Search

Osteoarthritis is a degenerative joint disorder characterized by breakdown of articular cartilage. Degradation of aggrecan, which together with type II collagen provides cartilage with its unique characteristics of compressibility and elasticity, is an early and sustained feature of osteoarthritis. The present work was set up to identify the enzyme(s) responsible for aggrecan breakdown in osteoarthritis. We found that the two cartilage aggrecanases, ADAM-TS4 and ADAM-TS5, are present in osteoarthritic cartilage and that they are responsible for aggrecan degradation without the participation of matrix metalloproteinases. This is based on 1) neoepitopes found on aggrecan fragments in osteoarthritis (OA) cartilage explants in vitro, 2) aggrecan fragments detected in synovial fluid of OA patients, 3) the observation that an aggrecanase inhibitor, BB-16, blocked aggrecan degradation in OA cartilage in vitro, whereas the matrix metalloproteinase inhibitor XS309 did not, and 4) the presence of mRNA and protein for ADAM-TS4 and ADAM-TS5 in OA cartilage. These results suggest that ADAM-TS4 and ADAM-TS5 represent a potential target for the treatment of osteoarthritis.

The human cytomegalovirus US2 gene product targets major histocompatibility class I molecules for degradation in a proteasome-dependent fashion. Degradation requires interaction between the endoplasmic reticulum (ER) lumenal domains of US2 and class I. While ER insertion of US2 is essential for US2 function, US2 lacks a cleavable signal peptide. Radiosequence analysis of glycosylated US2 confirms the presence of the NH2terminus predicted on the basis of the amino acid sequence, with no evidence for processing by signal peptidase. Despite the absence of cleavage, the US2 NH2-terminal segment constitutes its signal peptide and is sufficient to drive ER translocation of chimeric reporter proteins, again without further cleavage. The putative US2 signal peptide c-region is responsible for the absence of cleavage, despite the presence of a suitable −3,−1 amino acid motif for signal peptidase recognition. In addition, the US2 signal peptide affects the early processing events of the nascent polypeptide, altering the efficiency of ER insertion and subsequent N-linked glycosylation. To our knowledge, US2 is the first example of a membrane protein that does not contain a cleavable signal peptide, yet otherwise behaves like a type I membrane glycoprotein.

Human cytomegalovirus encodes two glycoproteins, US2 and US11, that target major histocompatibility complex (MHC) class I heavy chains for proteasomal degradation. We have developed a mRNA-dependent cell-free system that recapitulates US2- and US11-mediated degradation of MHC class I heavy chains. Microsomes support the degradation of MHC class I heavy chains in the presence of US2 or US11 in a cytosol-dependent manner. In vitro, the glycosylated heavy chain is exported from the microsomes. A deglycosylated breakdown intermediate of the heavy chain identical to that generated in intact cells accumulates in soluble form in the presence of proteasome inhibitors. Microsomes derived from the U373 astrocytoma cell line are far more effective than canine-derived membranes in supporting this US2- or US11-dependent reaction. In contrast, the HIV-encoded Vpu membrane protein can cause the destruction of CD4 from either human- or canine-derived membranes. Using the in vitrosystem, we show that a truncation mutant of US2 that lacks the cytosolic domain is unable to catalyze degradation, whereas a similar truncation of US11 continues to catalyze degradation of class I heavy chains. Therefore, US2 requires both transmembrane and cytosolic interactions to trigger dislocation of heavy chains, whereas US11 relies on the transmembrane domain to target heavy chains. US2 and US11 thus utilize different targeting mechanisms for class I degradation.

In many cell types including myoblasts, growth factors control proliferation and differentiation, in part, via the mitogen-activated protein kinase (MAPK) pathway (also known as the extracellular regulated kinase (Erk) pathway). In C2C12 myoblast cells, insulin-like growth factor-1 and basic fibroblast growth factor (bFGF) activate MAPK/Erk, and both growth factors promote myoblast proliferation. However, these factors have opposing roles with respect to differentiation; insulin-like growth factor-1 enhances muscle cell differentiation, whereas bFGF inhibits the expression of the muscle-specific transcription factors MyoD and myogenin. Cells treated with bFGF and PD98059, a specific inhibitor of the MAPK pathway, show enhanced expression of the muscle-specific transcription factors MyoD and myogenin as compared with cells not exposed to this inhibitor. Inhibiting MAPK activity also enhances myoblast fusion and the expression of the late differentiation marker myosin heavy chain. Basic FGF mediated repression of muscle-specific genes does not result from continued cell proliferation, since bFGF-treated cells progress through only one round of cell division. We have identified a critical boundary 16 to 20 h after plating during which bFGF induced MAPK activity is able to repress myogenic gene expression and differentiation. Thus, the targets of MAPK that regulate myogenesis are functional at this time and their identification is in progress.

