Aggrecan degrading metallo proteases

Details Aggrecan degrading metallo proteases Aggrecan degrading metallo proteases

2002-09-19

2004-06-22

Hutson, Richard (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S212000, C435S219000, C435S183000, C435S252300, C435S320100, C435S325000, C536S023100, C536S023200, C536S023500, C530S350000

Reexamination Certificate

active

06753176

ABSTRACT:

FIELD OF THE INVENTION
The invention is directed to the family of proteins that exhibit aggrecanase activity, the nucleic acids encoding such enzymes, processes for production of recombinant aggrecanases, compositions containing such enzymes, antibodies raised against these enzymes, and the use of these enzymes or antibodies in various assays and therapies.
BACKGROUND OF THE INVENTION
Aggrecan is the major proteoglycan of cartilage and provides this tissue with its mechanical properties of compressibility and elasticity. In arthritic conditions one of the earliest changes observed in cartilage morphology is the depletion of aggrecan [Mankin et al. (1970) J. Bone Joint Surg. 52A, 424-434], which appears to be due to an increased rate of degradation.
The aggrecan molecule is composed of two N-terminal globular domains, G1 and G2, which are separated by an approximately 150 residue interglobular domain (IGD), followed by a long central glycosaminoglycan (GAG) attachment region and a C-terminal globular domain, G3 [Hardingham et al. (1992) in Articular Cartilage and Osteoarthritis: Aggrecan, The Chondroitin Sulfate/Keratin Sulfate Proteoglycan from Cartilage (Kuettner et al.) pp. 5-20, Raven Press, New York and Paulson et al. (1987) Biochem. J. 245, 763-772]. These aggrecan molecules interact through the G1 domain with hyaluronic acid and a link protein to form large molecular weight aggregates which are trapped within the cartilage matrix [Hardingham et al. (1972) Biochim. Biophys. Acta 279, 401-405, Heinegard et al. (1974) J. Biol. Chem. 249, 4250-4256, and Hardingham, T. E. (1979) Biochem. J. 177, 237-247]. Loss of aggrecan from cartilage in arthritic conditions involves proteolytic cleavage of the aggrecan core protein within the IGD, producing a N-terminal G-1 fragment that remains bound to hyaluronic acid and the link protein within the matrix, releasing a large C-terminal GAG-containing aggrecan fragment that diffuses out of the cartilage matrix. Loss of the C-terminal fragment results in cartilage deficient in its mechanical properties. This deficiency arises because the GAGs are the components of aggrecan that impart the mechanical properties to the molecule through their high negative charge and water binding capacity.
Two major sites of proteolytic cleavage have been identified within the IGD, one between amino acid residues Asn
341
-Phe
342
and the other between amino acid residues Glu
373
-Ala
374
. Although G1 fragments formed by cleavage at the Asn
341
-Phe
342
site and at the Glu
373
-Ala
374
site have been identified within articular cartilage [Flannery et al. (1992) J. Biol. Chem. 267, 1008-1014], the N-terminus identified on the large GAG-containing aggrecan C-terminal fragments in synovial fluids of patients with osteoarthritis [Sandy et al. (1992) J. Clin. Invest. 69, 1512-1516], inflammatory joint disease [Lohmander et al. (1993) Arthritis Rheum. 36, 1214-1222] and in the media from cartilage explant and chondrocyte cultures stimulated with interleukin-1 or retinoic acid [Sandy et al. (1991) J. Biol. Chem. 266, 8198., Sandy et al. (1991) J. Biol. Chem. 266, 8683-8685., Loulakis et al. (1992) Biochem. J. 264, 589-593., Ilic et al. (1992) Arch. Biochem. Biophys. 294, 115-122., Lark et al. (1995) J. Biol. Chem. 270, 2550-2556.] was ARGSVIL, indicating that they were formed by cleavage between amino acid residues Glu
373
-Ala
374
. These observations suggest that cleavage at this site may be responsible for cartilage degradation.
Although many matrix metalloproteases (MMP-1, -2, -3, -7, -8, -9 and 13) have been shown to cleave in vitro at the Asn
341
-Phe
342
site, digestion of aggrecan with a number of these purified proteases has not resulted in cleavage at the Glu
373
-Ala
374
