Fetal Bovine Cartilage Research Articles

Decellularized Articular Cartilage Microgels as Microcarriers for Expansion of Mesenchymal Stem Cells.

Conventional microcarriers used for expansion of human mesenchymal stem cells (hMSCs) require detachment and separation of the cells from the carrier prior to use in clinical applications for regeneration of articular cartilage, and the carrier can cause undesirable phenotypic changes in the expanded cells. This work describes a novel approach to expand hMSCs on biomimetic carriers based on adult or fetal decellularized bovine articular cartilage that supports tissue regeneration without the need to detach the expanded cells from the carrier. In this approach, the fetal or adult bovine articular cartilage was minced, decellularized, freeze-dried, ground, and sieved to produce articular cartilage microgels (CMGs) in a specified size range. Next, the hMSCs were expanded on CMGs in a bioreactor in basal medium to generate hMSC-loaded CMG microgels (CMG-MSCs). Then, the CMG-MSCs were suspended in sodium alginate, injected in a mold, crosslinked with calcium chloride, and incubated in chondrogenic medium as an injectable cellular construct for regeneration of articular cartilage. The expression of chondrogenic markers and compressive moduli of the injectable CMG-MSCs/alginate hydrogels incubated in chondrogenic medium were higher compared to the hMSCs directly encapsulated in alginate hydrogels.

Proteomic analysis of Col11a1-associated protein complexes.

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 α1(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 α1(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 α1(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.

Derivation, characterization and expansion of fetal chondrocytes on different microcarriers

Fetal chondrocytes (FCs) have recently been identified as an alternative cell source for cartilage tissue engineering applications because of their partially chondrogenically differentiated phenotype and developmental plasticity. In this study, chondrocytes derived from fetal bovine cartilage were characterized and then cultured on commercially available Cytodex-1 and Biosilon microcarriers and thermosensitive poly(hydroxyethylmethacrylate)-poly(N-isopropylacrylamide) (PHEMA-PNIPAAm) beads produced by us. Growth kinetics of FCs were estimated by means of specific growth rate and metabolic activity assay. Cell detachment from thermosensitive microcarriers was induced by cold treatment at 4°C for 20min or enzymatic treatment was applied for the detachment of cells from Cytodex-1 and Biosilon. Although attachment efficiency and proliferation of FCs on PHEMA-PNIPAAm beads were lower than that of commercial Cytodex-1 and Biosilon microcarriers, these beads also supported growth of FCs. Detached cells from thermosensitive beads by cold induction exhibited a normal proliferative activity. Our results indicated that Cytodex-1 microcarrier was the most suitable material for the production of FCs in high capacity, however, 'thermosensitive microcarrier model' could be considered as an attractive solution to the process scale up for cartilage tissue engineering by improving surface characteristics of PHEMA-PNIPAAm beads.

Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms

The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo.

Chondromodulin-I: A Growth-Modulating Functional Matrix in Cartilage

Cartilaginous extracellular matrix (ECM) is a unique assembly of macromolecules such as type II collagen and proteoglycans. In association with a number of accessory molecules, it confers special biological functions on cartilage. Among these various accessory molecules, growth-modulating functions have been identified only in several instances, and in many cases functional properties therefore remain to be elucidated. Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein, which was initially identified in our laboratory as a cartilage-specific functional matrix component that stimulates the growth and matrix synthesis of primary chondrocytes in vitro. We subsequently purified an endothelial cell growth inhibitor from guanidinium extracts of fetal bovine cartilage, which was surprisingly found to be identical to ChM-I. Purified ChM-I was found to inhibit the proliferation of vascular endothelial cells as well as tube morphogenesis in vitro. The generation of a ChM-I null mouse has enabled us to examine the functional roles of this unique ECM component in vivo. In this review, we describe the identification and biological characterization of ChM-I as an example of a growth-modulating functional matrix in cartilage.

