Production and crystallization of the C-propeptide trimer from human procollagen III.

The C-propeptide domains of the fibrillar procollagens, which are present throughout the Metazoa in the form of ∼90 kDa trimers, play crucial roles in both intracellular molecular assembly and extracellular formation of collagen fibrils. The first crystallization of a C-propeptide domain, that from human procollagen III, is described. Following transient expression in mammalian 293T cells of both the native protein and a selenomethionine derivative, two crystal forms of the homotrimer were obtained: an orthorhombic form (P2(1)2(1)2(1)) that diffracted to 1.7 Šresolution and a trigonal form (P321) that diffracted to 3.5 Šresolution. Characterization by MALDI-TOF mass spectrometry allowed the efficiency of selenomethionine incorporation to be determined.

Development of a hemicornea from human primary cell cultures for pharmacotoxicology testing.

We report the reconstruction and characterization of a hemicornea (epithelialized stroma), using primary human cells, for use in research and as an alternative to the use of animals in pharmacotoxicology testing. To create a stromal equivalent, keratocytes from human corneas were cultured in collagen-glycosaminoglycan-chitosan foams. Limbal stem cell-derived epithelial cells were seeded on top of these, giving rise to hemi-corneas. The epithelium appeared morphologically similar to its physiological counterpart, as shown by the basal cell expression of p63 isoforms including, in some cases, the stem cell marker p63DeltaNalpha, and the expression of keratin 3 and 14-3-3sigma in the upper cell layers. In addition, the cuboidal basal epithelial cells were anchored to a basement membrane containing collagen IV, laminin 5, and hemidesmosomes. In the stromal part, the keratocytes colonized the porous scaffold, formed a network of interconnecting cells, and synthesized an ultrastructurally organized extracellular matrix (ECM) containing collagen types I, V, and VI. Electron microscopy showed the newly synthesized collagen fibrils to have characteristic periodic striations, with diameters and interfibril spacings similar to those found in natural corneas. Compared to existing models for corneal pharmacotoxicology testing, this new model more closely approaches physiological conditions by including the inducing effects of mesenchyme and cell-matrix interactions on epithelial cell morphogenesis.

Inhibition of lysyl oxidase activity can delay phenotypic modulation of chondrocytes in two-dimensional culture.

OBJECTIVE: Chondrocytes frequently de-differentiate in two-dimensional (2D) culture, especially in the presence of serum. To examine the role of lysyl oxidase (LOX) induced cross-linking in this phenomenon, the effect of the specific LOX inhibitor beta-aminopropionitrile (BAPN) was studied in 2D chondrocyte culture. DESIGN: Chick embryo sternal chondrocytes (both proliferative and hypertrophic, from caudal and cranial zones, respectively) were cultured in the presence and absence of BAPN. The production and activities of LOX and LOX-like (LOXL) were assessed by enzyme assay and the use of specific antibodies. Seventeen batches of serum of different origin were compared. Chondrocyte phenotype was assessed both morphologically and biochemically, the latter by quantitative analysis of production of radiolabeled cartilage collagens II, IX, X and XI, and the de-differentiation marker collagen I, for up to 4 weeks in culture. RESULTS: LOX and LOXL were identified, by Western blotting and immunofluorescence, and LO activity was measured in the medium, with both proliferative and hypertrophic chondrocytes. Inhibition of LO activity prevented or delayed chondrocyte de-differentiation, as characterized by changes in cell shape and synthesis of the five different collagen types, from the first days of culture for up to 4 weeks, depending on the origin of the serum added to the culture medium. CONCLUSION: LO activity may be involved in the control of chondrocyte phenotype, in addition to serum factors. Inhibition of LO activity by BAPN may be useful for the maintenance of the chondrocyte phenotype in 2D culture. Specific variations in the relative proportions of collagens II, IX and XI could be involved in the mechanism underlying these observations.

Intra-articular hyaluronate in experimental rabbit osteoarthritis can prevent changes in cartilage proteoglycan content.

