Proteomic analysis of Col11a1-associated protein complexes.
ABSTRACT: Cartilage plays an essential role during skeletal development within the growth plate and in articular joint function. Interactions between the collagen fibrils and other extracellular matrix molecules maintain structural integrity of cartilage, orchestrate complex dynamic events during embryonic development, and help to regulate fibrillogenesis. To increase our understanding of these events, affinity chromatography and liquid chromatography/tandem mass spectrometry were used to identify proteins that interact with the collagen fibril surface via the amino terminal domain of collagen ?1(XI) a protein domain that is displayed at the surface of heterotypic collagen fibrils of cartilage. Proteins extracted from fetal bovine cartilage using homogenization in high ionic strength buffer were selected based on affinity for the amino terminal noncollagenous domain of collagen ?1(XI). MS was used to determine the amino acid sequence of tryptic fragments for protein identification. Extracellular matrix molecules and cellular proteins that were identified as interacting with the amino terminal domain of collagen ?1(XI) directly or indirectly, included proteoglycans, collagens, and matricellular molecules, some of which also play a role in fibrillogenesis, while others are known to function in the maintenance of tissue integrity. Characterization of these molecular interactions will provide a more thorough understanding of how the extracellular matrix molecules of cartilage interact and what role collagen XI plays in the process of fibrillogenesis and maintenance of tissue integrity. Such information will aid tissue engineering and cartilage regeneration efforts to treat cartilage tissue damage and degeneration.
Project description:Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein/proteoglycan (SLRP) family found in vitreous humour and cartilage. It was first identified associated with the surface of vitreous collagen fibrils and several other SLRPs are also known to bind collagen fibrils and it some cases alter fibril morphology. The purpose of this study was to investigate the binding of opticin to the collagen II-containing fibrils found in vitreous and cartilage. Electron microscopic studies using gold labelling demonstrated that opticin binds vitreous and thin cartilage collagen fibrils specifically at a single site in the gap region of the collagen D-period corresponding to the e2 stain band; this is the first demonstration of the binding site of a class III SLRP on collagen fibrils. Opticin did not bind thick cartilage collagen fibrils from cartilage or tactoids formed in vitro from collagen II, but shows high specificity for thin, heterotypic collagen fibrils containing collagens II, and XI or V/XI. Vitreous collagen fibrils from opticin null and wild-type mice were compared and no difference in fibril morphology or diameter was observed. Similarly, in vitro fibrillogenesis experiments showed that opticin did not affect fibril formation. We propose that when opticin is bound to collagen fibrils, rather than influencing their morphology it instead hinders the binding of other molecules to the fibril surfaces and/or act as an intermediary bridge linking the collagen fibrils to other non-collagenous molecules.
Project description:BACKGROUND:In osteoarthritis (OA), cartilage matrix is lost despite vigorous chondrocyte anabolism. In this study, we attempted to determine whether altered matrix synthesis is involved in this paradox in disease progression through gene expression analysis and ultrastructural analysis of collagen fibrils within the cartilage matrix. METHODS:Cartilage tissues were obtained from 29 end-stage OA knees and 11 control knees. First, cDNA microarray analysis was performed and the expression of 9 genes involved in collagen fibrillogenesis was compared between OA and control cartilages. Then their expression was investigated in further detail by a quantitative polymerase chain reaction (qPCR) analysis combined with laser capture microdissection. Finally, collagen fibril formation was compared between OA and control cartilage by transmission electron microscopy. RESULTS:The result of the microarray analysis suggested that the expression of type IX and type XI collagens and fibrillogenesis-related small leucine-rich proteoglycans (SLRPs) may be reduced in OA cartilage relative to the type II collagen expression. The qPCR analysis confirmed these results and further indicated that the relative reduction in the minor collagen and SLRP expression may be more obvious in degenerated areas of OA cartilage. An ultrastructural analysis suggested that thicker collagen fibrils may be formed by OA chondrocytes possibly through reduction in the minor collagen and SLRP expression. CONCLUSIONS:This may be the first study to report the possibility of altered collagen fibrillogenesis in OA cartilage. Disturbance in collagen fibril formation may be a previously unidentified mechanism underlying the loss of cartilage matrix in OA.
