Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.     

Interaction of proteoglycans with the pericellular (1 alpha, 2 alpha, 3 alpha) collagens of cartilage

Interaction between cartilage proteoglycan and the collagen(s) composed of 1 alpha, 2 alpha, and 3 alpha chains was studied in vitro. Most of the collagen was insoluble under the conditions of assay (0.15 M NaCl, 0.008 M phosphate buffer, pH 7.4; 4 degrees C) and was in the form of fibrils 20 nm in diameter or thinner. The larger fibrils had 60-70 nm periodicity, characteristic of native collagens. Proteoglycan monomers which had been labeled by incubating cartilage slices in vitro with Na2 35SO4 were used to assay the interaction. The insoluble collagen fraction bound proteoglycan from solution. At proteoglycan:collagen ratios lower than 1:2, binding was rapid and linear, and the dissociation constant was 1.7 X 10(-9) M. At higher proteoglycan:collagen ratios, more proteoglycan was bound, but at a slower rate. Binding of proteoglycan to collagen did not require fibrils, since soluble 1 alpha, 2 alpha, and 3 alpha containing collagen also bound to proteoglycan and formed an insoluble complex. Denatured collagens did not bind proteoglycan or compete for binding with normal collagen. Optimum binding occurred with intact proteoglycan, but proteoglycan which had been treated with protease was also bound at low levels. Both protease-treated proteoglycan and free chondroitin sulfate competed with intact proteoglycan in the binding assays, but neither chondroitinase ABC-treated proteoglycan nor the oligosaccharides produced by digestion of chondroitin sulfate with testicular hyaluronidase altered the binding of proteoglycan to collagen. Hyaluronic acid did not compete with radioactive proteoglycan, but heparin and dextran sulfate were extremely effective inhibitors of binding. These data suggest a relatively nonspecific interaction between sulfated polyanions and 1 alpha, 2 alpha, and 3 alpha containing collagens. However, given the location of these collagens near the chondrocyte surface, the interaction of fibrillar 1 alpha, 2 alpha, 3 alpha collagen with proteoglycan is likely to occur and to be of biological importance.     

All three chains of 1 alpha 2 alpha 3 alpha collagen from hyaline cartilage resist human collagenase

A previous report that the 3 alpha collagen chain of hyaline cartilage was cleaved by human collagenase could not be confirmed when the 1 alpha 2 alpha 3 alpha collagen fraction was freed of all contaminating type II collagen. All three minor collagen chains, 1 alpha, 2 alpha and 3 alpha, were totally resistant to highly purified collagenases from both rheumatoid synovial and gastric mucosal tissues. This finding and CNBr-peptide patterns suggest that, despite the close homology with alpha 1 (II), the 3 alpha chain is a unique collagen component, possibly combined with 1 alpha and 2 alpha in heterotrimeric molecules. In contrast, a 3 alpha-like component from fibrocartilage was cleaved by collagenase and gave a CNBr-peptide pattern more typical of alpha 1 (II) than of the collagenase-resistant 3 alpha of hyaline cartilage.     

A study of the interaction in vitro between type I collagen and a small dermatan sulphate proteoglycan.

The interaction between a small dermatan sulphate proteoglycan isolated from human uterine cervix and collagen type I from human and rat skin was investigated by collagen-fibrillogenesis experiments. Collagen fibrillogenesis was initiated by elevation of temperature and pH after addition of proteoglycan, chondroitinase-digested proteoglycan or isolated side chains, and monitored by turbidimetry. Collagen-associated and unbound proteoglycan was determined by enzyme-linked immunosorbent assay after aggregation was complete. (1) The binding of proteoglycan to collagen could be explained by the presence of two mutually non-interacting binding sites, with Ka1 = 1.3 x 10(8) M-1 and Ka2 = 1.3 x 10(6) M-1. The number of binding sites per tropocollagen molecule was n1 = 0.11 and n2 = 1.1. The 0.1 high-affinity binding site per tropocollagen molecule indicates that the strong interaction between proteoglycan and collagen results from a concerted action of tropocollagen molecules in fibrils. Digestion of the proteoglycan with chondroitinase ABC did not affect these binding characteristics. (2) Proteoglycan did not affect the rate of fibrillogenesis, but increased the steady-state A400 by up to 90%. This increase was directly proportional to the saturation of the high-affinity type of binding sites. Neither isolated core protein nor isolated side chains induced a similar high increase in steady-state A400. (3) Electron micrographs showed that the fibril diameter was affected only to a minor extent, if at all, by the proteoglycan, whereas bundles of laterally aligned fibrils were common in the presence of proteoglycan. (4) Results obtained with human and rat collagen were similar.     