OBJECTIVE: The purpose of this study was to determine the effects of intra-articular injections of high molecular weight (2000 kDa) sodium hyaluronate (HA) on the progression of articular cartilage degeneration in a rabbit partial medial meniscectomy model of osteoarthritis. DESIGN: Six experimental groups included normal, sham operated, and operated and injected animals, the latter injected once-weekly (for two weeks or twelve weeks, beginning four weeks after surgery) with either 1% (w/v) HA or phosphate buffered saline (PBS). Following assessment of gross morphology, serial adjacent blocks of full-depth articular cartilage were prepared from the tibial condyle for analysis of total water, hydroxyproline, DNA and proteoglycan (uronic acid) content, as well as the ratio of galactosamine to glucosamine. Samples were sub-divided into inner (medial) and outer (lateral) regions. RESULTS: No morphological differences were recognized between joints injected with PBS and those receiving HA. When analysed biochemically, there were no significant differences in hydration, hydroxyproline or DNA content between the experimental groups. In contrast, HA injection did affect changes in proteoglycan content. Expressed per tissue dry weight, uronic acid content in the operated group injected with PBS for two weeks was lower than normal (P<0.02), a result not seen in the corresponding HA injected group. After 12 weeks of PBS injections, uronic acid content (per dry weight) was higher than normal (P<0.01), an effect again not observed in the corresponding HA injected group. Results for the galactosamine: glucosamine ratio showed a reduction after 12 weeks of injections, but no differences between PBS and HA injected groups. CONCLUSIONS: Once-weekly, intra-articular injection of high molecular weight HA can prevent changes in proteoglycan content in tibial condylar articular cartilage, compared to PBS injected controls, in the rabbit partial meniscectomy model of osteoarthritis.

Biophysical characterization of the C-propeptide trimer from human procollagen III reveals a tri-lobed structure.

Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule.

Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1.

Recently several cDNAs have been described encoding lysyl oxidase-like proteins. Their deduced amino acid sequences are characterized by a strong similarity in the C-terminal region, corresponding to the lysyl oxidase family catalytic domain, and by marked differences in the N-terminal regions. Different biological functions have been described for lysyl oxidases in addition to their traditionally assumed cross-linking role. To answer the question of whether these different functions are carried out by different lysyl oxidases, purified and active forms of these enzymes are required. At present only the classical form of lysyl oxidase has been purified and characterized. The purpose of this study was to isolate and characterize the lysyl oxidase-like protein. In view of the strong sequence homology with the C-terminal domain of other lysyl oxidases, we chose to purify the protein from bovine aorta using antibodies specific to the N-terminal domain of the proenzyme. We have isolated a 56-kDa protein identified by amino acid sequencing as the bovine lysyl oxidase-like precursor, which is cleaved at the Arg-Arg-Arg sequence at positions 89-91 by a furin-like activity, as revealed after deblocking of the N-terminal residue. The immunopurified protein was largely inactive, but further processing in vitro by bone morphogenetic protein-1 led to an enzyme that was active on elastin and collagen substrates.

Unhydroxylated triple helical collagen I produced in transgenic plants provides new clues on the role of hydroxyproline in collagen folding and fibril formation.

Human unhydroxylated homotrimeric triple-helical collagen I produced in transgenic plants was used as an experimental model to provide insights into the role of hydroxyproline in molecular folding and fibril formation. By using chemically cross-linked molecules, we show here that the absence of hydroxyproline residues does not prevent correct folding of the recombinant collagen although it markedly slows down the propagation rate compared with bovine fully hydroxylated homotrimeric collagen I. Relatively slow cis-trans-isomerization in the absence of hydroxyproline likely represents the rate-limiting factor in the propagation of the unhydroxylated collagen helix. Because of the lack of hydroxylation, recombinant collagen molecules showed increased flexibility as well as a reduced melting temperature compared with native homotrimers and heterotrimers, whereas the distribution of charged amino acids was unchanged. However, unlike with bovine collagen I, the recombinant collagen did not self-assemble into banded fibrils in physiological ionic strength buffer at 20 degrees C. Striated fibrils were only obtained with low ionic strength buffer. We propose that, under physiological ionic strength conditions, the hydroxyl groups in the native molecule retain water more efficiently thus favoring correct fibril formation. The importance of hydroxyproline in collagen self-assembly suggested by others from the crystal structures of collagen model peptides is thus confirmed experimentally on the entire collagen molecule.

Control of heterotypic fibril formation by collagen V is determined by chain stoichiometry.

