Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS‐4 (aggrecanase 1) or ADAMTS‐5 (aggrecanase 2)

Objective

To determine whether aggrecanase (ADAMTS) activities in articular cartilage can directly lead to the release of hyaluronan (HA) and hyaladherins (aggrecan G1 domain and link proteins), as may occur ex vivo during stimulation of cartilage explants with interleukin‐1 (IL‐1) or retinoic acid or in vivo in synovial joints during aging and joint pathology.

Methods

Bovine articular cartilage discs (live or freeze‐killed) were cultured in the presence of IL‐1 or were incubated in digestion buffer containing recombinant human ADAMTS‐4 (rHuADAMTS‐4; aggrecanase 1) or rHuADAMTS‐5 (aggrecanase 2). Culture media, digestion supernatants, and tissue extracts were assayed for sulfated glycosaminoglycan (sGAG) content and analyzed by Western blotting to detect aggrecanase‐generated G1 domain (using neoepitope monoclonal antibody AGG‐C1/anti‐NITEGE³⁷³) and link proteins (using monoclonal antibody 8‐A‐4), as well as by quantitative enzyme‐linked immunosorbent assays to detect aggrecanase‐generated G1 domain (G1‐NITEGE³⁷³) and HA.

Results

IL‐1 treatment of live cartilage explants induced a time‐dependent release of sGAG, aggrecanase‐generated G1 domain (G1‐NITEGE³⁷³), and HA into the culture media. Exposure of live or freeze‐killed articular cartilage discs to rHuADAMTS‐4 or rHuADAMTS‐5 resulted in a dose‐ and time‐dependent release of sGAG and hyaluronan from the tissue, accompanied by a concomitant release of functionally intact hyaladherins (aggrecan G1‐NITEGE³⁷³ and link proteins).

Conclusion

Coincident with aggrecanolysis, aggrecanase activities in articular cartilage may actuate the release of HA and associated hyaladherins, thereby further compromising the integrity of the cartilage matrix during degenerative joint diseases such as osteoarthritis.

     

Evidence of a novel aggrecan-degrading activity in cartilage: Studies of mice deficient in both ADAMTS-4 and ADAMTS-5

OBJECTIVE: To characterize aggrecan catabolism and the overall phenotype in mice deficient in both ADAMTS-4 and ADAMTS-5 (TS-4/TS-5 Delta-cat) activity. METHODS: Femoral head cartilage from the joints of TS-4/TS-5 Delta-cat mice and wild-type mice were cultured in vitro, and aggrecan catabolism was stimulated with either interleukin-1alpha (IL-1alpha) or retinoic acid. Total aggrecan release was measured, and aggrecanase activity was examined by Western blotting using neoepitope antibodies for detecting cleavage at EGE 373-374 ALG, SELE 1279-1280 GRG, FREEE 1467-1468 GLG, and AQE 1572-1573 AGEG. Aggrecan catabolism in vivo was examined by Western blotting of cartilage that had been extracted immediately ex vivo. RESULTS: TS-4/TS-5 Delta-cat mice were viable, fertile, and phenotypically normal. TS-4/TS-5 Delta-cat cartilage explants did not release aggrecan in response to IL-1alpha, and there was no detectable increase in aggrecanase neoepitopes. TS-4/TS-5 Delta-cat cartilage explants released aggrecan in response to retinoic acid. There was no retinoic acid-stimulated cleavage at either EGE 373-374 ALG or AQE 1572-1573 AGEG. There was a low level of cleavage at SELE 1279-1280 GRG and major cleavage at FREEE 1467-1468 GLG. Ex vivo, cleavage at FREEE 1467-1468 GLG was substantially reduced, but still present, in TS-4/TS-5 Delta-cat mouse cartilage compared with wild-type mouse cartilage. CONCLUSION: An aggrecanase other than ADAMTS-4 and ADAMTS-5 is expressed in mouse cartilage and is up-regulated by retinoic acid but not IL-1alpha. The novel aggrecanase appears to have different substrate specificity from either ADAMTS-4 or ADAMTS-5, cleaving E-G bonds but not E-A bonds. Neither ADAMTS-4 nor ADAMTS-5 is required for normal skeletal development or aggrecan turnover in cartilage.     