The proteoglycan aggrecan is an important major component of cartilage matrix that gives articular cartilage the ability to withstand compression. Increased breakdown of aggrecan is associated with the development of arthritis and is considered to be catalyzed by aggrecanases, members of the ADAM-TS family of metalloproteinases. Four endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate the activities of functional matrix metalloproteinases (MMPs), enzymes that degrade most components of connective tissue, but no endogenous factors responsible for the regulation of aggrecanases have been found. We show here that the N-terminal inhibitory domain of TIMP-3, a member of the TIMP family that has functional properties distinct from other TIMPs, is a strong inhibitor of human aggrecanases 1 and 2, with K(i) values in the subnanomolar range. This truncated inhibitor, which lacks the C-terminal domain that is responsible for interactions with molecules other than active metalloproteinases, is produced at high yield by bacterial expression and folding from inclusion bodies. This provides a starting point for developing a biologically available aggrecanase inhibitor suitable for the treatment of arthritis.

Aggrecan degradation involves proteolytic cleavage of the core protein within the interglobular domain. Because aggrecan is highly glycosylated with chondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether glycosylation affects digestion by aggrecanase at the Glu373–Ala374bond. Treatment of bovine aggrecan monomers to remove CS and KS resulted in loss of cleavage at this site, suggesting that glycosaminoglycans (GAGs) play a role in cleavage at the Glu373–Ala374bond. In contrast, MMP-3 cleavage at the Ser341–Phe342bond was not affected by glycosidase treatment of aggrecan. Removal of KS, but not CS, prevented cleavage at the Glu373–Ala374bond. Thus, KS residues may be important for recognition of this cleavage site by aggrecanase. KS glycosylation has been observed at sites adjacent to the Glu373-Ala374bond in steer aggrecan, but not in calf aggrecan (Barry, F. P., Rosenberg, L. C., Gaw, J. U., Gaw, J. U., Koob, T. J., and Neame, P. J. (1995) J. Biol. Chem.270, 20516–20524). Interestingly, although we found that aggrecanase degraded both calf and steer cartilage aggrecan, the proportion of fragments generated by cleavage at the Glu373–Ala374bond was higher in steer than in calf, consistent with our observations using aggrecan treated to remove KS. We conclude that the GAG content of aggrecan influences the specificity of aggrecanase for cleavage at the Glu373–Ala374bond and suggest that age may be a factor in aggrecanase degradation of cartilage.

Aggrecanase-1 (ADAMTS-4) is a member of theadisintegrin andmetalloprotease withthrombospondin motifs (ADAMTS) protein family that was recently identified. Aggrecanase-1 is one of two ADAMTS cartilage-degrading enzymes purified from interleukin-1-stimulated bovine nasal cartilage (Tortorella, M. D., Burn, T. C., Pratta, M. A., Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., and Arner, E.C. (1999) Science284, 1664–1666; 2 Abbaszade, I., Liu, R. Q., Yang, F., Rosenfeld, S. A., Ross, O. H., Link, J. R., Ellis, D. M., Tortorella, M. D., Pratta, M. A., Hollis, J. M., Wynn, R., Duke, J. L., George, H. J., Hillman, M. C., Jr., Murphy, K., Wiswall, B. H., Copeland, R. A., Decicco, C. P., Bruckner, R., Nagase, H., Itoh, Y., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Burn, T. C. (1999) J. Biol. Chem.274, 23443–23450). The aggrecan products generated by this enzyme are found in cartilage cultures stimulated with cytokines and in synovial fluid from patients with arthritis, suggesting that aggrecanase-1 may be important in diseases involving cartilage destruction. Here we demonstrate that the thrombospondin type-1 (TSP-1) motif located within the C terminus of aggrecanase-1 binds to the glycosaminoglycans of aggrecan. Data from several studies indicate that this binding of aggrecanase-1 to aggrecan through the TSP-1 motif is necessary for enzymatic cleavage of aggrecan. 1) A truncated form of aggrecanase-1 lacking the TSP-1 motif was not effective in cleaving aggrecan. 2) Several peptides representing different regions of the TSP-1 motif effectively blocked aggrecanase-1 cleavage of aggrecan by preventing the enzyme from binding to the substrate. 3) Aggrecanase-1 was not effective in cleaving glycosaminoglycan-free aggrecan. Taken together, these data suggest that the TSP-1 motif of aggrecanase-1 is critical for substrate recognition and cleavage.