Removal of O-linked and N-linked oligosaccharides is required for optimum detection of NITEGE neoepitope on ADAMTS4-digested fetal aggrecans: implications for specific N-linked glycan-dependent aggrecanolysis at Glu373–Ala374

We have observed that Western blot analysis with an anti-G1 antibody detects G1-NITEGE product in a disintegrin and metalloprotease with thrombospondin motifs-4 (ADAMTS4)-digested fetal and mature human and bovine aggrecan, but the neoepitope-specific anti-NITEGE antibody only detects this product in digests of mature aggrecan. Our objective was to determine whether enzymatic removal of O- and/or N-linked oligosaccharides from the fetal products would enable detection of the NITEGE neoepitope with anti-NITEGE antibody. Aggrecan was purified from fetal and mature human and bovine cartilage and digested with: (1) ADAMTS4, (2) ADAMTS4, sialidase II, and N-glycanase, (3) ADAMTS4, sialidase II, and O-glycanase, or (4) ADAMTS4, sialidase II, and both N- and O-glycanases. Western blot analysis was performed using anti-G1 and anti-NITEGE antibodies. When fetal G1-NITEGE products were treated with a combination of ADAMTS4, sialidase II, O-glycanase and N-glycanase, the resultant products migrated faster on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the NITEGE neoepitope was rendered detectable. It appears that the NITEGE neoepitope is blocked on Western blots by oligosaccharide structures present on Asn368 and Thr370 of fetal human and bovine aggrecans. Such masking structures do not appear to be present on mature aggrecans from these species. We suggest that when anti-NITEGE antibody is used in Western analysis, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), and immunohistochemistry (IHC), removal of oligosaccharides with appropriate glycosidases may unmask reactivity that would otherwise go undetected. The implications of these findings for the much-studied effect of Asn368-linked keratan sulfate (KS)-based structures on ADAMTS4 and ADAMTS5 activity are discussed.

Chondromodulin-I and Tenomodulin: The Negative Control of Angiogenesis in Connective Tissue

The negative regulation of angiogenesis may provide a promising therapeutic target for a number of lifestyle-related diseases, as the switch to an angiogenic phenotype in many tissues represents a critical step during the progression of such disorders. Cartilage is avascular and shows resistance to vascular invasion from the surrounding well-vascularized mesenchyme. Using guanidine extracts of fetal bovine cartilage, we have identified and purified chondromodulin-I (ChM-I) as an angiogenesis inhibitor. The cDNA sequence of this factor has revealed that the ChM-I precursor protein is a type II transmembrane glycoprotein (334 amino acids) and that mature ChM-I is encoded in the C-terminal region of the precursor. After cleavage of the ChM-I precursor at its processing site, mature ChM-I (120 amino acids) is secreted from chondrocytes into the extracellular matrix. Following on from the identification of ChM-I as an angiogenesis inhibitor in cartilage, we have also cloned both mouse and human tenomodulin (TeM), which share significant homology with ChM-I at their C-termini. Moreover, exogenous expression experiments in COS cells suggests that TeM is a type II transmembrane glycoprotein (317 amino acids). When overexpressed in HUVECs, the C-terminal domain (116 amino acids) of the TeM protein shows both anti-angiogenic and anti-tumorigenic activities at equivalent levels to mature ChM-I. In our present review, we discuss the structure, biological activities and localization of these anti-angiogenic molecules.

Cartilage-derived growth factor and antitumor factor: past, present, and future studies

We isolated growth-cartilage cells from rat and rabbit ribs and cultured them in vitro . Then we found that growth-cartilage cells in culture have a marked osteogenic potential, forming differentiated colonies abundant macromolecules such as proteoglycans and type II collagen in vitro . We also found that calcitonin and parathyroid hormone markedly enhanced proteoglycan synthesis in these cells. In addition to these systemic factors, extracellular matrix components have long been thought to play important roles in the control of cartilage development. Thus, we have searched for regulatory factors in cartilage and found that fetal bovine cartilage contains cartilage-specific growth modulating matrices that stimulate DNA synthesis of cartilage cells in the presence of bFGF. We named this unique principle chondromodulin- I (ChM- I ). The aim of this chapter is to survey various approaches to the study of recent advances in the regulation of cartilage development by locally generated factors.

Bovine chondrocyte behaviour in three-dimensional type I collagen gel in terms of gel contraction, proliferation and gene expression

This study evaluated the in vitro behaviour of bovine chondrocytes seeded in collagen gels, promising recently reported scaffolds for the treatment of full-thickness cartilage defects. To determine how chondrocytes respond to a collagen gel environment, 2×10 6 chondrocytes isolated from fetal, calf and adult bovine cartilage were seeded within type I collagen gels and grown for 12 days in both attached and floating (detached from the culture dish after polymerisation) conditions. Monolayer cultures were performed in parallel. All chondrocytes contracted floating gels to 55% of the initial size, by day 12. Contraction was dependent on initial cell density and inhibited by the presence of dihydrocytochalasin B as previously observed with fibroblasts. Gene expression was determined using conventional and real-time PCR. The chondrocyte phenotype was better maintained in floating gels compared to attached gels and monolayers. This was demonstrated by comparing the ratio of COL2A1/ COL1A2 mRNA and also of alpha10/alpha11 integrin mRNA. A strong up-regulation of MMP13 expression was measured at day 12 in floating gels. The composition of cartilage-like tissue obtained by growing chondrocytes in a collagen gel varied depending on the floating or attached conditions and initial cell density. It is thus important to consider these parameters when using this culture system in order to prepare a well-defined implant for cartilage repair.