Type X collagen, a product of hypertrophic chondrocytes.

The synthesis of collagen types IX and X by explants of chick-embryo cartilages was investigated. When sternal cartilage labelled for 24h with [3H]proline was extracted with 4M-guanidinium chloride, up to 20% of the 3H-labelled collagen laid down in the tissue could be accounted for by the low-Mr collagenous polypeptides (H and J chains) of type IX collagen; but no type X collagen could be detected. Explants of tibiotarsal and femoral cartilages were found to synthesize type IX collagen mainly in zones 1 and 2 of chondrocyte proliferation and elongation, whereas type X collagen was shown to be a product of the hypertrophic chondrocytes in zone 3. Pulse-chase experiments with tibiotarsal (zone-3) explants demonstrated a time-dependent conversion of type X procollagen into a smaller species whose polypeptides were of Mr 49 000. The processed chains [alpha 1(X) chains] were shown by peptide mapping techniques to share a common identity with the pro alpha 1(X) chains of Mr 59 000. No evidence for processing of type IX collagen was obtained in analogous pulse-chase experiments with sternal tissue. When chondrocytes from tibiotarsal cartilage (zone 3) were cultured on plastic under standard conditions for 4-10 weeks they released large amounts of type X procollagen into the medium. However, 2M-MgCl2 extracts of the cell layer were found to contain mainly the processed collagen comprising alpha 1(X) chains. The native type X procollagen purified from culture medium was shown by rotary shadowing to occur as a short rod-like molecule 148 nm in length with a terminal globular extension, whereas the processed species comprising alpha 1(X) chains of Mr 49 000 was detected by electron microscopy as the linear 148 nm segment.     

Effects of products from macrophages, blood mononuclear cells and or retinol on collagenase secretion and collagen synthesis in chondrocyte culture

Collagenase secretion was studied on cultures of rabbit articular chondrocytes. Differentiation of the cells was assessed by characterizing the type of 3H-labelled collagen produced during treatment with (1) conditioned media from rabbit peritoneal macrophages and human blood mononuclear cells, and (2) with retinol, a potent cartilage resorbing agent in tissue culture. Conditioned media stimulated collagenase secretion. Total collagen synthesis was reduced due to a decrease of synthesis of alpha 1 chains; the amount of alpha 2 chains synthesized was unchanged. This is thought to be due to a reduction in type II synthesis. Retinol did not stimulate collagenase secretion. Total collagen synthesis was reduced by retinol. alpha 2 chain synthesis, however, was significantly increased, suggesting a switch of collagen synthesis in favor of type I collagen, and therefore, dedifferentiation. These results demonstrate that dedifferentiation of chondrocytes with respect to collagen synthesis is not necessarily associated with a stimulation of collagenase secretion.     

Modulation of fibroblast functions by interleukin 1: increased steady- state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta

Interleukin-1 (IL-1) is synthesized by and released from macrophages in response to a variety of stimuli and appears to play an essential role in virtually all inflammatory conditions. In tissues of mesenchymal origin (e.g., cartilage, muscle, bone, and soft connective tissue) IL-1 induces changes characteristic of both destructive as well as reparative phenomena. Previous studies with natural IL-1 of varying degrees of purity have suggested that it is capable of modulating a number of biological activities of fibroblasts. We have compared the effects of purified human recombinant (hr) IL-1 alpha and beta on several fibroblast functions. The parameters studied include cell proliferation, chemotaxis, and production of collagen, collagenase, tissue inhibitor of metalloproteinase (TIMP), and prostaglandin (PG) E2. We observed that hrIL-1s stimulate the synthesis and accumulation of type I procollagen chains. Intracellular degradation of collagen is not altered by the hrIL-1s. Both IL-1s were observed to increase the steady-state levels of pro alpha 1(I) and pro alpha 2(I) mRNAs, indicating that they exert control of type I procollagen gene expression at the pretranslational level. We found that both hrIL-1 alpha and beta stimulate synthesis of TIMP, collagenase, PGE2, and growth of fibroblasts in vitro but are not chemotactic for fibroblasts. Although hrIl-1 alpha and beta both are able to stimulate production of PGE2 by fibroblasts, inhibition of prostaglandin synthesis by indomethacin has no measurable effect on the ability of the IL-1s to stimulate cell growth or production of collagen and collagenase. Each of the IL-1s stimulated proliferation and collagen production by fibroblasts to a similar degree, however hrIL-1 beta was found to be less potent than hrIL-1 alpha in stimulating PGE2 production. These observations support the notion that IL-1 alpha and beta may both modulate the degradation of collagen at sites of tissue injury by virtue of their ability to stimulate collagenase and PGE2 production by fibroblasts. Furthermore, IL-1 alpha and beta might also direct reparative functions of fibroblasts by stimulating their proliferation and synthesis of collagen and TIMP.     