Aggrecan, the major proteoglycan of cartilage that provides its mechanical properties of compressibility and elasticity, is one of the first matrix components to undergo measurable loss in arthritic diseases. Two major sites of proteolytic cleavage have been identified within the interglobular domain (IGD) of the aggrecan core protein, one between amino acids Asn341-Phe342which is cleaved by matrix metalloproteinases and the other between Glu373-Ala374that is attributed to aggrecanase. Although several potential aggrecanase-sensitive sites had been identified within the COOH terminus of aggrecan, demonstration that aggrecanase cleaved at these sites awaited isolation and purification of this protease. We have recently cloned human aggrecanase-1 (ADAMTS-4) (Tortorella, M. D., Burn, T. C., Pratta, M. A., Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Arner, E. C. (1999) Science284, 1664–1666) and herein demonstrate that in addition to cleavage at the Glu373-Ala374bond, this protease cleaves at four sites within the chondroitin-sulfate rich region of the aggrecan core protein, between G2 and G3 globular domains. Importantly, we show that this cleavage occurs more efficiently than cleavage within the IGD at the Glu373-Ala374bond. Cleavage occurred preferentially at the KEEE1667–1668GLGS bond to produce both a 140-kDa COOH-terminal fragment and a 375-kDa fragment that retains an intact G1. Cleavage also occurred at the GELE1480–1481GRGT bond to produce a 55-kDa COOH-terminal fragment and a G1-containing fragment of 320 kDa. Cleavage of this 320-kDa fragment within the IGD at the Glu373-Ala374bond then occurred to release the 250-kDa BC-3-reactive fragment from the G1 domain. The 140-kDa GLGS-reactive fragment resulting from the preferential cleavage was further processed at two additional cleavage sites, at TAQE1771-1772AGEG and at VSQE1871–1872LGQR resulting in the formation of a 98-kDa fragment with an intact G3 domain and two small fragments of ∼20 kDa. These data elucidate the sites and efficiency of cleavage during aggrecan degradation by aggrecanase and suggest potential tools for monitoring aggrecan cleavage in arthritis.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn341-Phe342and Glu373-Ala374. While several matrix metalloproteinases have been shown to cleave at Asn341-Phe342, an as yet unidentified protein termed “aggrecanase” is responsible for cleavage at Glu373-Ala374and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11(aggrecanase-2), which has extensive homology to ADAMTS4(aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu373-Ala374site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.

A method was developed for generating soluble, active "aggrecanase" in conditioned media from interleukin-1-stimulated bovine nasal cartilage cultures. Using bovine nasal cartilage conditioned media as a source of the aggrecanase enzyme, an enzymatic assay was established employing purified aggrecan monomers as a substrate and monitoring specific aggrecanase-mediated cleavage products by Western analysis using the monoclonal antibody, BC-3 (which recognizes the new N terminus, ARGS, on fragments produced by cleavage between amino acid residues Glu373 and Ala374). Using this assay we have characterized cartilage aggrecanase with respect to assay kinetics, pH and salt optima, heat sensitivity, and stability upon storage. Aggrecanase activity was inhibited by the metalloprotease inhibitor, EDTA, while a panel of inhibitors of serine, cysteine, and aspartic proteinases had no effect, suggesting that aggrecanase is a metalloproteinase. Sensitivity to known matrix metalloproteinase inhibitors as well as to the endogenous tissue inhibitor of metalloproteinases, TIMP-1, further support the notion that aggrecanase is a metalloproteinase potentially related to the ADAM family or MMP family of proteases previously implicated in the catabolism of the extracellular matrix.

