Collagen type XI is a constituent of the pericellular matrix of chondrocytes and plays a role in the regulation of fibrillogenesis. The amino-terminal domain of collagen type XI ␣1 chain is a noncollagenous structure that has been identified on the surface of cartilage collagen fibrils. The biochemical composition of the amino-terminal domain varies due to alternative splicing of the primary transcript. Recombinantly expressed ␣1(XI) aminoterminal domain isoforms were used in this study to investigate potential interactions. Purified products were analyzed for heparan sulfate binding properties. The results demonstrated that two additional binding sites exist within the ␣1(XI) aminoterminal domain, one within the amino propeptide and one within the variable region of the amino-terminal domain. Analysis of relative affinities indicated that the site located within the amino propeptide (site 1) was of similar affinity to sites that exist within the major triple helix of collagen type XI. Substitution of amino acid residues 147 to 152 within the amino propeptide by site-directed mutagenesis resulted in altered affinity for heparan sulfate. The binding site located within the variable region (site 2) demonstrated significantly higher affinity than other sites within the molecule. Displacement of collagen type XI within the pericellular matrix was observed in cell culture in the presence of excess heparan sulfate and by treatment with heparinase. These studies suggest two additional binding sites located within the noncollagenous amino-terminal domain that may play a role in the function of collagen type XI. The localization of collagen type XI within the pericellular matrix may be dependent upon interactions with heparan sulfate proteoglycans, and these are likely to take place in an isoform-specfic manner.Collagens represent an extensive family of proteins that are found in the extracellular matrix. Collagens are assembled from ␣ chains of which there have been reported at least 41 genetically distinct proteins comprising 28 different collagen types (1-3). All collagen family members are modular proteins characterized by regions of triple helix yet differ with respect to the combination and location of the nontriple helical domains present.Collagen type XI, composed of ␣1, ␣2, and ␣3 chains, appears to be concentrated pericellularly and plays a role in the regulation of fibrillogenesis (4, 5). The presence of collagen type XI correlates to the location of thin collagen fibrils within the pericellular matrix (6). The ␣1 and ␣2 chains of collagen type XI each contain homologous noncollagenous amino-terminal structures that comprise two separate regions: an amino propeptide (Npp) 2 and a variable region (VR). Together, the Npp and the variable region make up the amino-terminal domain (NTD) of the collagen ␣1(XI) chain. The ␣3 chain of collagen type XI is the gene product of Col2a1 and contains an amino-terminal domain that is distinct from the ␣1 and ␣2 chains (7).Within the NTD portion of ␣1 and ␣2, the Npp domain de...

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Collagen is a major component of the newly synthesized pericellular microenvironment of chondrocytes. Collagen types II, IX, and XI are synthesized and assembled into higher ordered complexes by a mechanism in which type XI collagen plays a role in nucleation of new fibrils, and in limiting fibril diameter. This study utilizes a cell line derived from the Swarm rat chondrosarcoma that allows the accumulation and assembly of pericellular matrix. Immunofluorescence and atomic force microscopy were used to assess early intermediates of fibril formation. Results indicate that this cell line synthesizes and secretes chondrocyte-specific pericellular matrix molecules including types II, IX, and XI collagen and is suitable for the study of newly synthesized collagen matrix under the experimental conditions used. AFM data indicate that small fibrils or assemblies of microfibrils are detectable and may represent precursors of the ~20 nm thin fibrils reported in cartilage. Treatment with hyaluronidase indicates that the dimensions of the small fibrils may be dependent upon the presence of hyaluronan within the matrix. This study provides information on the composition and organization of the newly synthesized extracellular matrix that plays a role in establishing the material properties and performance of biological materials such as cartilage.

Connective tissues such as cartilage, tendon, skin, bone, and arteries are composite bio-materials that contain predominantly water, collagen, proteoglycans and hyaluronic acid. Like any composite material, the components themselves and their interactions dictate the properties of the material. Fibrillar collagens are the principal structural molecules of the connective tissues and require regulated assembly and growth. Previous work from our lab indicates that the amino propeptide (Npp) domain of collagen type XI α1 chain regulates fibril diameter growth. Npp is a globular domain that is thought to sterically hinder the dense packing assembly of collagen molecules in fibrils. This mechanism of regulating collagen fibril assembly may be more complex than steric hindrance. We hypothesize that the Npp domain has a more dynamic role in establishing the structure/function relationship of collagen fibrils in connective tissues. In this study, the molecular structure of Npp was predicted by modeling. The model predicted putative binding sites for heparan sulfate and divalent cations. These predicted binding sites were evaluated empirically by fluorescence spectroscopy and surface plasmon resonance.