Variations in aggrecan structure modulate its susceptibility to aggrecanases.

Proteoglycan aggregates and purified aggrecan from adult and fetal bovine cartilage and adult and neonatal human cartilage were subjected to in vitro degradation by recombinant aggrecanase-1 and aggrecanase-2. The ability of the aggrecanases to cleave within the aggrecan IGD (interglobular domain) and CS2 domain (chondroitin sulphate-rich domain 2) was monitored by SDS/PAGE and immunoblotting. Aggrecanase-2 showed a similar ability to cleave within the IGD of adult and immature aggrecan, whereas aggrecanase-1 was less efficient in cleavage in the IGD of immature aggrecan, for both the bovine and the human substrates. Both aggrecanases showed a similar ability to cleave within the CS2 domain of bovine aggrecan irrespective of age, but showed a much lower ability to cleave within the CS2 domain of human aggrecan. Equivalent results were obtained whether aggrecan was present in isolation or as part of proteoglycan aggregates. When proteoglycan aggregates were used, neither aggrecanase was able to cleave link protein. Thus, for aggrecan cleavage by aggrecanases, variations in cleavage efficiency exist with respect to the species and age of the animal from which the aggrecan is derived and the type of aggrecanase being used.

Integrative articular cartilage repair: dependence on developmental stage and collagen metabolism

Objectives The objectives of this research were to determine whether the integrative repair of bovine cartilage explants was dependent on developmental stage, and whether observed differences in integration with developmental stage were related to deposition of newly synthesized collagen and lysyl oxidase-mediated collagen cross-linking.Methods Pairs of fetal, newborn calf, and adult bovine cartilage blocks were cultured in partial apposition for 2 weeks in medium supplemented with serum, ascorbate, and [3H]proline. Following culture, mechanical integration between apposed cartilage blocks was assessed by measuring adhesive strength in a single-lap shear configuration. Formation and stabilization of newly synthesized protein and collagen was investigated by determination of [3H]proline and [3H]hydroxyproline in tissue digests and guanidine extracts.Results Calf cartilage exhibited a relatively high integrative repair phenotype, achieving an adhesive strength that was three–four-fold that of adult or fetal specimens. The low and high integrative repair phenotypes appeared related in part to different levels of collagen biosynthesis, which was ∼four–five-fold higher in calf cartilage samples than in the adult. However, fetal cartilage also exhibited a high level of biosynthesis. The different integrative repair phenotypes were not associated with marked differences in the kinetics of chemical stabilization of newly synthesized collagen, as the proportion of incorporated [3H]proline and newly-formed [3H]hydroxyproline that was resistant to extraction by 4M guanidine-HCl following culture was similar for cartilage from all developmental stages. Integration of calf cartilage appeared to depend on lysyl oxidase-mediated collagen cross-link formation, since inclusion of beta-aminopropionitrile (BAPN) in the culture medium completely eliminated development of adhesive strength. BAPN treatment also increased the percentage of newly synthesized protein in the guanidine extracts from 10% to 36% of the total, and that of newly synthesized collagen from 2% to 20%, while having only slight inhibitory effects on overall protein and collagen biosynthesis.Conclusion The finding that cartilage exhibits enhanced integrative repair at a certain developmental stage suggests that it may ultimately be possible to enhance repair when needed in clinical situations.

Characterization of EHD4, an EH domain-containing protein expressed in the extracellular matrix.

