The C-Type Lectin of the Aggrecan G3 Domain Activates Complement

Excessive complement activation contributes to joint diseases such as rheumatoid arthritis and osteoarthritis during which cartilage proteins are fragmented and released into the synovial fluid. Some of these proteins and fragments activate complement, which may sustain inflammation. The G3 domain of large cartilage proteoglycan aggrecan interacts with other extracellular matrix proteins, fibulins and tenascins, via its C-type lectin domain (CLD) and has important functions in matrix organization. Fragments containing G3 domain are released during normal aggrecan turnover, but increasingly so in disease. We now show that the aggrecan CLD part of the G3 domain activates the classical and to a lesser extent the alternative pathway of complement, via binding of C1q and C3, respectively. The complement control protein (CCP) domain adjacent to the CLD showed no effect on complement initiation. The binding of C1q to G3 depended on ionic interactions and was decreased in D2267N mutant G3. However, the observed complement activation was attenuated due to binding of complement inhibitor factor H to CLD and CCP domains. This was most apparent at the level of deposition of terminal complement components. Taken together our observations indicate aggrecan CLD as one factor involved in the sustained inflammation of the joint.     

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.     

The Different Roles of Aggrecan Interaction Domains

The aggregating proteoglycans of the lectican family are important components of extracellular matrices. Aggrecan is the most well studied of these and is central to cartilage biomechanical properties and skeletal development. Key to its biological function is the fixed charge of the many glycosaminoglycan chains, that provide the basis for the viscoelastic properties necessary for load distribution over the articular surface. This review is focused on the globular domains of aggrecan and their role in anchoring the proteoglycans to other extracellular matrix components. The N-terminal G1 domain is vital in that it binds the proteoglycan to hyaluronan in ternary complex with link protein, retaining the proteoglycan in the tissue. The importance of the C-terminal G3 domain interactions has recently been emphasized by two different human hereditary disorders: autosomal recessive aggrecan-type spondyloepimetaphyseal dysplasia and autosomal dominant familial osteochondritis dissecans. In these two conditions, different missense mutations in the aggrecan C-type lectin repeat have been described. The resulting amino acid replacements affect the ligand interactions of the G3 domain, albeit with widely different phenotypic outcomes.     

Fibulin-1 is a ligand for the C-type lectin domains of aggrecan and versican.

The aggregating proteoglycans (aggrecan, versican, neurocan, and brevican) are important components of many extracellular matrices. Their N-terminal globular domain binds to hyaluronan, but the function of their C-terminal region containing a C-type lectin domain is less clear. We now report that a 90-kDa protein copurifies with recombinant lectin domains from aggrecan and versican, but not from the brain-specific neurocan and brevican. Amino acid sequencing of tryptic peptides from this protein identified it as fibulin-1. This extracellular matrix glycoprotein is strongly expressed in tissues where versican is expressed (blood vessels, skin, and developing heart), and also expressed in developing cartilage and bone. It is thus likely to interact with these proteoglycans in vivo. Surface plasmon resonance measurements confirmed that aggrecan and versican lectin domains bind fibulin-1, whereas brevican and neurocan do not. As expected for a C-type lectin, the interactions with fibulin-1 are Ca2+-dependent, with KD values in the low nanomolar range. Using various deletion mutants, the binding site for aggrecan and versican lectin domains was mapped to the epidermal growth factor-like repeats in domain II of fibulin-1. No difference in affinity was found for deglycosylated fibulin-1, indicating that the proteoglycan C-type lectin domains bind to the protein part of fibulin-1.     

The proteoglycans aggrecan and Versican form networks with fibulin-2 through their lectin domain binding

Aggrecan, versican, neurocan, and brevican are important components of the extracellular matrix in various tissues. Their amino-terminal globular domains bind to hyaluronan, but the function of their carboxyl-terminal globular domains has long remained elusive. A picture is now emerging where the C-type lectin motif of this domain mediates binding to other extracellular matrix proteins. We here demonstrate that aggrecan, versican, and brevican lectin domains bind fibulin-2, whereas neurocan does not. As expected for a C-type lectin, the interactions are calcium-dependent, with K(D) values in the nanomolar range as measured by surface plasmon resonance. Solid phase competition assays with previously identified ligands demonstrated that fibulin-2 and tenascin-R bind the same site on the proteoglycan lectin domains. Fibulin-1 has affinity for the common site on versican but may bind to a different site on the aggrecan lectin domain. By using deletion mutants, the interaction sites for aggrecan and versican lectin domains were mapped to epidermal growth factor-like repeats in domain II of fibulin-2. Affinity chromatography and solid phase assays confirmed that also native full-length aggrecan and versican bind the lectin domain ligands. Electron microscopy confirmed the mapping and demonstrated that hyaluronan-aggrecan complexes can be cross-linked by the fibulins.     