Macrophage-fibroblast interactions in collagenase production and cartilage degradation.

Rabbit bone-marrow macrophages and fibroblasts were cultured, independently or together, with pieces of 35S-labelled cartilage or at the surface of dried [14C]collagen gels. Each type of cell, cultivated alone, rapidly degraded the proteoglycan of cartilage, but only the fibroblasts degraded collagen. The co-culture of both types of cell had no consistent effect on the rate of proteoglycan degradation, but it stimulated the rate of collagen degradation. In parallel, the accumulation of collagenase in the culture fluid was enhanced but not that of neutral proteinase. Coinditioned media from macrophage cultures added to cultures of fibroblasts had the same effect as the living macrophages in stimulating the production of collagenase. Their action was itself enhanced when the macrophages had been activated by concanavalin A-stimulated spleen-cell factors. These data suggest that fibroblasts may act as effector cells in producing collagenase and degrading collagen in response to soluble factors released by macrophages under the control of lymphocyte factors.     

Isolation and characterization of cyanogen bromide peptides from the collagen of bovine articular cartilage

Significant amounts of native collagen can be extracted from bovine articular cartilage after removal of the acid mucopolysaccharides by controlled proteolysis. The fraction thus solubilized upon denaturation gives rise to three identical alpha chains. Cleavage of these chains with CNBr generated nine peptides, all of which contain glycine as one-third of their total amino acid residues. Two of the smaller peptides CB-1 and CB-2 contain partially hydroxylated proline. A similar CNBr digest of intact cartilage also gives a series of peptides identical with those obtained from the soluble cartilage collagen. The absence of cross-linking peptides, the fact that only few beta components are seen in articular cartilage collagen and the similarity in peptide pattern between the two collagen fractions investigated, suggests that this collagen is stabilized by a different cross-linking mechanism, possibly involving an association with the tissue proteoglycans.     

The solubilization of collagen and protein–polysaccharides from the developing cartilage of lathyrytic chicks

1. The solubilization of collagen and protein-polysaccharides from the developing cartilage of normal and lathyritic chicks was studied by using mild extraction procedures. One-third of the protein-polysaccharides could be solubilized in salt solutions at neutral pH from normal cartilage, whereas 95-100% could be extracted from the cartilage of animals that were severely lathyritic. Likewise, whereas in normal animals the collagen of cartilage was essentially insoluble in salt solutions at neutral pH, in lathyritic animals it was almost completely soluble. 2. The increased solubility of the collagen of cartilage from lathyritic animals enabled sufficient material to be collected so that the pure alpha1 chains of the collagen were isolated by repeated reconstitution, precipitation and CM-cellulose column chromatography. The purified alpha1 component was characterized by its relatively high content of hydroxylysine (14 residues/1000 amino acids). 3. About 37% of the collagen from the cartilage of normal chick embryos could be extracted as the gelatin at pH7.4 in lithium chloride solution. This was accompanied by the extraction of approx. 14% of the protein-polysaccharide content. 4. The protein-polysaccharides and the collagen from normal animals could be extracted from the cartilage relatively independently of one another under mild conditions. These same components obtained from lathyritic animals easily separated from one another after solubilization. This provided evidence that the two components are probably not covalently cross-linked. 5. The collagen of cartilage extracted as a gelatin from normal animals contained a high proportion of alpha chains compared with beta dimers, similar to the lathyritic collagen of cartilage and other tissues, and similar to the gelatin extracted from normal chick bone.     