To identify proteins that promote assembly of type VI collagen tetramers or stabilize type VI collagen filaments, a two-hybrid screen of a human placenta library was used and a new extracellular protein discovered. The cDNA sequence of the new protein encodes 541 amino acid residues. This cDNA sequence is identical to EHD4, a recently described member of the EH domain family of proteins. Two mRNAs of 4.4 and 3.0 kilobases were present in human skin fibroblasts and most tissues tested but were most prevalent in the heart. The chromosomal localization of the gene for this new protein was determined to be at 15q14-q15. Three polyclonal peptide antibodies were made against synthetic EHD4 peptides. The affinity-purified antibodies were used in immunofluorescent staining of developing limbs and matrices produced by human skin fibroblasts and mouse NIH3T3 fibroblasts in culture. Embryonic rat limb cartilage was strongly stained throughout development, and cultured fibroblasts deposited an extracellular filamentous network containing EHD4. In non-denaturing extracts of fetal bovine cartilage and in human skin fibroblast culture media, two components of approximately 220 and 158 kDa were observed, which, after reduction, migrated as a 56-kDa component on SDS-polyacrylamide gel electrophoresis. EHD4 is the first extracellular matrix protein described that contains an EH domain.

The ribosomal protein QM is expressed differentially during vertebrate endochondral bone development.

Endochondral ossification is a carefully coordinated developmental process that converts the cartilaginous model of the embryonic skeleton to bone with accompanying long bone growth. To identify genes that regulate this process we performed a complementary DNA (cDNA) subtractive hybridization of fetal bovine proliferative chondrocyte cDNA from epiphyseal cartilage cDNA. The subtracted product was used to screen a fetal bovine cartilage cDNA library. Ten percent of the clones identified encoded the bovine orthologue of the human ribosomal protein "QM." Northern and western blot analysis confirmed that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In contrast, no detectable difference in the expression of mRNA for the ribosomal protein S11 was detected. Immunohistochemical analysis of fetal bovine limb sections revealed that QM was not expressed by the majority of the epiphyseal chondrocytes but only by chondrocytes in close proximity to capillaries that had invaded the epiphyseal cartilage. Strongest QM expression was seen in osteoblasts in the diaphyseal region of the bone adjoining the growth plate, within the periosteum covering the growth plate and within secondary centers of ossification. Hypertrophic chondrocytes within the growth plate adjoining the periosteum also were positive for QM as were chondrocytes in the perichondrium adjoining the periosteum. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. The in vivo and in vitro expression pattern of QM suggests that this protein may have a role in cell differentiation before mineralization.

A New Type of Matrix Vesicles is Found in Fetal Bovine Tracheal Cartilage

Using 4-month-old fetal bovine tissue, the properties of the tibia epiphyseal cartilage matrix vesicles, a type of endochondral ossification tissue, were compared with those from tracheal cartilage. The matrix vesicle fractions, obtained by collagenase digestion and differential centrifugation, were subjected to sucrose-density-gradient centrifugation. Alkaline phosphatase activity, protease activity, and lacatate dehydrogenase activity were assayed for the marker enzyme of the matrix vesicles. Matrix vesicles containing alkaline phosphatase, metalloprotease, and lacatate dehydrogenase were found in the tibia epiphyseal cartilage at a density of 1.11 g/ml. In surprising contrast, we also found matrix vesicle-like vesicles with a high density of 1.24 g/ml in the tracheal cartilage. These also contained alkaline phosphatase and lactate dehydrogenase, but not metalloprotease. The electrophoretic profiles of the lactate dehydrogenase isoenzymes from the matrix vesicle and matrix vesicle-like vesicles were identical with those of chondrocyte cytosolic lactate dehydrogenase. Aldolase, aspartate: 2-oxoglutarate aminotransferase, alanine: 2-oxoglutarate aminotransferase, glucose-6-phosphatase, glutamate dehydrogenase, catalase, and cytosolic enzymes except for lactate dehydrogenase were not detected in these vesicles. These results suggest the presence of a mechanism for specific uptake of cytosolic lactate dehydrogenase in both vesicles.In this study, a new type of matrix vesicles without protease was found in the tracheal cartilage, a kind of permanent cartilage, but not in the tibia epiphyseal cartilage, which is replaced by bone tissue.

Generation of multiple transcripts from the chicken chondromodulin-I gene and their expression during embryonic development

Chondromodulin-I (ChM-I) is an angiogenesis inhibitor isolated from fetal bovine cartilage. Here, we report the nucleotide sequence of chicken ChM-I cDNA. Chicken mature ChM-I had a significantly larger N-terminal hydrophilic domain than its mammalian counterparts. Chicken embryos expressed multiple transcripts (3.3, 2.0 and 1.7 kb in size) due to the alternative utilization of polyadenylation signals, whereas only the 1.7 kb transcripts were detected in mammals. Although confined to cartilage and eye at a later stage of development, whole-mount in situ hybridization revealed the expression of ChM-I mRNA in somites, heart, bronchial arches, roof plate, retina and limb buds. The expression pattern of the gene suggests a role for ChM-I in the morphogenesis during embryonic development.