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.     

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.     

The matrilins: modulators of extracellular matrix assembly

The matrilins form a family of oligomeric extracellular adaptor proteins that are most strongly expressed in cartilage but also present in many other extracellular matrices. Matrilins bind to different types of collagen fibrils, to other noncollagenous proteins and to aggrecan. They thereby support matrix assembly by connecting fibrillar components and mediating interactions between these and the aggrecan gel. The binding avidity of a matrilin can be varied by alternative splicing, proteolytic processing and formation of homo- and heterooligomers. Such changes in matrilin structure may lead to a modulation of extracellular matrix assembly. Some matrilins bind weakly to α1β1 integrin and cell surface proteoglycans, but even though matrilins play a role in mechanotransduction and matrilin-3 activates the expression of osteoarthritis-associated genes the physiological relevance of matrilin-cell interactions is unclear. Matrilin knockout mice do not display pronounced phenotypes, which points to a redundancy within the protein family or with functionally related proteins. In man, dominant mutations in the von Willebrand factor A like domain of matrilin-3 lead to a protein retention in the endoplasmic reticulum that causes multiple epiphyseal dysplasia by initiating a cell stress response. In contrast, a mutation in an EGF domain of matrilin-3 that is associated with hand osteoarthritis and disc degeneration does not interfere with secretion but instead with extracellular assembly of matrix structures. In this review we summarize such information on matrilin structure and function that we believe is important for the understanding of extracellular matrix assembly and for deciphering pathophysiological mechanisms in diseases causing skeletal malformations or cartilage degeneration.     

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.     

Expression and characterization of a carbohydrate-binding fragment of rat aggrecan

The COOH-terminal portion of cartilage proteoglycan core protein,aggrecan, expressed by in vitro translation, binds carbohydrate-containingaffinity columns. The in vitro expression approach has beenused to define the sugar-binding protion of the core protein.The active fragment, which corresponds closely to the carbohydrate-recognitiondomains in the family of Ca²⁺-dependent (C-type) animal lectins,has been expressed in bacteria and characterized. The CD spectrumof the domain is very similar to the spectrum of the bindingdomain of serum mannose-binding protein, suggesting that itsoverall structure probably resembles the known three-dimensionalstructure of the mannose-binding domain. The binding specificityof the core protein fragment has been characterized using asolidphase assay. The results suggest that the monosaccharide-bindingsite is also similar to that in other C-type carbohydrate-recognitiondomains. binding carbohydrate recognition expression lectin proteoglycan     

Aggrecan: Beyond cartilage and into the brain

Aggrecan is well-studied in cartilage but its expression and function in the central nervous system has only recently begun to be appreciated. Aggrecan plays an important role in the organization of the neural extracellular space by binding and organizing hyaluronan to the cell surface through interactions with link protein and tenascins forming a large aggregated quaternary complex. While all members of the lectican family to which aggrecan belongs are thought to mediate similar roles in organizing the neural matrix, aggrecan is unique in that it is the only family member found almost exclusively in an enigmatic matrix substructure called the perineuronal net. Current work has established a critical role for perineuronal nets and aggrecan in regulating developmental neural plasticity and in the recover from injury. In this review we focus on the structure, expression and function of aggrecan in the central nervous system.     

Aggrecan domains expected to traffic through the exocytic pathway are misdirected to the nucleus

In this article, we report the misdirected targeting of expressed aggrecan domains. Aggrecan, the chondroitin sulfate (CS) proteoglycan of cartilage, normally progresses through the exocytic pathway. Proteins expressed from constructs containing the putative aggrecan signal sequence (i.e., the first 23 N-terminal amino acids), specified globular (G) domains G1 and/or G3, and a segment of the CS domain were detected in the endoplasmic reticulum (ER) and Golgi complex. Although proteins expressed from constructs containing the putative signal and G3, but lacking G1, were detected to a limited extent in the secretory pathway, they primarily accumulated in nuclei. Discrete nuclear inclusions were seen when G3 was expressed. Immunoelectron microscopic characterization of the inclusions suggested the association of nuclear G3 with other proteins. When signal-free G3 constructs and those with G3 immediately following the N-terminal signal were expressed, abundant dispersed accumulations filled the nucleoplasm. The data suggest first, that signal-free and signal-containing G3 proteins enter the nucleus from the cytosol, and second, that the entry of signal-containing G3 proteins into the ER lumen is inefficient. Hsp25, Hsp70, and ubiquitin were colocalized with nuclear G3, indicating the involvement of chaperones and the degradative machinery in the formation and/or attempted disposal of the abnormal nuclear inclusions. Overall, the results focus attention on (1) intracellular protein trafficking at the ER membrane and the nuclear envelope and (2) chaperone interactions and mechanisms leading to abnormal protein deposition in the nucleus.     