Cleavage of aggrecan core protein at the Glu373-Ala374 site by the unidentified enzyme, "aggrecanase," is thought to play an important role in cartilage degradation. To examine aggrecan cleavage by MMP-8 at this aggrecanase site, we evaluated the release of fragments with the N terminus ARGSVIL from freeze-thawed bovine nasal cartilage using the monoclonal antibody BC-3. Recombinant human MMP-8 catalytic domain cleaved native aggrecan in a concentration-related manner between 0.2 and 2 microg/ml, with complete release of glycosaminoglycan at 2 microg/ml or greater. Cleavage at the aggrecanase site was observed only at MMP-8 concentrations resulting in complete release of glycosaminoglycan from the cartilage, suggesting that preferential cleavage occurs at a different site. Time course studies indicated that only following depletion of substrate containing the preferred clip site did MMP-8 rapidly cleave at the aggrecanase site. Finally, MMP-8 resulted in a different pattern of BC-3-reactive fragments from that produced by endogenous aggrecanase in live cartilage, and SA751(N-(1(R)-carboxyethyl) -alpha-(S)-(4-phenyl-3-butynyl)glycyl-L-O-methyltyrosine, N-methylamide), a potent inhibitor of MMP-8 (Ki = 2 nM) which was effective in blocking cleavage by MMP-8 at the aggrecanase site with an IC50 in the nanomolar range, did not prevent aggrecan degradation or specific cleavage at this site by endogenously generated aggrecanase at concentrations up to 100 microM. Taken together these data suggest that MMP-8 does not represent cartilage aggrecanase.

Two primary cleavage sites have been identified within the interglobular domain of the cartilage aggrecan core protein: one is between amino acid residues Asn341and Phe342, where many matrix metalloproteinases (MMP) have been shown to cleave; and the other is between amino acid residues Glu373and Ala374. Although cleavage at the Glu373-Ala374site 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 (adisintegrin andmetalloproteinase) 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 NH2terminus ARGSV generated by cleavage at the aggrecanase site, and with the AF-28 antibody, which recognizes aggrecan fragments with the new NH2terminus FFGVG generated by cleavage at the MMP site. Cleavage at both the aggrecanase and MMP sites occurred in a concentration-dependent manner with 100 nmatrolysin 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-Ala374bond, 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.

Diphtheria toxin undergoes membrane insertion and translocation across membranes when exposed to low pH. In this study, the translocation of the toxin has been investigated by the binding of antibodies to two preparations of model membrane-inserted toxin. In one preparation, toxin was added externally to model membrane vesicles and then inserted by exposure to low pH. In the other preparation, toxin was entrapped in the vesicles at neutral pH, and then inserted by decreasing pH. At neutral pH, externally added antibodies could not bind to entrapped toxin, although they could bind to externally added native toxin. However, after low pH exposure, antibodies against all three toxin domains (catalytic (C), transmembrane (T), and receptor-binding (R)) could bind to entrapped toxin, and also to externally added membrane-inserted toxin. The binding to the entrapped toxin shows that all three domains of the toxin translocate to the trans face of the membrane after exposure to low pH. The observation that antibodies bind to both external and entrapped preparations of toxin after low pH exposure shows that toxin inserts in a mixed orientation. A difference in antibody binding to low pH-treated toxin in which the C domain is folded (Lr' conformation) or unfolded (Lr" conformation) was also observed. An increase in antibody binding to C and T domains in the Lr" conformation relative to binding to the Lr' conformation was found for entrapped toxin, suggesting that more of the C and T domains translocate across the bilayer in the Lr" conformation. These results suggest all three toxin domains insert in the membrane bilayer and participate in translocation in vitro. The C and R domains lack classical transmembrane hydrophobic sequences. However, they possess sequences that have the potential to form membrane-inserting beta-sheets.

Diphtheria toxin is a bacterial protein that undergoes a physiologically critical conformational change at low pH. This change involves a partial unfolding event forming a molten globule-like structure, which exposes hydrophobic regions and which allows the toxin to insert into, and translocate across, membranes. In this report, antibody binding was used to examine the regions of the toxin that undergo structural changes at low pH. Monoclonal antibodies specific to the catalytic (C), transmembrane (T), and receptor-binding (R) domains of diphtheria toxin were prepared and isolated. In addition, the binding of anti-peptide antibodies raised against peptides in the C and T domains to toxin was examined. Anti-C monoclonals and antipeptide antibodies were found to bind preferentially to low pH-treated toxin relative to native toxin. Anti-T and anti-R monoclonal binding ranged between preference for native toxin and preference for low pH-treated toxin. These results suggest that the C domain becomes more exposed to solution at low pH, and that both the T and R domains of the B chain undergo major conformational changes at low pH. Based on these results, a model in which low pH induces several coordinated changes in intra- and inter-domain interactions is suggested. The participation of the R domain in these changes is of particular significance because it suggests that the R domain plays a more important role in low pH-induced changes than previously realized.