Although the collagen V heterotrimer is known to be involved in the control of fibril assembly, the role of the homotrimer in fibrillar organization has not yet been examined. Here, the production of substantial amounts of recombinant collagen V homotrimer has allowed a detailed study of its role in homotypic and heterotypic fibril formation. After removal of terminal regions by pepsin digestion, both the collagen V heterotrimer and homotrimer formed thin homotypic fibrils, thus showing that diameter limitation is at least in part an intrinsic property of the collagen V triple helix. When mixed with collagen I, however, various complementary approaches indicated that the collagen V heterotrimer and homotrimer exerted different effects in heterotypic fibril formation. Unlike the heterotrimer, which was buried in the fibril interior, the homotrimer was localized as thin filamentous structures at the surface of wide collagen I fibrils and did not regulate fibril assembly. Its localization at the fibril surface suggests that the homotrimer can act as a molecular linker between collagen fibrils or macromolecules in the extracellular matrix or both. Thus, depending on their respective distribution in tissues, the different collagen V isoforms might fulfill specific biological functions.

Folding and activity of recombinant human procollagen C-proteinase enhancer.

Recombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus system and purified to homogeneity using a three-step procedure including heparin affinity chromatography. Heparin binding was dependent on the C-terminal netrin-like domain. The recombinant protein was found to be active, increasing the activity of procollagen C-proteinase/bone morphogenetic protein-1 on type I procollagen in a manner comparable to the native protein. Enhancing activity was dependent on intact disulfide bonding within the protein. By circular dichroism, the observed secondary structure of rPCPE was consistent with the known three-dimensional structures of proteins containing homologous domains.

Changes in proteoglycan content of articular cartilage during avian degenerative joint disease.

OBJECTIVE: To determine the biochemical changes in articular cartilage composition associated with the development of avian degenerative joint disease (DJD) in ad libitum fed broiler fowl, in comparison to feed-restricted broilers and J-lin fowl (non-susceptible to DJD). METHODS: Articular cartilage from the distal tibiotarsus (DTT) was characterised up to age 180 days. Proteoglycan content was determined by uronic acid and sulphated glycosaminoglycan analysis, cellularity by assay for DNA content, and collagen content and crosslinking by hydroxyproline and pyridinoline analysis, respectively. RESULTS: Disease development was accompanied by increased hydration and proteoglycan content (particularly sulphated proteoglycans) and decreased cellularity, with no significant differences in either total collagen content or in mature collagen cross-linking. CONCLUSION: The biochemical features of avian DJD are similar to those observed in other animal models. This bipedal model is exceptional however since cartilage alterations occur spontaneously and in a load-dependent manner.

Liquid crystalline ordering of procollagen as a determinant of three-dimensional extracellular matrix architecture.

The precise molecular mechanisms that determine the three-dimensional architectures of tissues remain largely unknown. Within tissues rich in extracellular matrix, collagen fibrils are frequently arranged in a tissue-specific manner, as in certain liquid crystals. For example, the continuous twist between fibrils in compact bone osteons resembles a cholesteric mesophase, while in tendon, the regular, planar undulation, or "crimp", is akin to a precholesteric mesophase. Such analogies suggest that liquid crystalline organisation plays a role in the determination of tissue form, but it is hard to see how insoluble fibrils could spontaneously and specifically rearrange in this way. Collagen molecules, in dilute acid solution, are known to form nematic, precholesteric and cholesteric phases, but the relevance to physiological assembly mechanisms is unclear. In vivo, fibrillar collagens are synthesised in soluble precursor form, procollagens, with terminal propeptide extensions. Here, we show, by polarized light microscopy of highly concentrated (5-30 mg/ml) viscous drops, that procollagen molecules in physiological buffer conditions can also develop long-range nematic and precholesteric liquid crystalline ordering extending over 100 microm(2) domains, while remaining in true solution. These observations suggest the novel concept that supra-fibrillar tissue architecture is determined by the ability of soluble precursor molecules to form liquid crystalline arrays, prior to fibril assembly.

Collagen XI nucleates self-assembly and limits lateral growth of cartilage fibrils.

Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of approximately 20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitro produced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils.

Abnormal collagen assembly, though normal phenotype, in alginate bead cultures of chick embryo chondrocytes.

