From Inflammatory Diseases Research, The DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880 and the § Microbiology Department, University of Virginia, Charlottesville, Virginia 22908
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two primary cleavage sites have been identified within the interglobular domain of the cartilage aggrecan core protein: one is between amino acid residues Asn 341 and Phe342, where many matrix metalloproteinases (MMP) have been shown to cleave; and the other is between amino acid residues Glu373 and Ala374. Although cleavage at the Glu373-Ala374 site is believed to play a critical role in cartilage aggrecan degradation in arthritic diseases, the enzyme responsible for cleavage at this site, "aggrecanase," has not been identified. Members of the ADAM (a disintegrin and metalloproteinase) family of proteins, which shows structural homology to the snake venom hemorrhagic metalloproteinases (reprolysins), have recently been demonstrated to be expressed in articular chondrocytes. Because many ADAM family members have a putative proteinase function, this raises the possibility that aggrecanase may be a member of this family of proteases. To examine whether reprolysins have the ability to cleave aggrecan at either the aggrecanase site or the MMP site, the snake venom hemorrhagic toxin metalloproteinase HT-d (atrolysin C) was tested for its ability to cleave bovine aggrecan monomer. Cleavage was monitored using the BC-3 antibody, which recognizes aggrecan fragments with the new NH2 terminus ARGSV generated by cleavage at the aggrecanase site, and with the AF-28 antibody, which recognizes aggrecan fragments with the new NH2 terminus FFGVG generated by cleavage at the MMP site. Cleavage at both the aggrecanase and MMP sites occurred in a concentration-dependent manner with 100 nM atrolysin C or greater. AF-28-reactive fragments were generated by 30 min of incubation, and levels were maximal by 8 h; BC-3-reactive fragments were detected at 2 h and continued to increase through 48 h, thus suggesting that atrolysin C can cleave at the MMP and aggrecanase sites. NH2-terminal aggrecan fragments generated by cleavage at the aggrecanase site were also detected using antisera recognizing the new COOH terminus, NITEGE, formed by cleavage at the Glu373-Ala374 bond, indicating that cleavage at this site does not require prior cleavage at the MMP site. These data provide the first demonstration that a reprolysin can cleave the core protein of aggrecan and the first example of a specific protease that can cleave at the aggrecanase site independent of cleavage at the MMP cleavage site.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aggrecan is the major proteoglycan of cartilage and provides this tissue with its mechanical properties of compressibility and elasticity. Aggrecan monomers interact with hyaluronic acid (HA)1 and are usually found as part of a large aggregate containing 10-100 monomers/HA molecule. The primary role of aggrecan is to swell and hydrate the framework of cartilage collagen fibrils thus providing cartilage with its properties of compressibility and elasticity. The NH2 terminus of the aggrecan monomer core protein is comprised of two globular domains called G1 and G2 which are separated by an interglobular domain that spans about 150 residues in length. The G2 region is followed by a long central glycosaminoglycan attachment region and a COOH-terminal globular domain, G3 (1, 2).
In cartilage degradation associated with diseases such as osteoarthritis and rheumatoid arthritis, aggrecan is one of the first matrix components to undergo measurable loss, which ultimately leads to loss of cartilage function. Proteolytic cleavage within the interglobular domain is believed to be responsible for the loss of aggrecan from cartilage. However, the enzyme responsible for aggrecan cleavage in these diseases has yet to be identified.
Two major sites of proteolytic cleavage have been identified within the interglobular domain, one between amino acids Asn341 and Phe342 and the other between Glu373 and Ala374. Many matrix metalloproteinases, including MMP-1, -2, -3, -7, -8, -9, and -13, have been shown to cleave aggrecan at the Asn341-Phe342 site, but to date attempts to generate cleavage at the Glu373-Ala374 site with a number of MMPs and other purified proteases have been relatively unsuccessful (3-7). Of the enzymes evaluated, only MMP-8 has shown the ability to clip at this site (8, 9), and cleavage was seen only after cleavage at the Asn341-Phe342 MMP site. Thus, cleavage at the Glu373-Ala374 site has been attributed to an as yet unidentified enzyme given the name "aggrecanase" because of its ability the cleave the aggrecan core molecule.