The G1 domain of aggrecan released from porcine articular cartilage forms stable complexes with hyaluronan/link protein

OBJECTIVE: To raise peptide antibodies recognizing the C-terminal amino acid sequence in the G1 domain of porcine aggrecan, generated by the action of either aggrecanase or neutral metalloproteinase(s), in rabbits and to use them to investigate the release of aggrecan from porcine articular cartilage. METHOD: An explant culture system was used to investigate the release of the G1 domain of aggrecan from porcine articular cartilage treated with retinoic acid or interleukin 1beta and to study how the activity of these agents is modified by the proteinase inhibitor, batimastat (BB94). RESULTS: Retinoic acid and interleukin 1beta induced both enzyme activities and the release of the G1 domain into the culture medium. Proteinase activity was significantly reduced when the tissue was incubated in the presence of BB94. The functional properties of the enzyme-generated G1 domain were studied using large-pore, agarose/polyacrylamide gel electrophoresis, and it was shown to interact with hyaluronan and link protein. CONCLUSIONS: The results show that there must be a mechanism for removing a functional G1 domain from aggrecan during tissue turnover using this culture system.     

The accumulation of intracellular ITEGE and DIPEN neoepitopes in bovine articular chondrocytes is mediated by CD44 internalization of hyaluronan

OBJECTIVE: A dramatic loss of aggrecan proteoglycan from cartilage is associated with osteoarthritis. The fate of residual G1 domains of aggrecan is unknown, but inefficient turnover of these domains may impede subsequent repair and retention of newly synthesized aggrecan. Thus, the objective of this study was to determine whether ITEGE- and DIPEN-containing G1 domains, generated in situ, are internalized by articular chondrocytes, and whether these events are dependent on hyaluronan (HA) and its receptor, CD44. METHODS: ITEGE and DIPEN neoepitopes were detected by immunofluorescence staining of bovine articular cartilage chondrocytes treated with or without interleukin-1alpha (IL-1alpha). Additionally, purified ITEGE- or DIPEN-containing G1 domains were aggregated with HA and then added to articular chondrocytes, articular chondrocytes transfected with CD44delta67, or COS-7 cells transfected with or without full-length CD44. Internalized epitopes were distinguished by their resistance to extensive trypsinization of the cell surface. RESULTS: Both ITEGE and DIPEN were visualized within the extracellular cell-associated matrix of chondrocytes as well as within intracellular vesicles. Following trypsinization, the intracellular accumulation of both epitopes was clearly visible. IL-1 treatment increased extracellular as well as intracellular ITEGE epitope accumulation. Once internalized, the ITEGE neoepitope became localized within the nucleus and displayed little colocalization with HA, DIPEN, or other G1 domain epitopes. The internalization of both ITEGE and DIPEN G1 domains was dependent on the presence of HA and CD44. CONCLUSION: One important mechanism for the elimination of residual G1 domains following extracellular degradation of aggrecan is CD44-mediated co-internalization with HA.     

Low molecular weight isoforms of the aggrecanases are responsible for the cytokine-induced proteolysis of aggrecan in a porcine chondrocyte culture system

OBJECTIVE: The major proteases responsible for aggrecan turnover in articular cartilage are the aggrecanases (ADAMTS-4 and ADAMTS-5). Although several studies have demonstrated C-terminal truncation of these aggrecanases, the mechanism and importance of this processing are poorly understood. The objective of this study was to further investigate ADAMTS-4 and ADAMTS-5 C-terminal truncation in a porcine model in vitro culture system. METHODS: Chondrocyte-agarose cultures with well-established extracellular matrices were treated with or without interleukin-1 (IL-1), for a variety of different culture time periods. Cultures were analyzed for release of sulfated glycosaminoglycan, aggrecanase-generated interglobular domain (IGD)-aggrecan cleavage, and the presence of ADAMTS-4 and ADAMTS-5 isoforms. Inhibition of aggrecanase activity with monoclonal antibodies, tissue inhibitor of metalloproteinases 3 (TIMP-3), and cycloheximide pretreatment were used to identify ADAMTS isoforms involved in IGD-aggrecan catabolism. RESULTS: Multiple isoforms, including possible zymogens, of ADAMTS-4 and ADAMTS-5 were sequestered within the extracellular matrix formed by 3-week chondrocyte-agarose cultures. IL-1 exposure induced production of a low molecular weight (37 kd) isoform of ADAMTS-4. This isoform was capable of degrading exogenous aggrecan at the IGD-aggrecanase site, was inhibited by TIMP-3, was blocked after preincubation with an antibody to a sequence in the catalytic domain of ADAMTS-4, and required de novo synthesis in the presence of IL-1 for its generation. CONCLUSION: In porcine chondrocyte-agarose cultures, a 37-kd ADAMTS-4 isoform appears to be the major matrix protease responsible for the IGD-aggrecanase activity detected in response to exposure to IL-1. This conclusion contradicts that of recent studies of transgenic knockout mice and highlights the need to determine the roles of the different aggrecanase(s) in human disease.     

