NC4 Domain of cartilage-specific collagen IX inhibits complement directly due to attenuation of membrane attack formation and indirectly through binding and enhancing activity of complement inhibitors C4B-binding protein and factor H

Collagen IX containing the N-terminal noncollagenous domain 4 (NC4) is unique to cartilage and a member of the family of fibril-associated collagens with both collagenous and noncollagenous domains. Collagen IX is located at the surface of fibrils formed by collagen II and a minor proportion of collagen XI, playing roles in tissue stability and integrity. The NC4 domain projects out from the fibril surface and provides sites for interaction with other matrix components such as cartilage oligomeric matrix protein, matrilins, fibromodulin, and osteoadherin. Fragmentation of collagen IX and loss of the NC4 domain are early events in cartilage degradation in joint diseases that precedes major damage of collagen II fibrils. Our results demonstrate that NC4 can function as a novel inhibitor of the complement system able to bind C4, C3, and C9 and to directly inhibit C9 polymerization and assembly of the lytic membrane attack complex. NC4 also binds the complement inhibitors C4b-binding protein and factor H and enhances their cofactor activity in degradation of activated complement components C4b and C3b. NC4 interactions with fibromodulin and osteoadherin inhibited binding to C1q and complement activation by these proteins. Taken together, our results suggest that collagen IX and its interactions with matrix components are important parts of a machinery that protects the cartilage from complement activation and chronic inflammation seen in diseases like rheumatoid arthritis.     

The extracellular matrix and inflammation: fibromodulin activates the classical pathway of complement by directly binding C1q

Components that propagate inflammation in joint disease may be derived from cartilage since the inflammation resolves after joint replacement. We found that the cartilage component fibromodulin has the ability to activate an inflammatory cascade, i.e. complement. Fibromodulin and immunoglobulins cause comparable deposition of C1q, C4b, and C3b from human serum. Using C1q and factor B-deficient sera in combination with varying contents of metal ions, we established that fibromodulin activates both the classical and the alternative pathways of complement. Further studies revealed that fibromodulin binds directly to the globular heads of C1q, leading to activation of C1. However, deposition of the membrane attack complex and C5a release were lower in the presence of fibromodulin as compared with IgG. This can be explained by the fact that fibromodulin also binds complement inhibitor factor H. Factor H and C1q bind to non-overlapping sites on fibromodulin, but none of the interactions is mediated by the negatively charged keratan sulfate substituents of fibromodulin. C1q but not factor H binds to an N-terminal fragment of fibromodulin previously implicated to be affected in cartilage stimulated with the inflammatory cytokine interleukin 1. Taken together our observations indicate fibromodulin as one factor involved in the sustained inflammation of the joint.     

The factor H variant associated with age-related macular degeneration (His-384) and the non-disease-associated form bind differentially to C-reactive protein, fibromodulin, DNA, and necrotic cells

Recently, a polymorphism in the complement regulator factor H (FH) gene has been associated with age-related macular degeneration. When histidine instead of tyrosine is present at position 384 in the seventh complement control protein (CCP) domain of FH, the risk for age-related macular degeneration is increased. It was recently shown that these allotypic variants of FH, in the context of a recombinant construct corresponding to CCPs 6-8, recognize polyanionic structures differently, which may lead to altered regulation of the alternative pathway of complement. We show now that His-384, corresponding to the risk allele, binds C-reactive protein (CRP) poorly compared with the Tyr-384 form. We also found that C1q and phosphorylcholine do not compete with FH for binding to C-reactive protein. The interaction with extracellular matrix protein fibromodulin, which we now show to be mediated, at least in part, by CCP6-8 of FH, occurs via the polypeptide of fibromodulin and not through its glycosaminoglycan modifications. The Tyr-384 variant of FH bound fibromodulin better than the His-384 form. Furthermore, we find that CCP6-8 is able to interact with DNA and necrotic cells, but in contrast the His-384 allotype binds these ligands more strongly than the Tyr-384 variant. The variations in binding affinity of the two alleles indicate that complement activation and local inflammation in response to different targets will differ between His/His and Tyr/Tyr homozygotes.     

A Missense Mutation in the Aggrecan C-type Lectin Domain Disrupts Extracellular Matrix Interactions and Causes Dominant Familial Osteochondritis Dissecans

Osteochondritis dissecans is a disorder in which fragments of articular cartilage and subchondral bone dislodge from the joint surface. We analyzed a five-generation family in which affected members had autosomal-dominant familial osteochondritis dissecans. A genome-wide linkage analysis identified aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo.     