A novel family of metalloproteinases named ADAMs (a disintegrin and metalloproteinase) has been described (10-14). These proteins are structurally related to the snake venom hemorrhagic toxins (reprolysins) and contain minimally a metalloproteinase domain, and some members have additional domain structures such as NH2-terminal propeptide domains, disintegrin domains, and cysteine-rich domains. Cell-associated ADAMs also contain an epidermal growth factor-like transmembrane domain and cytoplasmic domain at the COOH terminus of the molecule. Recent studies have demonstrated that human articular chondrocytes express mRNA for three members of the ADAMs family, ADAM-10, -12, and -15 (15). In addition, some ADAM proteins have been shown to be up-regulated at the mRNA level by interleukin-1 (16), suggesting that their expression may be inflammation-associated. Members of the reprolysin family of enzymes have been shown to degrade basement membranes and cleave the extracellular matrix molecules, type IV collagen, laminin, and fibronectin (17). X-ray crystal structures of reprolysins show that their structure is similar to that of the astacin family and the MMPs, especially within the active site region (18). Taken together these data open the possibility that ADAM family members may be involved in the proteolytic cleavage of the cartilage extracellular matrix.
In this study we investigated the ability of a reprolysin, atrolysin C, to cleave the aggrecan core protein within the interglobular domain. Using the monoclonal antibody BC-3, which recognizes the NH2-terminal neoepitope ARGSVIL, generated by cleavage at the Glu373-Ala374 aggrecanase site (19), and the monoclonal antibody AF-28, which recognizes the NH2-terminal neoepitope FFGVG, generated by cleavage at the Asn341-Phe342 MMP site (20) (Fig. 1), we demonstrate that atrolysin C is capable of cleaving cartilage aggrecan at both the aggrecanase site and the MMP site. Our results also show that these cleavages occur concomitantly, unlike cleavage by MMP-8, which required complete depletion of the preferred Asn341-Phe342 MMP site to clip at the aggrecanase site (8, 9). These findings establish that a reprolysin is able to cleave aggrecan at the aggrecanase site and open the possibility that the as yet unidentified cartilage aggrecanase enzyme may be a member of the ADAM family of proteins.
|
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials-- The snake venom hemorrhagic toxin HT-d (atrolysin C) was purified from rattlesnake venom (21). Purity of the atrolysin C was demonstrated by silver stain analysis where only one band was detected after loading 100 ng of total protein on a 10% gel. The activity of the atrolysin C sample could be blocked completely by the metalloproteinase inhibitor EDTA and by a specific hydroxamate MMP inhibitor confirming the matrix metalloproteinase nature of this enzyme. Antibody BC-3, which recognizes the new NH2 terminus ARGSVIL on aggrecan fragments produced by aggrecanase cleavage, was obtained from Dr. Bruce Caterson (University of Wales, Cardiff, U. K.). Antibody AF-28, which recognizes the new NH2 terminus FFGVG on aggrecan fragments produced by MMP cleavage, was provided by Dr. Amanda Fosang (University of Melbourne, Parkville, Australia). NITEGE antibody, which recognizes the new COOH terminus NITEGE on aggrecan fragments produced by aggrecanase cleavage, was from Dr. Michael Lark and Merck Research Laboratories (Rahway, NJ). Monoclonal antibody MAB2005, which recognizes a protein-related epitope associated with the binding region of chondroitin sulfate chains, was purchased from Chemicon International (Temecula, CA). MAB2005 reacts with the protein-related epitope only after chondroitinase ABC digestion, and it has been shown not to react with any carbohydrate structural elements (22). Biotinylated hyaluronic acid (bHA), which recognizes the functional intact aggrecan G1 domain (23), was provided by Dr. James Melrose (University of Sydney, Australia). Sites recognized by these reagents are shown in Fig. 1. Chondroitinase ABC lyase (Proteus vulgaris) (EC 4.2.2.4), keratanase (Pseudomonas sp.) (EC 3.2.1.103), and keratanase II (Bacillus sp.) were from Seikaguku (Kogyo, Japan). Polyvinylidene difluoride membrane filters used for Western transfer and 8-16% Tris-glycine gels used to separate aggrecan products were from Novex (San Diego, CA). Goat anti-mouse IgG alkaline phosphatase conjugate and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color development used for Western detection were from Promega (Madison, WI). Streptavidin-alkaline phosphatase was purchased from Life Technologies, Inc.