Induction of aggrecanase 1 (ADAM-TS4) by interleukin-1 occurs through activation of constitutively produced protein

OBJECTIVE: To study the production of aggrecanase 1 (ADAM-TS4) in monolayer chondrocytes, capsular fibroblasts, and cartilage. METHODS: Bovine nasal and articular cartilage, monolayer chondrocytes, and capsular fibroblasts were incubated in the absence and presence of interleukin-1 (IL-1). ADAM-TS4 production was evaluated by immunofluorescence or by Western blot analysis. Aggrecanase activity was measured in cells grown on an immobilized peptide substrate, and peptide cleavage was monitored by enzyme-linked immunosorbent assay. RESULTS: There was constitutive production of ADAM-TS4 in both cells and tissue. The protein was associated with the extracellular matrix based on the observation that the staining could be reduced following treatment of chondrocytes with heparin or exposure to chondroitinase ABC. Interestingly, there was no detectable change in the abundance of ADAM-TS4 in response to IL-1. Western blot analysis of cell lysates from IL-1-stimulated chondrocytes showed no evidence of increased ADAM-TS4 production, but resulted in activation of ADAM-TS4. The activation was associated with an increased generation in the aggrecanase neoepitope NITEGE in nasal cartilage in response to IL-1. These data suggest that induction of aggrecanase activity both in cells and in cartilage by IL-1 may involve the stimulation of an activator of ADAM-TS4. Consistent with this observation, culture of chondrocytes on a solid support containing a peptide substrate resulted in the generation of aggrecanase-mediated cleavage that could be blocked by selective inhibitors of ADAM-TS4. CONCLUSION: These data support the hypothesis that ADAM-TS4 is constitutively produced in these cells and tissue, and that stimulation by IL-1 results in aggrecanase activation. Thus, the activator could be a potential target by which to control aggrecanase-mediated degradation in arthritic diseases.     

G1 domain of aggrecan cointernalizes with hyaluronan via a CD44-mediated mechanism in bovine articular chondrocytes

OBJECTIVE: To determine whether aggrecan fragments bound to hyaluronan (HA) can be retained and internalized by articular chondrocytes and whether these events are dependent on HA and its receptor, CD44. An additional objective was to determine whether partial degradation of aggrecan is a prerequisite for internalization. METHODS: Binding and internalization of a variety of fluorescein isothiocyanate (FITC)- or biotin-labeled HA/proteoglycan probes were investigated on normal bovine articular cartilage chondrocytes, bovine articular chondrocytes transfected with a dominant-negative construct of CD44, or COS-7 cells transfected with wild-type CD44. The probes were defined as being internalized by the presence of label associated with the cells following extensive trypsinization of the cell surface. RESULTS: Biotinylated aggrecan fragments bound to FITC-HA were cointernalized in bovine articular chondrocytes or COS-7 cells transfected with CD44. Intracellular vesicles containing FITC-HA colocalized with a fluorescent probe for lysosomes. The internalization of the aggrecan fragments was dependent on the presence of HA as well as the presence of functional CD44. Intact aggrecan/FITC-HA complexes bound to the cell surface but were not internalized. However, following brief trypsin digestion of the aggrecan/HA complex, the remaining proteoglycan fragments were bound and internalized. CONCLUSION: Partially degraded aggrecan fragments (e.g., aggrecan G1 domains bound to HA) can be internalized by articular chondrocytes via a mechanism involving HA/CD44-mediated endocytosis. Further, the presence of an intact aggrecan monomer bound to HA inhibits the internalization of HA as well as HA-bound fragments.     

Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin-1 and the selective inhibition of type II collagen cleavage by collagenase

OBJECTIVE: To compare interleukin-1alpha (IL-1alpha)-induced degradation of nasal and articular cartilages in terms of proteoglycan loss and type II collagen cleavage, denaturation, and release; to examine the temporal relationship of these changes; and to investigate the effects of an inhibitor of collagenase 2 and collagenase 3 on these catabolic processes. METHODS: Discs of mature bovine nasal and articular cartilages were cultured with or without human IL-1alpha (5 ng/ml) with or without RS102,481, a selective synthetic inhibitor of collagenase 2 and collagenase 3 (matrix metalloproteinase 8 [MMP-8] and MMP-13, respectively) but not of collagenase 1 (MMP-1). Immunoassays were used to measure collagenase-generated type II collagen cleavage neoepitope (antibody COL2-3/4C(short)) and denaturation (antibody COL2-3/4m), as well as total type II collagen content (antibody COL2-3/4m) in articular cartilage and culture media. A colorimetric assay was used to measure total proteoglycan concentration (principally of aggrecan) as sulfated glycosaminoglycans (sGAG). RESULTS: IL-1alpha initially induced a decrease in tissue proteoglycan content in nasal cartilage. A progressive loss of proteoglycan was noted during culture in articular cartilages, irrespective of the presence of IL-1alpha. In both cartilages, proteoglycan loss was followed by IL-1alpha-induced cleavage of type II collagen by collagenase, which was often reflected by increased denaturation. The inhibitor RS102,481 had no clear effect on the reduction in proteoglycan content (measured by sGAG) and collagen denaturation in either cartilage, but at 10 nM it inhibited the enhanced cleavage of type II collagen, partially in nasal cartilage and completely in articular cartilage. CONCLUSION: IL-1alpha-induced cleavage and denaturation of type II collagen is observed in both hyaline cartilages and is secondary to proteoglycan loss. It probably involves different collagenases, since there is no evidence of a rate-limiting role for collagenase 1 in articular cartilage, unlike the case for nasal cartilage. Inhibitors of this kind may be of value in the treatment of cartilage damage in arthritis. Also, the ability to detect the release of type II collagen collagenase-generated fragments from degraded cartilage offers the potential to monitor cartilage collagen damage and its control in vivo.     

Inhibitors of hyaluronan export prevent proteoglycan loss from osteoarthritic cartilage

OBJECTIVE: Osteoarthritis (OA) is characterized by cartilage erosion, proteolysis of aggrecan and collagen, and disturbed synthesis rates of aggrecan and hyaluronan by chondrocytes. The hypothesis is tested that hyaluronan overproduction contributes to aggrecan loss from osteoarthritic cartilage. METHODS: Human chondrocytes or bovine cartilage explants were incubated with interleukin 1beta (IL-1beta) to induce upregulation of hyaluronan and downregulation of aggrecan. OA was induced by injection of iodoacetate into the synovial cavity in rat knees. Hyaluronan export was inhibited by ATP-binding cassette transporter inhibitors such as the multidrug resistance (MDR) inhibitors valspodar or verapamil. The concentration of aggrecan was measured in cell culture media or visualized histochemically in cartilage tissue sections. RESULTS: Valspodar inhibited hyaluronan export from human chondrocytes in cell culture selectively without reducing aggrecan secretion. Valspodar and other MDR inhibitors prevented loss of aggrecan from osteoarthritic cartilage explants in culture. Verapamil prevented loss of aggrecan from cartilage in osteoarthritic rat knees. CONCLUSION: Hyaluronan is synthesized at plasma membranes and exported out of the cell. We recently identified an ATP-binding cassette transport system that is responsible for hyaluronan export. A number of ATP-binding cassette transport inhibitors are known and are in use clinically. These inhibitors were used here to inhibit hyaluronan export and to prevent aggrecan loss from arthritic cartilage. New drugs for treatment of arthritis are suggested by these studies.     