Changes in the types of collagen synthesized during chondrogenesis of the mouse otic capsule

We have investigated the temporal relationship between the morphological differentiation of the mouse otic capsule and the pattern of collagen synthesis by mouse otocyst-mesenchyme complexes labeled in vitro. In 10.5- to 12-day embryos the mesenchyme surrounding the otocyst was loosely organized except for a few lateroventral condensations; explants from these embryos synthesized only small amounts of collagen. Collagen synthesis by whole explants increased by more than 50% between 12 and 13 days concomitant with metachromatic staining of the lateral periotic mesenchyme. Cartilage specific type II collagen was the predominant collagen synthesized by these explants as confirmed by SDS-PAGE, densitometry, CNBr cleavage, and V8 protease digestion. This biochemical expression of the cartilage phenotype preceded morphologic recognition of otic capsular cartilage by almost 2 days. Type II collagen synthesis continued to increase and predominate through Day 16 of gestation by which time the otic labyrinth was surrounded by mature cartilage. The minor cartilage collagen chains, 1 alpha, 2 alpha, and 3 alpha, first appeared on different days of gestation. The 1 alpha, and 3 alpha chains were synthesized by explants from 11-day embryos while the 2 alpha chain appeared during Day 13, just before overt differentiation of mature cartilage. These results suggested that the 1 alpha, 2 alpha, and 3 alpha chains may not form heterotrimers containing all three chains and that synthesis of the 2 alpha chain may be associated with stabilization of the cartilaginous matrix. Comparison of these data with the patterns of collagen production by mutant, diseased, or experimentally manipulated inner ear tissues may provide insights into the molecular basis of chondrogenic tissue interactions.     

Nonenzymatic glycation of type I collagen. The effects of aging on preferential glycation sites.

The present study was designed to investigate the effects of aging on preferential sites of glucose adduct formation on type I collagen chains. Two CNBr peptides, one from each type of chain in the type I tropocollagen molecule, were investigated in detail: alpha 1(I)CB3 and alpha 2CB3-5. Together these peptides comprise approximately 25% of the total tropocollagen molecule. The CNBr peptides were purified from rat tail tendon, obtained from animals aged 6, 18, and 36 months, by ion exchange chromatography, gel filtration, and high-performance liquid chromatography (HPLC). Sugar adducts were radiolabeled by reduction with NaB3H4. Glycated tryptic peptides were prepared from tryptic digests of alpha 2CB3-5 and alpha 1(I)CB3 by boronate affinity chromatography and HPLC. Peptides were identified by sequencing and by compositional analysis. Preferential sites of glycation were observed in both CB3 and alpha 2CB3-5. Of the 5 lysine residues in CB3, Lys-434 was the favored glycation site. Of the 18 lysine residues and 1 hydroxylysine residue in alpha 2CB3-5, 3 residues (Lys-453, Lys-479, and Lys-924) contained more than 80% of the glucose adducts on the peptide. Preferential glycation sites were highly conserved with aging. In collagen that had been glycated in vitro, the relative distribution of glucose adducts in old animals differed from that of young animals. In vitro experiments suggest that primary structure is the major determinant of preferential glycation sites but that higher order structure may influence the relative distribution of glucose adducts among these preferred sites.     

Isolation and partial characterization of collagen chains dimerized by sugar-derived cross-links

Incubation of tail tendon from a young rat in solutions containing D-ribose resulted in attachment of the monosaccharide to collagen and subsequent cross-link formation at a rate much faster than found for glucose. The collagen rapidly became resistant to solubilization and showed increasing fluorescence. Ribose bound to all major CNBr peptides of collagen, with some preference for the alpha 2-CB3,5 peptide and the triple-helical region of alpha 1-CB6, and was incorporated into higher molecular weight material. Extensive pepsin digestion permitted isolation of dimers of alpha chains cross-linked in triple-helical regions as a result of incubation with ribose. The dimers were identified as beta 11, beta 12, and beta 22 components, and the limited degree of heterogeneity of these components indicated that cross-linking occurred at several sites, some of which must be intermolecular. Isolated beta components were strongly fluorescent with a spectrum similar to that of collagen in aged tissues. Fluorescent dimers with similar characteristics were found in pepsin digests of tail tendons from older rats.     

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.     