Aggrecan Isolation--
Nasal septa were removed from bovine
noses obtained fresh from slaughter. The cartilage was sliced, and
aggrecan was extracted by stirring at 4 °C for 48 h in 10 volumes of 4 M guanidine-HCl in 0.05 M sodium
acetate, pH 5.8, containing the protease inhibitors 0.01 M
EDTA, 0.1 M 6-aminohexanoic acid, 2 mM
phenymethanesulfonyl fluoride, and 0.05 M benzamidine-HCl.
Aggrecan monomers were isolated by equilibrium density gradient
centrifugation in cesium chloride (24, 25), and the bottom of this
gradient (d > 1.54 g/ml) containing the monomers was
dialyzed at 4 °C against water, lyophilized, and stored at
20 °C.
Enzyme Digestion-- Digestions were carried out in 100 µl of 50 mM Tris-HCl buffer, pH 7.5, containing 100 mM NaCl and 10 mM CaCl2. Purified bovine aggrecan (500 nM) was incubated with atrolysin C at 37 °C for the indicated times and concentrations. The reactions were quenched with EDTA and analyzed by labeling with bHA or by immunolocation with MAB2005, BC-3, NITEGE, or AF-28 antibodies in a Western blot analysis.
Analysis of Aggrecan Products--
For analysis of fragments by
Western blot, aggrecan was enzymatically deglycosylated with
chondroitinase ABC (0.1 unit/10 µg of aggrecan) for 1 h at
37 °C and then with keratanase (0.1 unit/10 µg of aggrecan) and
keratanase II (0.002 unit/10 µg of aggrecan) for 2 h at 37 °C
in buffer containing 50 mM sodium acetate, 0.1 M Tris-HCl, pH 6.5. After digestion the aggrecan was
precipitated with 5 volumes of acetone and reconstituted in 30 µl of
Tris-glycine-SDS sample buffer containing 2.5% -mercaptoethanol and
heated for 3 min at 100° C. Samples to be analyzed in the bHA
Western were not reduced and not heated before SDS-polyacrylamide gel
electrophoresis to maintain the structural integrity of the G1
domain.
Western Blot Analysis-- 20 µg of glycosaminoglycan from each sample was loaded on an 8-16% Tris-glycine gel and separated by SDS-polyacrylamide gel electrophoresis under reducing conditions. The separated proteins were transferred to polyvinylidene difluoride membranes and immunolocated with a 1:500 dilution of the BC-3 antibody, a 1:1000 dilution of the AF-28 antibody, a 1:5,000 dilution of the NITEGE antibody, a 1:2,000 dilution of the MAB2005 antibody, or with 2 µg/ml bHA. Membranes that were analyzed in the bHA Western for a functional G1 domain were washed more thoroughly with Tris-buffered saline to remove any residual SDS to allow the aggrecan fragments to renature to their native conformation before the addition of bHA. Subsequently, the membranes were incubated with a 1:5,000 dilution of goat anti-mouse IgG alkaline phosphatase conjugate for the BC-3, AF-28, NITEGE, and MAB2005 analysis or a 1:8,000 dilution of streptavidin alkaline phosphatase conjugate for the bHA analysis, and the aggrecan cleavage products were visualized by developing the blots in nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color developing reagent. Overnight transfer resulted in complete transfer of both low and high molecular fragments, and the densitometric response was found to be linear over the density ranges required for the blots. BC-3-, NITEGE-, and AF-28-reactive fragments were quantified by scanning densitometry.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aggrecan Cleavage by Atrolysin C-- Atrolysin C (0.5 µM) cleaved aggrecan in a time-dependent manner at several sites, resulting in the generation of five chondroitin sulfate-containing fragments, as detected by MAB2005 Western analysis (Fig. 2A). A major band at ~350 kDa, seen at time 0, prior to digestion, represents intact aggrecan. In as little as 30 min the 350 kDa intact aggrecan band was completely converted to a doublet of ~215 kDa. The top band of the doublet was lost by 4 h, and the bottom band showed a time-dependent increase in intensity up to 4 h and then began to lose intensity at 8 h and was completely lost by 24 h. At 2 h a band at ~185 kDa appeared, increased in intensity at 4 h, and was present up to 8 h but was lost by 24 h of incubation. Finally, two bands, one at ~190 kDa and the other at ~160 kDa, appeared at 4 h. The 190 kDa band was present up to 8 h but then was lost by 24 h, and the 160 kDa band showed a time-dependent increase in intensity up to 48 h.