site [Fosang et al. (1992) J. Biol. Chem. 267, 19470-19474., Flannery et al. (1992) J. Biol. Chem. 267, 1008-1014., Fosang et al. (1993) Biochem. J. 295, 273-276., Fosang et al. (1996) FEBS Lett. 380, 17-20., Flannery et al. (1993) Orthop. Trans. 17, 677., and Fosang et al. (1994) Biochem. J. 305, 347-351]. Therefore, cleavage at this site has been attributed to a novel, proteolytic activity, “aggrecanase”.
In addition to the Glu
373
-Ala
374
bond within the interglobular domain of aggrecan, four potential aggrecanase-sensitive sites have been identified within the C-terminus of the aggrecan core protein [Loulakis et al. (1992) Biochem. J. 264, 589-593. and Sandy et al. (1995) Acta Orhtop Scand (Suppl 266) 66, 26-32]. Although cleavage at these sites which are not within the interglobular domain would not be expected to release the major portion of the aggrecan molecule from the matrix, they may be involved in earlier processing of aggrecan within the matrix.
SUMMARY OF THE INVENTION
The invention encompasses a novel family of biologically active aggrecan degrading metallo proteases (“ADMP”) capable of cleaving the aggrecan monomer core protein at the Glu
373
-Ala
374
aggrecanase site, as isolated and purified polypeptides. An object of the invention covers novel sequences of nucleic acids which encode for members of the ADMP family, and to expression vectors containing cDNA which encodes for novel members of the ADMP family. Another object of the invention is host cells that have been transfected or transformed with expression vectors which contain cDNA that encodes for the ADMP family of polypeptides, and processes for producing members of the ADMP family by culturing such host cells under conditions conducive to expression of an ADMP. Another object of the invention is probes containing nucleic acid sequences that hybridize to a native ADMP nucleotide sequence and the use of these probes for detection of message for an ADMP in biological samples. A further object of the invention is antibodies raised against an ADMP, which may be created as a result of the purification and isolation of members of the ADMP family and the use of such antibodies for the detection of ADMPs in biological samples. Assays utilizing an ADMP to screen for its potential inhibitors are another object of this invention. Members of the ADMP family used to design novel inhibitors of proteases exhibiting aggrecanase activity are also part of the instant invention.
Members of the ADMP family are capable of cleaving the aggrecan monomer core protein at the Glu
373
-Ala
374
site, but do not readily cleave aggrecan at the Asn
341
-Phe
342
, MMP-sensitive cleavage site, and the zymogen form of the protein consists of the following domains: a propeptide domain containing a furin site, a metalloprotease domain, a disintegrin-like domain and a thrombospondin homologous domain.
As used herein, the term “zymogen” refers to the latent, full-length protein synthesized by the cells and further processed to a catalytically active form, the term “propeptide domain” refers to the N-terminal region of the molecule which contains a cysteine residue involved in latency of the protein, the term “furin cleavage site” refers to a region of the molecule containing a tetra basic sequence of amino acids susceptible to cleavage by furin or furin-like proteases, the term “metalloprotease domain” refers to a region of the molecule which contains a zinc-binding motif with the consensus sequence, HExxHxxGxxH, responsible for the catalytic activity of the protein, the term “disintegrin-like domain” refers to a region of the molecule which exhibits sequence similarity to the disintegrin family of anti-coagulant peptides found in snake venoms, which are characterized by a high cysteine content and have the ability to disrupt cell-matrix interaction, and the term “thrombospondin homologous domain” refers to a region of the molecule containing one or more thrombospondin type 1 (TSP1) motifs with sequence homologous to the amino acid sequence of TSP1 repeats which are conserved in thrombospondin 1 and 2 and have been implicated in the interaction of thrombospondin with sulfated glycoconjugates such as heparin and heparan sulfate.
The first isolated and p

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