Analysis of the catabolism of aggrecan in cartilage explants by quantitation of peptides from the three globular domains

A method has been developed for the production, isolation, and quantitation of 15 marker peptides from the three globular domains (G1, G2, and G3) and the interglobular domain of bovine aggrecan (aggregating cartilage proteoglycan). Three of the peptides are from G1, two are from the interglobular domain, four are from G2, and six are from G3. The method involves separation of tryptic peptides by sequential anion-exchange, cation-exchange, and reversed-phase high performance liquid chromatography and quantitation by absorbance at 220 nm. The values obtained (peak area per microgram of core protein) were a function of the molar yield and also the size and aromatic residue content of individual peptides. This procedure has been applied to aggrecan purified from fresh calf articular cartilage and to aggrecan isolated from the medium and tissue compartments of cartilage explant cultures, maintained in basal medium for 15 days without and with interleukin-1 alpha. These analyses indicate that aggrecan which is released into explant medium has a reduced content of the G1 domain, but has a normal content of the G2 domain, the COOH-terminal region of the interglobular domain, and also the G3 domain. On the other hand, aggrecan which is retained by the cartilage during 15 days of culture has a normal content of G1, interglobular domain, and G2 domains, but, in the presence of interleukin-1 alpha, it has a reduced content of the G3 domain. The percentage of medium molecules which retained the G1 domain was higher in control cultures (about 35%) than in interleukin cultures (about 20%), and this was consistent with the relative aggregability of these samples. Taken together these results suggest that catabolism of aggrecan in articular cartilage involves a specific proteolysis of the core protein at a site which is within the interglobular domain and NH2-terminal to the sequence LPGG. This process occurs in control cultures but is accelerated by the addition of interleukin-1 alpha. Degraded molecules which lack the G1 domain are released preferentially into the medium; however, these molecules carry both the G2 and G3 domains, indicating that these domains do not confer strong matrix binding properties on aggrecan. The method described here for the isolation of peptides from bovine aggrecan should have wide application to structural and biosynthetic studies on this molecule in species such as human and rat, since many of the marker peptides are from highly conserved regions of the aggrecan core protein.     

Complement activation by phospholipids: the interplay of factor H and C1q

Complement proteins in blood recognize charged particles. The anionic phospholipid (aPL) cardiolipin binds both complement proteins C1q and factor H. C1q is an activator of the complement classical pathway, while factor H is an inhibitor of the alternative pathway. To examine opposing effects of C1q and factor H on complement activation by aPL, we surveyed C1q and factor H binding, and complement activation by aPL, either coated on microtitre plates or in liposomes. Both C1q and factor H bound to all aPL tested, and competed directly with each other for binding. All the aPL activated the complement classical pathway, but negligibly the alternative pathway, consistent with accepted roles of C1q and factor H. However, in this system, factor H, by competing directly with C1q for binding to aPL, acts as a direct regulator of the complement classical pathway. This regulatory mechanism is distinct from its action on the alternative pathway. Regulation of classical pathway activation by factor H was confirmed by measuring C4 activation by aPL in human sera in which the C1q:factor H molar ratio was adjusted over a wide range. Thus factor H, which is regarded as a down-regulator only of the alternative pathway, has a distinct role in downregulating activation of the classical complement pathway by aPL. A factor H homologue, β2-glycoprotein-1, also strongly inhibits C1q binding to cardiolipin. Recombinant globular domains of C1q A, B and C chains bound aPL similarly to native C1q, confirming that C1q binds aPL via its globular heads.     

Link protein has greater affinity for versican than aggrecan

The function of link protein in stabilizing the interaction between aggrecan and hyaluronan to form aggrecan aggregates, via the binding of link protein to the aggrecan G1 domain and hyaluronan, is well established. However, it is not known whether link protein can function with similar avidity with versican, another member of the large hyaluronan-binding proteoglycan family that also binds to hyaluronan via its G1 domain. To address this issue, we have compared the interaction of the versican and aggrecan G1 domains with link protein and hyaluronan using recombinant proteins expressed in insect cells and BIAcore analysis. The results showed that link protein could significantly improve the binding of both G1 domains to hyaluronan and that its interaction with VG1 is of a higher affinity than that with AG1. These observations suggest that link protein may function as a stabilizer of the interaction, not only between aggrecan and hyaluronan in cartilage, but also between versican and hyaluronan in many tissues.     

Specific inhibition of the classical complement pathway by C1q-binding peptides.