|
Aggrecan Cleavage at the Asn341-Phe342 MMP Site by Atrolysin C-- Atrolysin C cleaved aggrecan at the Asn341-Phe342 MMP site (Fig. 1) in a concentration-dependent manner after a 24-h incubation, as detected by AF-28 Western analysis (Fig. 3A). Three AF-28-reactive bands were detected, a doublet at ~180 kDa and a single band at ~80 kDa. With as little as 0.1 µM atrolysin C, cleavage at the MMP site was observed, with total band intensity increasing through 0.5 µM. However, the banding pattern shifted with increasing enzyme concentration. The larger AF-28-reactive fragment of the 180 kDa doublet was no longer detected at concentrations of 0.5 µM or higher, whereas at 1 and 5 µM total band intensity decreased, suggesting that at higher concentrations these fragments were degraded further to smaller fragments that ran off the gel.
|
Aggrecan Cleavage at the Glu373-Ala374 Aggrecanase Site by Atrolysin C-- Atrolysin C cleaved purified aggrecan monomer at the Glu373-Ala374 aggrecanase site (Fig. 1) in a concentration-related manner as detected by BC-3 Western analysis (Fig. 4A) after a 24-h incubation. Four BC-3-reactive bands were detected, a doublet at ~150 kDa, and a doublet at ~64 kDa. With as low as 100 nM atrolysin C, cleavage at the aggrecanase site was observed, with total band intensity increasing through 1 µM. However, with increasing enzyme concentration the intensity of the bands at 150 kDa decreased in intensity, suggesting that these larger fragments were cleaved further at the COOH terminus resulting in conversion to fragments represented by the 64 kDa doublet or to smaller fragments that ran off the gel.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the work reported here, we demonstrate that atrolysin C at concentrations as low as 0.1 µM cleaves aggrecan at both at the Asn341-Phe342 MMP site and at the Glu373-Ala374 aggrecanase site. The ability to cleave aggrecan between amino acids Glu373 and Ala374 makes atrolysin C the only enzyme to date, other than neutrophil collagenase (MMP-8), to be shown capable of cleaving aggrecan at the aggrecanase site (8, 9). We have demonstrated previously that recombinant human MMP-8 cleaves native aggrecan in freeze-thawed bovine nasal cartilage at the Glu373-Ala374 aggrecanase site at concentrations similar to those effective in these studies with atrolysin C. However, MMP-8 was found to cleave preferentially at the Asn341-Phe342 MMP site and only cleaved at the Glu373-Ala374 aggrecanase site when complete depletion of substrate containing the preferred cleavage site was achieved (9). In contrast, atrolysin C appears to cleave simultaneously at both the Glu373-Ala374 aggrecanase site and the Asn341-Phe342 MMP site. This is supported by the ability of atrolysin C to generate the G1-NITEGE species. Both the AF-28-reactive species generated by cleavage at the MMP site and the NITEGE-reactive species generated by cleavage at the aggrecanase site were observed after a 30-min incubation with atrolysin C. The appearance of the NITEGE epitope prior to the appearance of the ARGSVIL epitope, which is present on the COOH-terminal fragment generated by this same cleavage, is most likely caused by the different affinities of the BC-3 and NITEGE antibodies for their respective antigens. Since the MMP site (Asn341-Phe342) is between the G1 at the NH2 terminus of the aggrecan core protein and the aggrecanase site (Glu373-Ala374), prior cleavage at the MMP site would preclude the generation of a G1-NITEGE species. Thus, cleavage at the aggrecanase site by atrolysin C could not require previous cleavage at the MMP site as is the case with MMP-8. Generation of all three species, ARGSVIL, FFGVG, and NITEGE, by atrolysin C is only consistent with this protease independently clipping at the MMP and aggrecanase sites or clipping first at the aggrecanase site and then further processing some of the COOH-terminal fragments by cleavage at the MMP site. Cleavage of aggrecan by atrolysin C differs from that by aggrecanase generated in chondrocyte cultures stimulated with interleukin-1 or retinoic acid where aggrecanase is apparently capable of cleaving at the Glu373-Ala374 bond in the complete absence of any detectable cleavage at the MMP site (26).