The collagens produced by chick embryo chondrocytes cultured in alginate beads were investigated both biochemically and ultrastructurally. The cartilage phenotype is maintained for at least 14 days, as indicated by the production of the cartilage-specific collagens II, IX, and XI and the absence of collagen I. There were differences in the distributions of collagens among the three different compartments analyzed (cells and their associated matrix, further-removed matrix (released by alginate solubilization), and culture medium), with large amounts of collagen IX (mainly in proteoglycan form) in the culture medium. Inhibition of lysyl oxidase activity by beta-aminopropionitrile led to an overall decrease in collagen production. In contrast to the biochemical observations, collagen ultrastructure in the extracellular matrix of alginate cultures was not in the form of the expected 64-nm banded fibrils, but rather in the form of segment-long-spacing-like crystallites. This abnormal structure is likely to be a result of alginate disrupting normal assembly. We conclude that, in this system, the native fibrillar structure of the collagenous matrix is not essential for the maintenance of the differentiated phenotype of chondrocytes.

Articular cartilage proteoglycan metabolism in avian degenerative joint disease: effects of strain selection and body weight.

The effects of strain selection and body weight on proteoglycan metabolism and the onset of degenerative joint disease (DJD) were investigated in avian articular cartilage. Samples from the hock joint (proximal tarsometatarsus, PTM; distal tibiotarsus, DTT) of rapidly growing broiler fowl, fed either ad libitum or on a restricted-diet, were compared with those from a slow growing, light and non-selected strain (J-line). Synthesis and degradation of proteoglycans were investigated by radioactive pulse-chase studies, determination of total sulphated glycosaminoglycans and electrophoretic analysis. By gross morphology, degenerative changes in articular cartilage occurred solely in the DTT from ad libitum-fed broiler fowl, after 13 weeks. Differences in proteoglycan metabolism were also observed, most markedly in the DTT, where the rate of proteoglycan synthesis in the ad libitum-fed group was less than in age-matched J-line cartilage, and the proportions of both newly synthesised and resident proteoglycans released into the culture medium were greater. Results with the feed-restricted group were intermediate between ad libitum-fed and J-line. Electrophoretic analysis of proteoglycans in the culture media showed evidence of degradation solely in the ad libitum-fed group, with earliest onset in the DTT. The results indicate that proteoglycan metabolism in avian articular cartilage is similar to that in mammalian cartilage during the development of DJD, and that the onset of cartilage degeneration is linked with excessive load bearing.

Effects of body mass and genotype on avian degenerative joint disease pathology and articular cartilage proteoglycan distribution.

OBJECTIVE: The objective of this study was to investigate the role of body mass and genotype in the development of avian degenerative joint disease (DJD). METHODS: Layer strain and broiler strain fowl, fed either ad libitum or on a restricted diet, were kept under identical conditions for up to a year. At various time points cartilage samples were taken from the distal tibiotarsus (DTT), proximal tarsometatarsus, antitrochanter and proximal humerus. All samples were assessed for gross morphology and histopathology, and in some samples the cartilage proteoglycan distribution was investigated by Safranin O staining. RESULTS: Layer strain fowl did not develop DJD. Heavy ad libitum fed broiler strain fowl developed DJD earlier and more severely than lighter, feed restricted, broiler strain fowl. The articular surface of the DTT was worst affected by DJD. Safranin O staining of DTT samples (age 180 days) from the ad libitum fed broilers revealed variable proteoglycan distribution in the articular cartilage. Some areas were intensely stained throughout all zones, whereas other areas showed no staining in any zone. Age matched, non-diseased DTT samples from feed restricted broilers showed a more consistent staining pattern with little staining in the surface zone and more in the middle and deep zones. CONCLUSIONS: These data indicate that avian DJD is body mass mediated in broiler strain fowl, and that proteoglycan distribution is altered in diseased cartilage.

Structural requirements for fibromodulin binding to collagen and the control of type I collagen fibrillogenesis--critical roles for disulphide bonding and the C-terminal region.