Effects of glutathione depletion on the synthesis of proteoglycan and collagen in cultured chondrocytes

We studied the effect of the depletion of glutathione on the synthesis of proteoglycan and collagen in cultured chick chondrocytes. When the cultured chondrocytes were incubated with 1 mM buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase, the intracellular glutathione level markedly dropped within 12 h with no loss of cell viability. Incorporation of 35SO2-4 into proteoglycan was lowered in the presence of BSO. When the 35S-labeled proteoglycans were separated into two fractions by glycerol density gradient centrifugation, the inhibitory effect of BSO on the synthesis of proteoglycan was greater in the fast-sedimenting proteoglycan fraction, which consisted mainly of cartilage specific large proteoglycan (PG-H), than in the slowly sedimenting proteoglycan fraction. The inhibition by BSO of the synthesis of core protein-free glycosaminoglycan chains primed by p-nitrophenyl-beta-D-xyloside was smaller than the inhibition of the synthesis of proteoglycan. Analysis of glycosaminoglycans labeled with [3H]glucosamine indicated that the treatment of chondrocytes with BSO resulted in a small increase in the proportion of synthesis of hyaluronic acid to the synthesis of total glycosaminoglycan. The incorporation of [3H]proline into collagen was also inhibited by BSO. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the 3H-labeled collagen showed that, in the presence of BSO, processing of Type II collagen appeared to slow down and the proportion of Type X collagen synthesis was reduced.     

Proteochondroitin sulfate synthesized in cartilages induced in vivo and in vitro by bone matrix gelatin.

Implanted allogeneic demineralized bone matrix gelatin induced sequential development of cartilage and bone in the recipient rat muscle tissue. Proteoglycans of the implants labeled in vivo with [35S]sulfate at different stages of development were analyzed by sucrose density gradient centrifugation. The major proteoglycan synthesized in day-5 implant, just prior to onset of chondrogenesis, was a dermatan sulfate-containing proteoglycan with relatively slow sedimentation rate. Additionally, a small amount of a faster sedimenting component could be detected. The faster sedimenting proteoglycan, in which chondroitin 4-sulfate accounted for 85% of total radioactivity, became predominant in day-10 sample when cartilage formation was maximal. By day 30, when cartilage had been replaced by newly formed bone, the synthesis of this faster sedimenting component had ceased. A similar, if not identical, proteoglycan was found to be a major one synthesized by the in vitro-induced cartilage. This proteoglycan was smaller in overall size and shorter in length of its chondroitin sulfate chains than a major proteoglycan component obtained from neonatal rat epiphyseal cartilage. Concurrent with these changes in proteoglycan type, there appeared to be a change in collagen type, since type II collagen, in addition to type I collagen, was synthesized in day-10 implant. These results indicate that the proteoglycan can be used as a molecular marker for chondrogenesis by bone matrix gelatin.     

The purification of bovine cathepsin B1 and its mode of action on bovine collagens

1. Cathepsin B1 was isolated from bovine spleen by autolysis, (NH(4))(2)SO(4) fractionation and chromatography on Amberlite IRC-50. Two isoenzyme forms were purified to homogeneity by chromatography on CM-cellulose and DEAE-Sephadex. 2. A collagenolytic cathepsin was separated from cathepsin B1 during purification. The remaining collagenolytic activity of the purified cathepsin-B1 isoenzymes was no greater than 0.3 unit/unit of cathepsin B1 compared with about 5.0 unit/unit of cathepsin B1 in the autolysed spleen extracts. 3. The cathepsin B1 isoenzymes lowered the viscosity of gelatin at 37 degrees C. Optimum activity was at pH4-5. 4. At 28 degrees C the interchain cross-links in native tropocollagen were cleaved most effectively at pH4-5. Insoluble tendon collagen was digested at pH3.5 and 28 degrees C to yield mainly alpha-chain components, with the loss of a short N-terminal sequence. 5. Electron-microscope studies of collagen fibrils showed that cathepsin B1 caused longitudinal splitting and dissociation of the protofilaments. The effect was not general but occurred at selected sites. 6. The isoenzymes of cathepsin B1 cleaved the telopeptide region of calf skin tropocollagen between the lysine-derived cross-link and the triple helix. The CB1 peptide fragments obtained from enzyme-degraded alpha1 chains were hydrolysed at Gly(12)-Ile(13) and Ser(14)-Val(15). The residual alpha2 CB1 peptides were hydrolysed at Ala(8)-Asp(9) and Asp(9)-Phe(10).     