Undesired activation of the complement system is a major pathogenic factor contributing to various immune complex diseases and conditions such as hyperacute xenograft rejection. We aim for prevention of complement-mediated damage by specific inhibition of the classical complement pathway, thus not affecting the antimicrobial functions of the complement system via the alternative pathway and the lectin pathway. Therefore, 42 peptides previously selected from phage-displayed peptide libraries on basis of C1q binding were synthesized and examined for their ability to inhibit the function of C1q. From seven peptides that showed inhibition of C1q hemolytic activity but no inhibition of the alternative complement pathway, one peptide (2J) was selected and further studied. Peptide 2J inhibited the hemolytic activity of C1q from human, chimpanzee, rhesus monkey, rat, and mouse origin, all with a similar dose-response relationship (IC(50) 2-6 microM). Binding of C1q to peptide 2J involved the globular head domain of C1q. In line with this interaction, peptide 2J dose-dependently inhibited the binding of C1q to IgG and blocked activation of C4 and C3 and formation of C5b-9 induced via classical pathway activation, as assessed by ELISA. Furthermore, the peptide strongly inhibited the deposition of C4 and C3 on pig cells following their exposure to human xenoreactive Abs and complement. We conclude that peptide 2J is a promising reagent for the development of a therapeutic inhibitor of the earliest step of the classical complement pathway, i.e., the binding of C1q to its target.     

G3 domains of aggrecan and PG-M/versican form intermolecular disulfide bonds that stabilize cell-matrix interaction

The extracellular matrix plays a critical role in maintaining tissue integrity. Among the matrix molecules, the large aggregating chondroitin sulfate proteoglycans are the major structural molecules and are the primary contributors to the stability for some tissues such as cartilage. The notable exceptions are nanomelic cartilage and arthritic cartilage: the former contains a point mutation leading to a stop codon before translating to the C-terminal G3 domain; the latter contains a large proportion of aggrecan from which the G3 domain has been cleaved. These phenomena suggest that the G3 domain may be important in cartilage stability. Here, we demonstrated for the first time that the G3 domains of aggrecan and another proteoglycan, PG-M/versican, formed intermolecular disulfide bonds, and all subdomains were involved. Further studies indicated that each of the 10 cysteine residues of the aggrecan G3 domain could potentially form intermolecular disulfide bonds in vitro. The disulfide bonds were disrupted in the presence of reducing reagent beta-mercaptoethanol and dithiothreitol. As a result, normal chondrocyte-matrix interaction was disrupted, and the structure of the extracellular matrix was altered. Furthermore, disruption of disulfide bonds also reduced the role of PG-M/versican G3 domain in mediating cell adhesion. Our study provides strong evidence of the importance of proteoglycan interactions through intermolecular disulfide bonds in cartilage firmness and cell-matrix stability.     

A Recessive Skeletal Dysplasia, SEMD Aggrecan Type, Results from a Missense Mutation Affecting the C-Type Lectin Domain of Aggrecan

Analysis of a nuclear family with three affected offspring identified an autosomal-recessive form of spondyloepimetaphyseal dysplasia characterized by severe short stature and a unique constellation of radiographic findings. Homozygosity for a haplotype that was identical by descent between two of the affected individuals identified a locus for the disease gene within a 17.4 Mb interval on chromosome 15, a region containing 296 genes. These genes were assessed and ranked by cartilage selectivity with whole-genome microarray data, revealing only two genes, encoding aggrecan and chondroitin sulfate proteoglycan 4, that were selectively expressed in cartilage. Sequence analysis of aggrecan complementary DNA from an affected individual revealed homozygosity for a missense mutation (c.6799G → A) that predicts a p.D2267N amino acid substitution in the C-type lectin domain within the G3 domain of aggrecan. The D2267 residue is predicted to coordinate binding of a calcium ion, which influences the conformational binding loops of the C-type lectin domain that mediate interactions with tenascins and other extracellular-matrix proteins. Expression of the normal and mutant G3 domains in mammalian cells showed that the mutation created a functional N-glycosylation site but did not adversely affect protein trafficking and secretion. Surface-plasmon-resonance studies showed that the mutation influenced the binding and kinetics of the interactions between the aggrecan G3 domain and tenascin-C. These findings identify an autosomal-recessive skeletal dysplasia and a significant role for the aggrecan C-type lectin domain in regulating endochondral ossification and, thereby, height.     

Variability in the G3 domain content of bovine aggrecan from cartilage extracts and chondrocyte cultures.

