Effects of procollagen peptides on the translation of type II collagen messenger ribonucleic acid and on collagen biosynthesis in chondrocytes

Type II procollagen messenger ribonucleic acid (mRNA) was isolated from chick sternum and rat chondrosarcoma cells and translated in a reticulocyte lysate cell-free system. A high molecular weight band was identified as type II procollagen by gel electrophoresis, collagenase digestion, and specific immunoprecipitation. The translation of type II mRNA was specifically inhibited by addition of type I procollagen amino-terminal extension peptide. When this peptide was added to the media of cultured fetal calf chondrocytes, chick sternal chondrocytes, or chick tendon fibroblasts, no inhibition of collagen synthesis was evident. These data suggest a general regulation of collagen biosynthesis by these peptides in the cell-free translation system. However, as indicated by the cell culture experiments, cellular characteristics and evolutionary divergence of animal species seem to restrict the effect of the peptides.     

Intermediates in the limited proteolytic conversion of procollagen to collagen.

The conversion of chick bone procollagen to collagen proceeds in a stepwise fashion to produce a limited number of intermediates. Initial proteolytic cleavages remove NH2-terminal nonhelical extensions and yield an intermediate which remains disulfide-bonded via COOH-terminal extensions. Subsequent stepwise scission of one or two chains of the triple-stranded molecule in its COOH-terminal domain produces intermediates which can only be distinguished after dissociation of the noncovalently bonded alpha chains. A final cleavage in this region produces the collagen molecule and a disulfide-bonded triple-stranded fragment which represents the COOH-terminal domain. In all likelihood the endopeptidases which effect cleavage in the NH2- and COOH-terminal regions differ. More than two enzymes may be required for conversion of procollagen to collagen if the nonhelical domains are not released in an en bloc fashion.     

Characterization of the amino-terminal segment in type III procollagen.

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.     

The role of glycosylation in the enzymatic conversion of procollagen to collagen: studies using tunicamycin and concanavalin A

The enzymatic conversion of chick embryo cranial bone procollagen was studied in vitro using procollagen proteases isolated from the culture medium of chick tendon fibroblasts. During the normal conversion process, chains intermediate in length between proα and α chains, as well as the COOH-terminal extension peptides, can be identified. Underglycosylated procollagen, synthesized by bones treated with an inhibitor of protein glycosylation (tunicamycin), was processed by these proteases in a manner similar to that of intact procollagen. However, medium from cells cultured with tunicamycin lacked the COOH-terminal procollagen protease activity; this did not result from a direct inhibition of the protease by the drug. Concanavalin A also inhibited the conversion of procollagen to collagen by fibroblasts in culture. In an in vitro system, Concanavalin A inhibited the COOH-terminal procollagen protease, and this inhibition was reversed by methyl-α-d-glucopyranoside. These data suggest that the COOH-terminal procollagen protease contains oligosaccharide side chains that are recognized by concanavalin A and that tunicamycin affects the secretion, activity, or activation of the enzyme.     

Biosynthetic and structural properties of endothelial cell type VIII collagen

A highly unusual endothelial cell collagen (Sage, H., Pritzl, P., and Bornstein, P., (1980) Biochemistry 19, 5747-5755) has been characterized in greater detail. Pulse-chase experiments with bovine aortic endothelial cells revealed two nondisulfide-bonded collagens, of apparent chain Mr = 177,000 and 125,000, with an estimated synthesis and secretion time of 75 min. Stepwise, quantitative processing to stable lower molecular weight forms as described for type I procollagen was not observed. Endothelial collagen was secreted over a temperature range of 24-37 degrees C and, prior to heat denaturation, did not display affinity for a gelatin-binding fragment of fibronectin coupled to Sepharose. The presence of a pepsin-resistant domain (Mr = 50,000) in both the soluble and cell layer-associated forms of this protein was shown by ion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Endothelial collagen was cleaved by vertebrate collagenase into several discrete fragments that differed in molecular weight from the characteristic alpha A and alpha B fragments generated from the interstitial collagens. Nontriple helical domains corresponding to the NH2- and COOH-terminal propeptides of other procollagen types were not found after incubation of endothelial collagen with bacterial collagenase. Additional evidence for the lack of extended noncollagenous sequences was provided by studies with mast cell proteases, which convert native procollagen to collagen but are unreactive toward native interstitial collagens. Endothelial collagen was not cleaved by these enzymes at 37 degrees C, but, as observed for interstitial collagen alpha chains, required prior heating at elevated temperatures for cleavage to occur. In view of this unique set of structural characteristics, and a distribution that is not restricted to the endothelium, we have designated this protein as type VIII collagen.     

Collagen synthesis by bovine aortic endothelial cells in culture.