Enhancement of Cell Adhesion and Spreading by a Cartilage-specific Noncollagenous Protein, Cartilage Matrix Protein (CMP/Matrilin-1), via Integrin α1β1

Cartilage matrix protein (CMP; also known as matrilin-1), one of the major noncollagenous proteins in most cartilages, binds to aggrecan and type II collagen. We examined the effect of CMP on the adhesion of chondrocytes and fibroblasts using CMP-coated dishes. The CMP coating at 10-20 micrograms/ml enhanced the adhesion and spreading of rabbit growth plate, resting and articular chondrocytes, and fibroblasts and human epiphyseal chondrocytes and MRC5 fibroblasts. The effect of CMP on the spreading of chondrocytes was synergistically increased by native, but not heated, type II collagen (gelatin). The monoclonal antibody to integrin alpha1 or beta1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin alpha2, alpha3, alpha5, beta2, alpha5beta1, or alphaVbeta5 had little effect on cell adhesion or spreading. The antibody to integrin alpha1, but not to other subunits, coprecipitated 125I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin alpha1 subunit. Unlabeled CMP competed for the binding to integrin alpha1 with 125I-CMP. These findings suggest that CMP is a potent adhesion factor for chondrocytes, particularly in the presence of type II collagen, and that integrin alpha1beta1 is involved in CMP-mediated cell adhesion and spreading. Since CMP is expressed almost exclusively in cartilage, this adhesion factor, unlike fibronectin or laminin, may play a special role in the development and remodeling of cartilage.

Structural Analysis of Cross-linking Domains in Cartilage Type XI Collagen: INSIGHTS ON POLYMERIC ASSEMBLY

The collagen framework of hyaline cartilage is based on copolymers of types II, IX, and XI collagens. Previous studies have established specific covalent interactions between types II and IX collagens. The present study examined cross-linking sites in type XI collagen to define better the full heteropolymeric assembly. Pepsinsolubilized type XI collagen was purified from fetal bovine cartilage. The cross-linking amino acids in the preparation were primarily divalent, borohydride-reducible structures; pyridinoline residues were essentially absent. Individual alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains were resolved by high performance liquid chromatography. Telopeptides still attached by cross-links to helical sites were released by periodate oxidation and identified by microsequencing. Analysis of cross-linked peptides isolated from trypsin digest of each alpha-chain identified the attachment helical sites for the telopeptides. A high degree of interchain specificity was evident in the cross-linking between N-telopeptides and the COOH terminus of the triple-helix, consistent with a head-to-tail interaction of molecules staggered by 4D (D = 67 nm) periods. In addition, alpha 1(II) C-telopeptide was linked to the amino-terminal site of the alpha 1(XI) triple helix. In summary, the results show that type XI collagen molecules are primarily cross-linked to each other in cartilage, implying that a homopolymer is initially formed. Links to type II collagen are also indicated, consistent with an eventual cofibrillar assembly. Analysis of cartilage extracts showed that all three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI), had at least in part retained their N-propeptides in cartilage matrix and that the alpha 3 (XI) chain was the IIB splicing variant product of the COL2A1 gene. Of particular note was the finding that the N-telopeptide cross-linking site in both alpha 1(XI) and alpha 2(XI) is located amino-terminal to the putative N-propeptidase cleavage site. This structural feature provides a potential mechanism for the proteolytic depolymerization of type XI collagen by proteases that can cleave between the cross-link and the triple helix (e.g. stromelysin).

Native cartilage matrix protein (CMP). A compact trimer of subunits assembled via a coiled-coil alpha-helix.

Cartilage matrix protein (CMP), a major component of many types of cartilage, is a noncollagenous glycoprotein with a molecular mass of 148 kDa consisting of three identical subunits. With the aim of performing a more comprehensive characterization, we purified CMP in a native conformation from fetal bovine rib cartilage avoiding the denaturing solvents previously used. CMP could be selectively extracted with EDTA-containing buffer which indicates a divalent cation-dependent anchorage in the cartilage matrix. Determination of the amino-terminal sequence of the bovine protein confirmed its identity when compared with published cDNA sequences of chicken and human CMP. Electron microscopy revealed the presence of three ellipsoid subunits which are connected at one end. Sequence analysis indicated the presence of a coiled-coil alpha-helical assembly domain formed by the COOH-terminal end of the subunits. The trimeric structure was retained after complete reduction under native conditions which shows that the coiled-coil domain is stable also in the absence of interchain disulfide bonds.