The MAB2005 Western and bHA analysis suggest that atrolysin C first cleaves aggrecan between the G2 and G3 globular domains, to produce three large fragments with an intact G1 domain, a 215 kDa doublet and a 190 kDa band. These fragments are then processed further via cleavage within the interglobular domain at the MMP Asn341-Phe342 and aggrecanase Glu373-Ala374 sites to produce fragments that lack a G1 domain. Cleavage of aggrecan by atrolysin C at these two sites can be achieved at concentrations as low as 0.1 µM. Thus, concentrations of atrolysin C effective in cleaving aggrecan at both the Asn341-Phe342 and Glu373-Ala374 sites are within the range of concentrations of MMP-1 and MMP-3 (0.03-0.6 µM) reported to be present in synovial fluids of patients with arthritis (27, 28).
The pattern of BC-3-reactive fragments generated upon cleavage of aggrecan by atrolysin C was different from that which we previously found to be produced upon cleavage of cartilage aggrecan in response to interleukin-1 stimulation of bovine nasal cartilage (9). Although several bands of BC-3-reactive aggrecan fragments were seen in response to both endogenously generated aggrecanase and to atrolysin C, the high molecular mass band at ~230 kDa, which represents the COOH-terminal aggrecan fragment formed upon initial cleavage by aggrecanase within the interglobular domain, was not generated by cleavage with atrolysin C. These data suggest that although atrolysin C has the ability to cleave at the Glu373-Ala374 bond, it does not exhibit the same preference as the cartilage aggrecanase for cleavage sites within the aggrecan molecule.
Although atrolysin C is a snake venom metalloproteinase and not found in many species, ADAM family members, which have been demonstrated in mammalian cell types, are related both in amino acid sequence and in domain structure to the reprolysin family of enzymes. ADAM-10,-12, and -15, which are present on the membranes of articular chondrocytes (15), contain a zinc binding domain in an HEXXHXXGXXH conformation suggesting that they may have metalloproteinase activity, and catalytic activity has been demonstrated for ADAM 10 (29). Thus, these ADAMs in chondrocytes may have the ability to function in aggrecan degradation. In addition, hydroxamates designed as inhibitors of MMPs have been shown to inhibit reprolysins (30), and some of these inhibitors are also effective in blocking stimulated cartilage aggrecan degradation in vitro.
Our findings that a reprolysin can cleave aggrecan and, more importantly, has the ability to cleave at the Glu373-Ala374 bond, opens the possibility that the unidentified cartilage aggrecanase may be a member of the reprolysin/ADAM family of proteins. Further studies are under way to determine if any of the known ADAM proteins are capable of cleaving aggrecan specifically at the aggrecanase site.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Amanda Fosang and Dr. James Melrose for the kind gifts of the AF-28 monoclonal antibody and the bHA, respectively. We are grateful to Dr. Ada Cole and Barbara Schumacher for NITEGE Western blot analysis.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Inflammatory Diseases
Research, The DuPont Merck Pharmaceutical Company, Experimental Station
E400/4243, P. O. Box 400, Wilmington, DE 19880-0400. Tel.: 302-695-7081; Fax 302-695-7873.
1 The abbreviations used are: HA, hyaluronic acid; MMP, matrix metalloproteinase; bHA, biotinylated hyaluronic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|