Characterization of and osteoarthritis susceptibility in ADAMTS-4-knockout mice

OBJECTIVE: To determine the importance of the enzymatic activity of ADAMTS-4 in normal growth and development and to evaluate the role of ADAMTS-4 in the progression of osteoarthritis (OA). METHODS: We generated catalytic domain-deleted ADAMTS-4-transgenic mice and performed extensive gross and histologic analyses of various organs. The mice were challenged by surgical induction of joint instability leading to OA, to determine the importance of the enzymatic activity of ADAMTS-4 in the progression of the disease. The response of wild-type (WT) and ADAMTS-4-knockout (ADAMTS-4-KO) articular cartilage to interleukin-1 and retinoic acid challenge in vitro was also evaluated. RESULTS: ADAMTS-4-KO mice up to 1 year of age exhibited no gross or histologic abnormalities in 36 tissue sites examined. Despite evidence of ADAMTS-4 expression and activity in growth plates of WT mice, catalytic silencing of this proteinase caused no abnormalities in skeletal development, growth, or remodeling. There was no effect of ADAMTS-4 knockout on the progression or severity of OA 4 weeks or 8 weeks after surgical induction of joint instability. Enzymatic cleavage of aggrecan at the TEGE(373-374)ARGS site was clearly evident after exposure of articular cartilage from ADAMTS-4-KO mice to inflammatory cytokines. CONCLUSION: Although expression of the ADAMTS-4 gene has been found in many tissues throughout the body, deletion of enzymatic activity did not appear to have any effect on normal growth and physiology. Our study provides evidence that ADAMTS-4 is the primary aggrecanase in murine growth plates; however, deletion of its enzymatic activity did not affect normal long bone remodeling. Our results also lead to the hypothesis that, in the mouse, ADAMTS-4 is not the primary enzyme responsible for aggrecan degradation at the TEGE(373-374)ARGS site. The elucidation of the relative importance of ADAMTS-4 in the pathologic process of human OA will require examination of human OA tissues and evidence of disease modification in patients following therapeutic intervention.     

Calcium pentosan polysulfate inhibits the catabolism of aggrecan in articular cartilage explant cultures

OBJECTIVE: The catabolism of aggrecan and loss of aggrecan fragments from articular cartilage is a key event in the pathogenesis of arthritic diseases such as osteoarthritis. The catabolism of aggrecan is mediated by the specific proteolytic activity termed aggrecanase. The aim of this study was to investigate the effect of the chondroprotective agent calcium pentosan polysulfate (CaPPS) on the aggrecanase-mediated catabolism of aggrecan. METHODS: The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans (GAGs) were investigated using bovine articular cartilage explant cultures maintained in medium containing varying concentrations of CaPPS (1-100 microg/ml) in the presence or absence of 10(-6)M retinoic acid or 7 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha). In addition, the effect of CaPPS on the degradation of aggrecan monomers by aggrecanase activity present in conditioned medium from joint capsule explant cultures was investigated. RESULTS: CaPPS inhibited the catabolism of 35S-labeled aggrecan in a dose-dependent manner, particularly when retinoic acid or rHuIL-1alpha was used to stimulate aggrecan catabolism. These effects were reflected in the tissue levels of GAG remaining in these cultures at the end of the experiment. CaPPS inhibited the degradation of aggrecan monomers by soluble aggrecanase activity. CONCLUSION: CaPPS inhibits the catabolism of aggrecan by articular cartilage in a dose-dependent manner, particularly when the processes responsible for aggrecan loss are stimulated. This effect occurs, at least in part, through direct inhibition of aggrecanase activity. CaPPS did not adversely affect overall chondrocyte metabolism, as shown by the incorporation of 35S-sulfate and 3H-leucine into macromolecules and by lactate production in cartilage explant cultures.     

G1 domain of aggrecan cointernalizes with hyaluronan via a CD44‐mediated mechanism in bovine articular chondrocytes

Objective

To determine whether aggrecan fragments bound to hyaluronan (HA) can be retained and internalized by articular chondrocytes and whether these events are dependent on HA and its receptor, CD44. An additional objective was to determine whether partial degradation of aggrecan is a prerequisite for internalization.

Methods

Binding and internalization of a variety of fluorescein isothiocyanate (FITC)– or biotin‐labeled HA/proteoglycan probes were investigated on normal bovine articular cartilage chondrocytes, bovine articular chondrocytes transfected with a dominant‐negative construct of CD44, or COS‐7 cells transfected with wild‐type CD44. The probes were defined as being internalized by the presence of label associated with the cells following extensive trypsinization of the cell surface.

Results

Biotinylated aggrecan fragments bound to FITC‐HA were cointernalized in bovine articular chondrocytes or COS‐7 cells transfected with CD44. Intracellular vesicles containing FITC‐HA colocalized with a fluorescent probe for lysosomes. The internalization of the aggrecan fragments was dependent on the presence of HA as well as the presence of functional CD44. Intact aggrecan/FITC‐HA complexes bound to the cell surface but were not internalized. However, following brief trypsin digestion of the aggrecan/HA complex, the remaining proteoglycan fragments were bound and internalized.

Conclusion

Partially degraded aggrecan fragments (e.g., aggrecan G1 domains bound to HA) can be internalized by articular chondrocytes via a mechanism involving HA/CD44‐mediated endocytosis. Further, the presence of an intact aggrecan monomer bound to HA inhibits the internalization of HA as well as HA‐bound fragments.