Identification of cross-linking sites in bovine cartilage type IX collagen reveals an antiparallel type II-type IX molecular relationship and type IX to type IX bonding

Type IX collagen functions in covalent cross-linkage to type II collagen in cartilage (Eyre, D. R., Apone, S., Wu, J. J., Ericsson, L. H., and Walsh, K. A. (1987) FEBS Lett. 220, 337-341). To understand this molecular relationship better, an analysis of all cross-linking sites labeled by [3H]borohydride was undertaken using the protein prepared from fetal bovine cartilage. Sequence analysis of tryptic peptides containing the 3H-labeled cross-links showed that each of the chains of type IX collagen, alpha 1(IX), alpha 2(IX), and alpha 3(IX), contained a site of cross-linking at the amino terminus of the COL2 triple-helix to which the alpha 1(II)N-telopeptide could bond. The alpha 3(IX)COL2 domain alone also had an attachment site for the alpha 1(II)C-telopeptide. The distance between the alpha 1(II)N-telopeptide and alpha 1(II)C-telopeptide interaction sites, 137 residues, is equal to the length of the hole zone (0.6D) in a type II collagen fibril. This implies an antiparallel type II to type IX cross-linking relationship. Peptide analysis also revealed an unknown amino acid sequence linked to the COL2 cross-linking domains in both the alpha 1(IX) and alpha 3(IX) chains. Using antibodies to this novel peptide, its origin in the collagen alpha 3(IX)NC1 domain was established. In summary, the results confirm extensive covalent cross-linking between type IX and type II collagen molecules and reveal the existence of type IX-type IX bonding. These data provide a molecular basis for the proposed function of type IX collagen as a critical contributor to the mechanical stability and resistance to swelling of the collagen type II fibril framework of cartilage.

Characterization of collagen types XII and XIV from fetal bovine cartilage.

The structurally related type XII-like collagen molecules TL-A and TL-B were recently identified in fetal bovine epiphyseal cartilage and subsequently shown to be collagen types XII and XIV, respectively. By indirect immunofluorescent staining of cartilage using monoclonal antibodies to the NC3 domains of each molecule, it was shown that type XII collagen was present predominantly around cartilage canals, the articular surface, subperichondrial margins, and the perichondrium, was less so in the remaining cartilage matrix, and was absent from the growth plate region. In the permanent cartilage of trachea, type XII stained somewhat more intensely in the margins beneath the loose connective tissue. Type XIV collagen localized more uniformly throughout the articular cartilage and was also absent from the growth plate region, whereas in tracheal cartilage, its distribution was similar to type XII. We have characterized the structure of these cartilage molecules and compared them with those from fetal bovine skin. Extraction of cartilage with 1 M NaCl and differential NaCl precipitation yields a fraction enriched for these two collagens. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies to the large amino-terminal non-triple-helical domain, NC3, revealed the presence in cartilage of two forms of type XII collagen: type XIIB, the molecule previously identified in chick and bovine tissues, and type XIIA, a much larger form equivalent to the molecule recently identified in WISH-transformed epithelial cell culture medium (Lunstrum, G. P., McDonough, A. M., Marinkovich, M. P., Keene, D. R., Morris, N. P., and Burgeson, R. E. (1992) J. Biol. Chem. 267, 20087-20092). Digestion with bacterial collagenase shows that the increased mass is present in the NC3A domain. Additional purification by velocity sedimentation and observation of rotary-shadowed images demonstrates molecules with extended non-triple-helical arms approximately 80 nm in length analogous to the WISH cell molecules. Electrophoretic mobilities of bands corresponding to type XIIA, but not type XIIB, are sensitive to chondroitinase ABC, indicating that type XIIA is a chondroitin sulfate proteoglycan and that modification occurs predominantly within the NC3A domain distal to NC3B. Neither type XIIB from skin nor type XIIA from WISH cells are chondroitinase-sensitive. By similar analysis, a portion of the type XIV collagen chains in cartilage was also sensitive to chondroitinase digestion. Chondroitin sulfate is apparently not located on its NC3 domain. As in skin, collagen types XII and XIV have subtly different distributions within cartilage and type XII may have a tissue-specific structure.