Isolation and properties of a preparation of arterial collagen solubilized by pretreatment with alkali (author's transl)]

The acid-soluble, highly cross-linked aorta collagen, of which about 30% can be converted into a soluble form by alkali treatment, followed by extraction with aetic acid, was obtained predominantly in the form of monomeric, helical molecules, as indicated by the value for the intrinsic viscosity and its behaviour in sodium dodecylsulphate disc electrophoresis. Apart from decreased values for tyrosine (0.26%), arginine (4.4%) and aspartic acid (3.9%), the amino acid composition of the aorta collagen fraction was similar to that of the acid-soluble calf skin collagen. This finding, together with the cyanogen bromide peptide pattern, shows that the collagen extracted from the artery is predominantly type I. Treatment with alkali probably shortens the alpha1-CB6-peptide by about 45 amino acids. The collagen extracted from artery was compared with acid soluble skin collagen by sodium dodecylsulphate polyacrylamide electrophoresis. The arterial collagen showed a marked increase in the rations alpha1 to alpha2 (4:1), alpha to beta (3:1) and beta11 to beta12 (2.5:1). Compared with acid soluble skin collagen, the aorta collagen contained twice as much galactose and glucose (13.5 and 9.6 nmol/mg protein respectively), which are bound to hydroxylysine. 50% of the hydroxylysine residues are unsubstituted, 15% are present as galactosyl hydroxylysine, and 35% as glucosyl-galactosyl hydroxylysine. On the basis of its reported properties, arterial collagen obtained by the method of Fujii appears to be a suitable substrate for the study of the enzymic synthesis and enzymic degradation of hydroxylysine glycosides of native arterial collagen.     

Proteochondroitin sulfate synthesized in cartilages induced in vivo and in vitro by bone matrix gelatin

Implanted allogeneic demineralized bone matrix gelatin induced sequential development of cartilage and bone in the recipient rat muscle tissue. Proteoglycans of the implants labeled in vivo with [³⁵S]sulfate at different stages of development were analyzed by sucrose density gradient centrifugation. The major proteoglycan synthesized in day-5 implant, just prior to onset of chondrogenesis, was a dermatan sulfate-containing proteoglycan with relatively slow sedimentation rate. Additionally, a small amount of a faster sedimenting component could be detected. The faster sedimenting proteoglycan, in which chondroitin 4-sulfate accounted for 85% of total radioactivity, became predominant in day-10 sample when cartilage formation was maximal. By day 30, when cartilage had been replaced by newly formed bone, the synthesis of this faster sedimenting component had ceased. A similar, if not identical, proteoglycan was found to be a major one synthesized by the in vitro-induced cartilage. This proteoglycan was smaller in overall size and shorter in length of its chondroitin sulfate chains than a major proteoglycan component obtained from neonatal rat epiphyseal cartilage. Concurrent with these changes in proteoglycan type, there appeared to be a change in collagen type, since type II collagen, in addition to type I collagen, was synthesized in day-10 implant. These results indicate that the proteoglycan can be used as a molecular marker for chondrogenesis by bone matrix gelatin.     

Appearance and persistence of fibronectin in cartilage. Specific interaction of fibronectin with collagen type II

Binding of fibronectins (FN) to collagen types I-IV were studied using polyclonal antibodies against human and chicken FNs, proteoglycan monomers, collagen type II and monoclonal antibodies reacting with both soluble and insoluble forms of human FN. Plasma fibronectin and type II collagen were shown to interact specifically in a homologous system. Type II collagen, however, proved to be less effective in inhibition assays compared to other types of collagen. In high density cultures of chicken limb bud cells, fibronectin was first localized within the fibroblast-like cells of 4 hr cultures and an extensive extracellular filamentous network developed by the end of day 1. Fibronectin was present in the newly formed cartilage nodules although it seemed to disappear by day 6, when the proteoglycan accumulation became more intensive. Enzyme treatments (testicular hyaluronidase, chondroitinase ABC) helped to localize FN at this stage of development of chicken cartilage, in microdroplet high density cultures of human fetal chondrocytes and in articular cartilage. Fibronectin was localized only in the pericellular ring of intact human articular cartilage using monoclonal antibodies with the biotin-avidin system.