Project description:Collagen type XI is a minor constituent of heterotypic collagen fibrils of developing cartilage and plays a regulatory role in fibril diameter. Collagen type XI is a heterotrimer composed of the alpha1, alpha2 and alpha3 chains. The mRNA encoding exons 6a, 6b and 8 of the alpha1 chain are expressed alternatively to generate six possible isoforms. The 6b-containing isoform has the most restricted distribution of all isoforms. It is first localized in the developing long bone, where mineralized tissue initially forms, and is later restricted to regions of cartilage that will be subsequently converted into bone. Bone morphogenetic protein 1 (BMP-1) and related proteins cleave procollagens I-III, V and VII, yielding triple-helical molecules that associate into collagen fibrils. The present study demonstrates that the alpha1 chain of collagen type XI can serve as a substrate for BMP-1. In addition, the efficiency with which BMP-1 processes different isoforms of the alpha1 chain varies. The amino acid sequence adjacent to the processing site influences the rate and extent of processing, as do sequences further away. Smaller fragments identified from cartilage extracts indicated that processing by BMP-1, in combination with other processing enzymes, generates small fragments of p6b-containing isoforms.
Project description:Fibrillar collagens are the principal structural molecules of connective tissues. The assembly of collagen fibrils is regulated by quantitatively minor fibrillar collagens, types V and XI. A unique amino-terminal propeptide domain of these collagens has been attributed this regulatory role. The structure of the amino terminal propeptide has yet to be determined. Low sequence similarity necessitated a secondary structure-based method to carry out homology modeling based upon the determined structure of LNS family members, named for a common structure in the laminin LG5 domain, the neurexin 1B domain and the sex hormone binding globulin. Distribution of amino acids within the model suggested glycosaminoglycan interaction and calcium binding. These activities were tested experimentally. Sequence analyses of existing genes for collagens indicate that 16 known collagen alpha chains may contain an LNS domain. A similar approach may prove useful for structure/function studies of similar domains in other collagens with similar domains. This will provide mechanistic details of the organization and assembly of the extracellular matrix and the underlying basis of structural integrity in connective tissues. The absolute requirement for collagen XI in skeletal growth is indicated by collagen XI deficiencies such as chondrodystrophies found in the cho/cho mouse and in humans with Stickler syndrome.
Project description:Collagens V and XI comprise a single regulatory type of fibril-forming collagen with multiple isoforms. Both co-assemble with collagen I or II to form heterotypic fibrils and have been implicated in regulation of fibril assembly. The objective of this study was to determine the roles of collagens V and XI in the regulation of tendon fibrillogenesis. Flexor digitorum longus tendons from a haplo-insufficient collagen V mouse model of classic Ehlers Danlos syndrome (EDS) had decreased biomechanical stiffness compared with controls consistent with joint laxity in EDS patients. However, fibril structure was relatively normal, an unexpected finding given the altered fibrils observed in dermis and cornea from this model. This suggested roles for other related molecules, i.e. collagen XI, and compound Col5a1(+/-),Col11a1(+/-) tendons had altered fibril structures, supporting a role for collagen XI. To further evaluate this, transcript expression was analyzed in wild type tendons. During development (E18-P10) both collagen V and XI were comparably expressed; however, collagen V predominated in mature (P30) tendons. The collagens had a similar expression pattern. Tendons with altered collagen V and/or XI expression (Col5a1(+/-); Col11a1(+/-); Col5a1(+/-),Col11a1(+/-); Col11a1(-/-); Col5a1(+/-),Col11a1(-/-)) were analyzed at E18. All genotypes demonstrated a reduced fibril number and altered structure. This phenotype was more severe with a reduction in collagen XI. However, the absence of collagen XI with a reduction in collagen V was associated with the most severe fibril phenotype. The data demonstrate coordinate roles for collagens V and XI in the regulation of fibril nucleation and assembly during tendon development.