The content of the globular domains G1, G2 and G3 on the core protein of high-density (A1D1) aggrecan isolated from newborn and mature bovine cartilage and from cultures of bovine chondrocytes was examined. Quantitation based on the 220 nm absorbance of tryptic marker peptides from each domain isolated by reversed-phase HPLC showed that while the content of G1 and G2 was essentially the same for all samples, the content of G3 varied markedly. The molar yield of G3 and G1 marker peptides indicated that approximately 55% of the G1-bearing aggrecan from immature cartilage carried the G3 domain, while for mature cartilage this figure was markedly reduced, at about 35%. Aggrecan prepared from the cell layer matrix of calf chondrocyte cultures had an apparent G3 content similar to newborn cartilage (55%), whereas aggrecan prepared from the medium of these cultures had a markedly higher G3 content, at about 80%. The high content of G3 in cell medium samples compared to cartilage extracts was supported by electron microscopic analysis of A1D1 preparations. The G3 content of the two subpopulations of aggrecan present in mature cartilage and separable by flat bed agarose gel electrophoresis was also determined at about 45% (Band I) and 20% (Band II) respectively. These results are discussed in terms of the likely origin of the marked variability in the G3 domain content of aggrecan.     

Structural basis for interactions between tenascins and lectican C-type lectin domains: evidence for a crosslinking role for tenascins

The C-terminal G3 domains of lecticans mediate crosslinking to diverse extracellular matrix (ECM) proteins during ECM assembly, through their C-type lectin (CLD) subdomains. The structure of the rat aggrecan CLD in a Ca(2+)-dependent complex with fibronectin type III repeats 3-5 of rat tenascin-R provides detailed support for such crosslinking. The CLD loops bind Ca2+ like other CLDs, but no carbohydrate binding is observed or possible. This is thus the first example of a direct Ca(2+)-dependent protein-protein interaction of a CLD. Surprisingly, tenascin-R does not coordinate the Ca2+ ions directly. Electron microscopy confirms that full-length tenascin-R and tenascin-C crosslink hyaluronan-aggrecan complexes. The results are significant for the binding of all lectican CLDs to tenascin-R and tenascin-C. Comparison of the protein interaction surface with that of P-selectin in complex with the PGSL-1 peptide suggests that direct protein-protein interactions of Ca(2+)-binding CLDs may be more widespread than previously appreciated.     

Islet amyloid polypeptide triggers limited complement activation and binds complement inhibitor C4b-binding protein, which enhances fibril formation

Islet amyloid polypeptide (IAPP) is synthesized in pancreatic β-cells and co-secreted with insulin. Aggregation and formation of IAPP-amyloid play a critical role in β-cell death in type 2 diabetic patients. Because Aβ-fibrils in Alzheimer disease activate the complement system, we have here investigated specific interactions between IAPP and complement factors. IAPP fibrils triggered limited activation of complement in vitro, involving both the classical and the alternative pathways. Direct binding assays confirmed that IAPP fibrils interact with globular head domains of complement initiator C1q. Furthermore, IAPP also bound complement inhibitors factor H and C4b-binding protein (C4BP). Recombinant C4BP mutants were used to show that complement control protein (CCP) domains 8 and 2 of the α-chain were responsible for the strong, hydrophobic binding of C4BP to IAPP. Immunostaining of pancreatic sections from type 2 diabetic patients revealed the presence of complement factors in the islets and varying degree of co-localization between IAPP fibrils and C1q, C3d, as well as C4BP and factor H but not membrane attack complex. Furthermore, C4BP enhanced formation of IAPP fibrils in vitro. We conclude that C4BP binds to IAPP thereby limiting complement activation and may be enhancing formation of IAPP fibrils from cytotoxic oligomers.     

A recombinant homotrimer, composed of the alpha helical neck region of human surfactant protein D and C1q B chain globular domain, is an inhibitor of the classical complement pathway

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of IgG or IgM. The globular heads of C1q (gC1q domain) are located C-terminal to the six triple-helical stalks present in the molecule, each head being composed of the C-terminal halves of one A, one B, and one C chain. The gC1q modules are also found in a variety of noncomplement proteins, such as type VIII and X collagens, precerebellin, hibernation protein, multimerin, Acrp-30, and saccular collagen. In several of these proteins, the chains containing these gC1q modules appear to form a homotrimeric structure. Here, we report expression of an in-frame fusion of a trimerizing neck region of surfactant protein D with the globular head region of C1q B chain as a fusion to Escherichia coli maltose binding protein. Following cleavage by factor Xa and removal of the maltose binding protein, the neck and globular region, designated ghB(3), formed a soluble, homotrimeric structure and could inhibit C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. The functional properties of ghB(3) indicate that the globular regions of C1q may adopt a modular organization in which each globular head of C1q may be composed of three structurally and functionally independent domains, thus retaining multivalency in the form of a heterotrimer. The finding that ghB(3) is an inhibitor of C1q-mediated complement activation opens up the possibility of blocking activation at the first step of the classical complement pathway.     