Fibromodulin belongs to the family of small, leucine-rich proteoglycans which have been reported to interact with collagens and to inhibit type I collagen fibrillogenesis. Decorin and fibromodulin exhibit a noticeable degree of sequence similarity. However, as previously reported [Font, B., Eichenberger, D., Rosenberg, L. M. & van der Rest, M. (1996) Matrix Biol. 15, 341-348] the domains of these molecules implicated in the interactions with type XII and type XIV collagens are different, these being the dermatan sulphate/chondroitin sulphate chain for decorin and the core protein for fibromodulin. At the present time the fibromodulin domains implicated in the interactions with fibrillar collagens remain unknown. In experiments reported here, we have sought to identify the structural requirements for fibromodulin interaction with collagen and for the control of type I collagen fibrillogenesis. Circular dichroism spectra and fibrillogenesis inhibition studies show that fibromodulin structure and its collagen fibrillogenesis control function are strictly dependent on the presence of intact disulphide bridge(s). In addition, we show that the binding of fibromodulin (or fibromodulin-derived fragments) to type I collagen is not necessarily correlated with fibrillogenesis inhibition. To isolate fibromodulin domains, the native proteoglycan was submitted to mild proteolysis. We have isolated an alpha-chymotrypsin-resistant fragment which contains the bulk of the N-terminal and central region of the molecule including the leucine-rich repeats 4 and 6 reported for decorin to be involved in type I collagen binding. This fragment does not bind to type I collagen. Using enzymes with different specificities, a number of large fragments of fibromodulin were obtained, suggesting a compact structure for this molecule which is relatively resistant to proteolysis. None of these N-glycosylated fragments were able to bind to type I collagen in co-sedimentation experiments. Taken together these results suggest that fibromodulin-type I collagen interactions leading to fibrillogenesis inhibition require more than one binding domain. One of these domains could be the C-terminal end of the molecule containing the disulphide loop which is absent in the chymotrypsin-resistant fragment.

Degenerative joint disease in poultry--differences in composition and morphology of articular cartilage are associated with strain susceptibility.

The morphology and basic biochemical composition of articular cartilage from two strains of fowl were examined. Broiler breeder fowl are considered susceptible to degenerative joint disease (DJD); histological examination of one-year-old broiler breeders showed in some samples, articular cartilage thinning, fibrillation and chondrocyte cluster formation, features considered typical of DJD. Examination of similar samples from laying strain fowl showed only minor age-related changes such as some slight cartilage thinning and very mild fibrillation. The articular cartilage from the broiler breeder birds was significantly more hydrated with a higher uronic acid content than that of the laying strain birds. In addition, unloaded articular surfaces such as the proximal humerus had significantly higher amounts of uronic acid than the loaded cartilage surfaces of the proximal tarsometatarsus and the distal tibiotarsus; this suggested that the joint loading may have a role in any biochemical differences found between joints and between strains of fowl. These findings concur with other reports in mammals that showed increased hydration and uronic acid in association with early DJD and in models of osteoarthritis (OA). Thus, despite some differences between avian and mammalian articular cartilage, studies on avian DJD may give insights into mammalian disease.

The CUB domains of procollagen C-proteinase enhancer control collagen assembly solely by their effect on procollagen C-proteinase/bone morphogenetic protein-1.

Procollagen C-proteinase enhancer (PCPE) is a 55 kDa glycoprotein that increases the activity of procollagen C-proteinase (PCP)/bone morphogenetic protein-1 (BMP-1) during C-terminal processing of fibrillar collagen precursors. Here we show that the 36 kDa, active fragment of PCPE enhances the activity of both the short (mouse) and long (chick) forms of PCP/BMP-1. The activity of PCPE is not associated with the formation of sedimentable procollagen aggregates. In addition, PCPE (36 kDa) has no effect in vitro on N-terminal procollagen processing by highly purified procollagen N-proteinase. Finally, when the amount of PCP is adjusted so that the rate of C-terminal processing remains constant, PCPE (36 kDa) has no effect on the assembly of collagen or pN-collagen in vitro following C-terminal processing of the corresponding precursors.

Expression of active, human lysyl oxidase in Escherichia coli.

Lysyl oxidase (LO) is a copper amine oxidase of the extracellular matrix which initiates covalent cross-linking in collagens and elastin. Human LO was expressed in Escherichia coli. At 37 degrees C, large amounts of protein were obtained, but in the form of insoluble aggregates. Lowering the growth temperature, and reducing the amount of inducer, resulted in the production of soluble LO, which was active on a degrees [3H]lysine-labeled elastin substrate. LO was also targeted to the periplasm as a fusion protein with the pelb signal peptide. The periplasmic enzyme was soluble, active and inhibited by beta-aminopropionitrile. Production of the carbonyl co-factor is therefore not a limitation in the expression of active LO in bacteria.