Project description:Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The "cell interaction domain" is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The "matrix interaction domain" may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.
Project description:In cartilage collagen type XI exists as heterotrimeric molecules composed of alpha 1(XI), alpha 2(XI) and alpha 3(XI) subunits. Messenger RNAs for some of the alpha chains of collagen type XI have also been found in non-chondrogenic tissues but the chain composition of the molecule in these sites is not known. Some non-chondrogenic tissues also contain heterotrimers containing collagen alpha 2(V) and alpha 1(XI) chains. We have explored the possibility that collagen type XI could exist in differing trimeric forms in non-chondrogenic tissues and aimed to predict the subunit composition of this collagen in those tissues. The distribution and relative levels of expression of collagen alpha 1(XI), alpha 2(XI) and alpha 3(XI)/alpha 1(II) mRNAs in different human fetal tissues were studied. Expression of mRNAs for all three genes of collagen type XI is not restricted to cartilage but is widespread. However, in some non-chondrogenic tissues, the mRNAs for all three alpha chains of collagen type XI were not co-expressed, but collagen alpha 1(XI) and alpha 2(XI) mRNAs were found either singly or without collagen alpha 3(XI) transcripts. Collagen type XI may therefore exist as homotrimers and/or heterotrimers composed of two collagen alpha(XI) chains in some tissues. The distribution of mRNAs for collagen alpha 2(V) and alpha 1(I) were also studied. Co-expression of collagen type XI, alpha 2(V) and alpha 1(I) mRNAs was found for many tissues. These findings have implications for the possibility of additional chain associations for collagen types XI and V in cross-type heterotrimers within heterotypic fibrils.
Project description:Type IX collagen is covalently bound to the surface of type II collagen fibrils within the cartilage extracellular matrix. The N-terminal, globular noncollagenous domain (NC4) of the ?1(IX) chain protrudes away from the surface of the fibrils into the surrounding matrix and is available for molecular interactions. To define these interactions, we used the NC4 domain in a yeast two-hybrid screen of a human chondrocyte cDNA library. 73% of the interacting clones encoded fibronectin. The interaction was confirmed using in vitro immunoprecipitation and was further characterized by surface plasmon resonance. Using whole and pepsin-derived preparations of type IX collagen, the interaction was shown to be specific for the NC4 domain with no interaction with the triple helical collagenous domains. The interaction was shown to be of high affinity with nanomolar K(d) values. Analysis of the fibronectin-interacting clones indicates that the constant domain is the likely site of interaction. Type IX collagen and fibronectin were shown to co-localize in cartilage. This novel interaction between the NC4 domain of type IX collagen and fibronectin may represent an in vivo interaction in cartilage that could contribute to the matrix integrity of the tissue.
Project description:Marshall syndrome is a rare, autosomal dominant skeletal dysplasia that is phenotypically similar to the more common disorder Stickler syndrome. For a large kindred with Marshall syndrome, we demonstrate a splice-donor-site mutation in the COL11A1 gene that cosegregates with the phenotype. The G+1-->A transition causes in-frame skipping of a 54-bp exon and deletes amino acids 726-743 from the major triple-helical domain of the alpha1(XI) collagen polypeptide. The data support the hypothesis that the alpha1(XI) collagen polypeptide has an important role in skeletal morphogenesis that extends beyond its contribution to structural integrity of the cartilage extracellular matrix. Our results also demonstrate allelism of Marshall syndrome with the subset of Stickler syndrome families associated with COL11A1 mutations.
Project description:Type XI collagen is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the -530-bp promoter segment of the alpha 2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Krüppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRAB-zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.