Structural stability and heat-induced conformational change of two complement inhibitors: C4b-binding protein and factor H

The complement inhibitors C4b-binding protein (C4BP) and factor H (FH) both consist of complement control protein (CCP) domains. Here we examined the secondary structure of both proteins by circular dichroism and Fourier-transform infrared technique at temperatures ranging from 30 degrees C-90 degrees C. We found that predominantly beta-sheet structure of both proteins was stable up to 70 degrees C, and that a reversible conformational change toward alpha-helix was apparent at temperatures ranging from 70 degrees C to 90 degrees C. The ability of both proteins to inhibit complement was not impaired after incubation at 95 degrees C, exposure to extreme pH conditions, and storage at room temperature for several months. Similar remarkable stability was previously observed for vaccinia virus control protein (VCP), which is also composed of CCP domains; it therefore seems to be a general property of CCP-containing proteins. A typical CCP domain has a hydrophobic core, which is wrapped in beta-sheets and stabilized by two disulphide bridges. How the CCP domains tolerate harsh conditions is unclear, but it could be due to a combination of high content of prolines, hydrophobic residues, and the presence of two disulphide bridges within each domain. These findings are of interest because CCP-containing complement inhibitors have been proposed as clinical agents to be used to control unwanted complement activation that contributes to many diseases.     

Activation of the classical complement pathway by Bacillus anthracis is the primary mechanism for spore phagocytosis and involves the spore surface protein BclA

Interactions between spores of Bacillus anthracis and macrophages are critical for the development of anthrax infections, as spores are thought to use macrophages as vehicles to disseminate in the host. In this study, we report a novel mechanism for phagocytosis of B. anthracis spores. Murine macrophage-like cell line RAW264.7, bone marrow-derived macrophages, and primary peritoneal macrophages from mice were used. The results indicated that activation of the classical complement pathway (CCP) was a primary mechanism for spore phagocytosis. Phagocytosis was significantly reduced in the absence of C1q or C3. C3 fragments were found deposited on the spore surface, and the deposition was dependent on C1q and Ca(2+). C1q recruitment to the spore surface was mediated by the spore surface protein BclA, as recombinant BclA bound directly and specifically to C1q and inhibited C1q binding to spores in a dose-dependent manner. C1q binding to spores lacking BclA (ΔbclA) was also significantly reduced compared with wild-type spores. In addition, deposition of both C3 and C4 as well as phagocytosis of spores were significantly reduced when BclA was absent, but were not reduced in the absence of IgG, suggesting that BclA, but not IgG, is important in these processes. Taken together, these results support a model in which spores actively engage CCP primarily through BclA interaction with C1q, leading to CCP activation and opsonophagocytosis of spores in an IgG-independent manner. These findings are likely to have significant implications on B. anthracis pathogenesis and microbial manipulation of complement.     

Surfactant protein A regulates complement activation.

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.     

Human pentraxin 3 binds to the complement regulator c4b-binding protein

The long pentraxin 3 (PTX3) is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP). A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.     

Chondrocyte Apoptosis Induced by Aggrecan G1 Domain as a Result of Decreased Cell Adhesion

A major feature of cartilage deterioration during joint injury and disease is aggrecan degradation and the loss of proteoglycan. Most of the degraded fragments are released into the circulatory system except the G1 domain which accumulates locally in the synovial fluid and cartilage because of its hyaluronan-binding ability. In this study, our objective was to investigate the effects of G1 accumulation on chondrocyte function. We chose to mimic the accumulation of G1 domain by developing a method to express G1 in chondrocytes. We transiently and stably expressed aggrecan G1 domain in the cells and tested the effects of G1 in cell adhesion and apoptosis. Overexpression of the G1 construct induced apoptosis in adherent chondrocytes but not in chondrocytes maintained in suspension cultures. Higher levels of G1 expression caused greater reduction in cell-substratum interaction and induced more cell death. The effect was dose dependent. To corroborate our findings, the role of G1 in reducing adhesion and inducing apoptosis was further investigated in fibroblasts. We found that low adherent cultures also had high levels of apoptosis. Our results suggest that G1 induced apoptosis by destabilizing cell-substratum interaction.