Galactose recognition by a tetrameric C-type lectin, CEL-IV, containing the EPN carbohydrate recognition motif

CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galβ1-3/4(Fucα1-3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity.     

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.     

Aggrecan core protein is expressed in membranous bone of the chick embryo. Molecular and biomechanical studies of normal and nanomelia embryos.

The recessive mutation nanomelia blocks the synthesis of a large aggregating proteoglycan (aggrecan) by avian embryo chondrocytes. Lack of aggrecan is associated with short stature, multiple morphological defects in cartilage, and embryo lethality. Bony defects have also been described, but were assumed to be a secondary consequence of the cartilage defect. However, two lines of evidence presented in this paper indicate that the aggrecan deficiency directly affects intramembranous bone. First, the morphology (i.e. projected area and shape) of certain membranous bones of nanomelia embryos was abnormal. Second, membranous bone from nanomelia embryos proved to be significantly stiffer in biomechanical tests that measured functional properties of the extracellular matrix. These findings were unexpected because intramembranous bones normally develop from mesenchyme and not from a cartilage intermediate, and they prompted a search for evidence of aggrecan expression in the bone of normal chick embryos. We report that: 1) aggrecan mRNA was identified by PCR analysis of total RNA isolated from day-13 chick embryo calvarium, 2) the PCR method successfully amplified aggrecan mRNA from primary chick embryo osteoblasts in culture, 3) in situ hybridization of membranous bone tissue sections demonstrated aggrecan expression by chick embryo osteoblasts in vivo, and 4) the aggrecan message was identified in Northern blots of calvarial mRNA probed at high stringency. The results of the molecular and biomechanical studies provide evidence that aggrecan is indeed expressed in membranous bone as well as cartilage. Altogether, these results suggest that aggrecan may contribute to the functional properties and the normal growth and development of avian membranous bone.     

Interaction of cartilage oligomeric matrix protein/thrombospondin 5 with aggrecan

Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix (ECM) of the musculoskeletal system. Its importance is underscored by its association with several growth disorders. In this report, we investigated its interaction with aggrecan, a major component of cartilage ECM. We also tested a COMP/TSP5 mutant, designated MUT3 that accounts for 30% of human pseudoachondroplasia cases, to determine if the mutation affects function. Using a solid-phase binding assay, we have shown that COMP/TSP5 can bind aggrecan. This binding was decreased with MUT3, or when COMP/TSP5 was treated with EDTA, indicating the presence of a conformation-dependent aggrecan binding site. Soluble glycosaminoglycans (GAGs) partially inhibited binding, suggesting that the interaction was mediated in part through aggrecan GAG side chains. Using affinity co-electrophoresis, we showed that COMP/TSP5, in its calcium-replete conformation, bound to heparin, chondroitin sulfates, and heparan sulfate; this binding was reduced with EDTA treatment of COMP/TSP5. MUT3 showed weaker binding than calcium-repleted COMP/TSP5. Using recombinant COMP/TSP5 fragments, we found that the "signature domain" could bind to aggrecan, suggesting that this domain can mediate the interaction of COMP/TSP5 and aggrecan. In summary, our data indicate that COMP/TSP5 is an aggrecan-binding protein, and this interaction is regulated by the calcium-sensitive conformation of COMP/TSP5; interaction of COMP with aggrecan can be mediated through the GAG side chains on aggrecan and the "signature domain" of COMP/TSP5. Our results suggest that COMP/TSP5 may function to support matrix interactions in cartilage ECM.     

Monoclonal antibodies directed against epitopes within the core protein structure of the large aggregating proteoglycan (aggrecan) from the swarm rat chondrosarcoma.