     

Cartilage proteoglycan aggregate: structure and function

The proteoglycan aggregate is the major structural component of the extracellular matrix of the cartilage, composed of aggrecan, hyaluronan (HA) and link protein (LP). The aggregates provide cartilage with unique gel-like property and resistance to deformation through water absorption. Natural knockout mice of aggrecan, termed cartilage matrix deficiency, and LP-null mice exhibit decreased levels of aggrecan depositon in cartilage and correspond well with their phenotypes such as dwarfism and spinal misalignment, demonstrating in vivo roles of the aggregate in cartilage development and homeostasis. Our recent studies demonstrate that versican/PG-M, another member of aggrecan family with a similar domain structure, binds both HA and LP, forming an aggregate. Further functional analyses of domains and subdomains showed distinct interaction of versican/PG-M with LP from aggrecan, suggesting that the versican/PG-M aggregate may have different function in cartilage.     

Aggrecanase cleavage in juvenile idiopathic arthritis patients is minimally detected in the aggrecan interglobular domain but robust at the aggrecan C‐terminus

Objective

To investigate aggrecan degradation in juvenile idiopathic arthritis (JIA).

Methods

The pattern and abundance of aggrecan fragments in synovial fluid (SF) aspirates from JIA patients were analyzed and compared with aggrecan fragments in SF from patients with other arthritides, children with knee injury, and a knee‐healthy reference group. Concentrations of sulfated glycosaminoglycan (sGAG) in SF were measured by Alcian blue precipitation assay. Aggrecan fragments were purified by dissociative CsCl density‐gradient centrifugation, deglycosylated, and analyzed by Western blot using antibodies specific for either aggrecanase‐derived ARGS, SELE, and KEEE neoepitopes or the aggrecan G3 domain.

Results

The concentration of sGAG in SF from patients with JIA was significantly lower compared with that in SF from patients with osteoarthritis (OA) (P < 0.001), patients with juvenile knee injury (P = 0.006), and knee‐healthy controls (P = 0.022). Western blot analysis revealed KEEE, SELE, and G3 fragments generated by aggrecanase cleavage in the chondroitin sulfate–rich region of aggrecan in patients with JIA. The pattern of aggrecan fragments in JIA patients was not identical to that in pooled OA SF, although there were notable similarities. Surprisingly, aggrecanase‐derived ARGS fragments were barely detectable in JIA SF, in marked contrast to levels in OA SF.

Conclusion

Aggrecanases appear to cleave minimally in the interglobular domain of aggrecan in JIA patients despite robust levels of cleavage in the chondroitin sulfate–rich region. These results suggest that in JIA, unlike other arthritides, aggrecanase cleavage in the aggrecan interglobular domain might not be a major pathogenic event.

     

The structure of aggrecan fragments in human synovial fluid. evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease,joint injury,and osteoarthritis

Objective. To determine the proteolytic fragmentation patterns and N-terminal sequence of aggrecan fragments in human synovial fluid from patients with inflammatory arthritides, joint injury, or osteoarthritis (OA). Methods. Knee synovial fluid was obtained from patients with joint injury, OA, acute pyrophosphate arthritis (pseudogout), reactive arthritis, psoriatic arthritis, or juvenile rheumatoid arthritis. Chondroitin sulfate–substituted aggrecan fragments present in the fluid were purified by cesium chloride gradient centrifugation and enzymatically deglycosylated. Core protein species were determined by N-terminal analysis and by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with electroblotting and detection with monoclonal antibody 3B3. Results. Samples from patients with joint injury, OA, and inflammatory joint disease all showed a similar 3-band pattern, with core sizes of approximately 200 kd, 170 kd, and 135 kd. In all samples, diffuse immuno-reactive products were also seen, with an apparent size of >250 kd. N-terminal analysis of core preparations of all samples showed a consistent single predominant sequence, beginning at alanine 374 of the human aggrecan core protein. Conclusion. The aggrecan fragments present in joint fluids from patients with various inflammatory arthritides, joint injury, or OA result from a predominant cleavage of the human aggrecan core protein at the glutamate 373–alanine 374 bond within the interglobular domain, between the G1 and G2 domains. The consistent pattern of fragments seen on SDS-PAGE and the single predominant N-terminal sequence suggest a common degradative mechanism of aggrecan in these different joint conditions. The identity of the proteolytic agent (aggrecanase), however, remains unknown. These results appear to have important implications with regard to the development of therapies to protect cartilage from degradation in patients with joint disease.