinant substrate for aggrecanase;
(b) incubating the reaction mixture of step (a); and
(c) detecting the presence or absence of aggrecanase
Bartnik Eckart
Buettner Frank
Caterson Bruce
Eidenmueller Bernd
Hughes Clare
Aventis Pharma Deutschland GmbH
Foley & Lardner
Stole Einar
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