The core protein of the large hyaline cartilage proteoglycan, aggrecan, is composed of six distinct domains: globular 1 (G1), interglobular, globular 2 (G2), keratan sulfate attachment, chondroitin sulfate (CS) attachment, and globular 3 (G3). Monoclonal antibodies that recognize epitopes in these domains were raised against Swarm rat chondrosarcoma aggrecan that was either denatured through reduction and alkylation or partially deglycosylated through chondroitinase ABC digestion or alkali elimination, the latter with or without sulfite addition. Monoclonal antibodies were further characterized for reactivity to purified aggrecan substructures including rat chondrosarcoma G1 and CS attachment domains, a recombinant rat chondrosarcoma G3 domain fusion protein, bovine articular cartilage G2 domain, and rat chondrosarcoma link protein (LP). Biochemical characterization of the specificities of these monoclonal antibodies indicated that one (1C6) recognized an epitope shared by both the G1 and the G2 domains; one (5C4) recognized an epitope shared by both LP and the G1 domain; one (7D1) recognized an epitope shared by both the G1 and the CS attachment domains; two (14A1 and 15B2) recognized epitopes in the CS attachment domain; one (14B4) recognized an epitope in the G3 domain; and one (13D1) recognized a ubiquitous epitope shared by the G1, G2, G3, and CS attachment domains of aggrecan and also LP. Collectively the specificities of these antibodies confirm the occurrence of multiple repeated epitopes (both carbohydrate and protein in nature) throughout the different domain structures of aggrecan. These antibodies have been proven to be useful for identifying aggrecan-like molecules in several connective tissues other than cartilage.     

Aggrecan protects cartilage collagen from proteolytic cleavage

The matrix components responsible for cartilage mechanical properties, type II collagen and aggrecan, are degraded in osteoarthritis through proteolytic cleavage by matrix metalloproteinases (MMPs) and aggrecanases, respectively. We now show that aggrecan may serve to protect cartilage collagen from degradation. Although collagen in freeze-thawed cartilage depleted of aggrecan was completely degraded following incubation with MMP-1, collagen in cartilage with intact aggrecan was not. Using interleukin-1-stimulated bovine nasal cartilage explants where aggrecan depletion occurs during the first week of culture, followed by collagen loss during the second week, we evaluated the effect of selective MMP and aggrecanase inhibitors on degradation. A selective MMP inhibitor did not block aggrecan degradation but caused complete inhibition of collagen breakdown. Similar inhibition was seen with inhibitor addition following aggrecan depletion on day 6-8, suggesting that MMPs are not causing significant collagen degradation prior to the second week of culture. Inclusion of a selective aggrecanase inhibitor blocked aggrecan degradation, and, in addition, inhibited collagen degradation. When the inhibitor was introduced following aggrecan depletion, it had no effect on collagen breakdown, ruling out a direct effect through inhibition of collagenase. These data suggest that aggrecan plays a protective role in preventing degradation of collagen fibrils, and that an aggrecanase inhibitor may impart overall cartilage protection.     

Order within disorder: Aggrecan chondroitin sulphate‐attachment region provides new structural insights into protein sequences classified as disordered

Structural investigation of proteins containing large stretches of sequences without predicted secondary structure is the focus of much increased attention. Here, we have produced an unglycosylated 30 kDa peptide from the chondroitin sulphate (CS)‐attachment region of human aggrecan (CS‐peptide), which was predicted to be intrinsically disordered and compared its structure with the adjacent aggrecan G3 domain. Biophysical analyses, including analytical ultracentrifugation, light scattering, and circular dichroism showed that the CS‐peptide had an elongated and stiffened conformation in contrast to the globular G3 domain. The results suggested that it contained significant secondary structure, which was sensitive to urea, and we propose that the CS‐peptide forms an elongated wormlike molecule based on a dynamic range of energetically equivalent secondary structures stabilized by hydrogen bonds. The dimensions of the structure predicted from small‐angle X‐ray scattering analysis were compatible with EM images of fully glycosylated aggrecan and a partly glycosylated aggrecan CS2‐G3 construct. The semiordered structure identified in CS‐peptide was not predicted by common structural algorithms and identified a potentially distinct class of semiordered structure within sequences currently identified as disordered. Sequence comparisons suggested some evidence for comparable structures in proteins encoded by other genes (PRG4, MUC5B, and CBP). The function of these semiordered sequences may serve to spatially position attached folded modules and/or to present polypeptides for modification, such as glycosylation, and to provide templates for the multiple pleiotropic interactions proposed for disordered proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.