Endothelial cells isolated from bovine aorta synthesize and secrete type III procollagen in culture. The procollagen, which represents the major collagenous protein in culture medium, was specifically precipitated by antibodies to bovine type III procollagen and was purified by diethyl-aminoethylcellulose chromatography. Unequivocal identification of the pepsin-treated collagen was made by direct comparison with type III collagen isolated by pepsin digestion of bovine skin, utilizing peptide cleavage patterns generated by vertebrate collagenase, CNBr, and mast cell protease. The type III collagen was hydroxylated to a high degree, having a hydroxyproline/proline ratio of 1.5:1.0. Pulse-chase studies indicated that the procollagen was not processed to procollagen intermediates or to collagen. Pepsin treatment of cell layers, followed by salt fractionation at acidic and neutral pH, produced several components which were sensitive to bacterial collagenase and which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with alpha A, alpha B, and type IV collagen chains purified from human placenta by similar techniques. Bovine aortic endothelial cells also secreted fibronectin and a bacterial collagenase-insensitive glycoprotein which, after reduction, had a molecular weight of 135,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (using procollagen molecular weight standards) and which was not precipitable by antibodies to cold-insoluble globulin or to alpha 2-macroglobulin. Collagen biosynthesis by these cells provides an interesting model system for studying the polarity of protein secretion and the attachment of cells to an extracellular matrix. The presence of type III collagen in the subendothelium and the specific interaction of this protein with fibronectin and platelets suggest the involvement of this collagen in thrombus formation following endothelial cell injury.     

EFFECTS OF ANTIMICROTUBULAR AGENTS ON THE SECRETION OF COLLAGEN : A Biochemical and Morphological Study

Embryonic chick cranial bone was cultured in the presence of the antimicrotubular agents, colchicine and vinblastine, and with a number of other compounds known from previous studies to affect the cellular handling of collagen. Secretion of procollagen, quantitated by light microscope autoradiography, was correlated with the extent of conversion of procollagen to collagen and with rates of collagen and noncollagen-protein synthesis. Colchicine inhibited procollagen secretion and conversion to collagen and specifically inhibited collagen synthesis. Cells exposed to colchicine revealed an increased number of dilated Golgi-associated vacuoles and vesicles, some of which contained parallel aggregates of filamentous structures. These observations suggest that the pathway of at least a fraction of procollagen secretion by osteoblasts includes the Golgi complex. Disruption of microtubules may interfere with the movement of Golgi-derived vesicles, and the resulting accumulation of collagen precursors in the Golgi complex may lead secondarily to an inhibition of synthesis. Although vinblastine also inhibited both procollagen secretion and conversion to collagen, the observed reduction in general protein synthesis and striking changes in the ultrastructure of the rough endoplasmic reticulum complicated interpretation of the effects. Interpretation of the effects of cytochalasin B was limited by the finding that the cellular response in cranial bone was markedly heterogeneous and that, contrary to some previous reports, the drug caused an inhibition in the incorporation of radiolabeled amino acids into both collagen and noncollagen protein.     

Assembly and processing of procollagen type III in chick embryo blood vessels

The processing of [3H]proline-labeled procollagen III in excised chick embryo blood vessels was found to differ significantly from that of procollagen I in the same tissue. While first the amino propeptides and then the carboxyl propeptides were fairly rapidly cleaved from procollagen I, only the carboxyl propeptides were split off procollagen III, leaving pN-collagen III. This intermediate, which is only slowly converted to collagen III by loss of amino propeptides, was characterized by its sedimentation properties, isolation of the amino propeptide, and reaction with purified antibodies that are specific against bovine amino propeptide III. It is interchain disulfide-linked, both through the amino propeptide and the carboxyl ends of the collagen chains. The conversion of procollagen III to pN-collagen III either in blood vessels, or after isolation by a carboxyl procollagen peptidase obtained from chick tendon fibroblast cultures, is inhibited by 50 mM arginine. Underhydroxylated procollagen III was isolated from blood vessels treated with alpha, alpha'-dipyridyl. Its amino propeptides reacted with the above antibodies but were not linked to each other. In contrast, its carboxyl propeptides were interchain disulfide-bridged, supporting previous suggestions that the carboxyl propeptides play a role in the assembly of procollagen trimer.     

The immunology of procollagen. I. Development of antibodies to determinants unique to the proalpha l chain

Rabbits immunized with proα1 chains derived from embryonic chick cranial bone procollagen developed antibodies directed specifically to the NH₂-terminal non-triple-helical sequence unique to the precursor chain. Antibodies were detected by a sensitive radioimmunoassay which employed radiolabeled chick proα1 chains, rabbit antisera absorbed with an excess of α1 chain, and a sheep anti-rabbit γG globulin serum. The specificity of such rabbit antisera was demonstrated by inhibition assays with unlabeled proα1 chains and with CNBr and collagenase fragments obtained from the additional sequence in the precursor chain. The availability of antibodies to this relatively immunogenic region of procollagen provides a means of assaying for the collagen precursor in biosynthetic experiments and may permit the intracellular localization of the macromolecule by antibody-labeling techniques.     

Isolation and partial characterization of genomic clones coding for a human pro-alpha 1 (II) collagen chain and demonstration of restriction fragment length polymorphism at the 3' end of the gene

We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene.     

Type I collagen messenger RNA levels in experimental granulation tissue and silicosis in rats

A complementary DNA (cDNA) clone was constructed for chick pro alpha 2(I) collagen mRNA. This and previously constructed cDNA clones for chick and human pro alpha 1(I) collagen mRNAs were used to measure levels of type I procollagen messenger RNAs in two experimental models: viscose cellulose sponge-induced experimental granulation tissue and silica-induced experimental lung fibrosis in rats. Both Northern RNA blot and RNA dot hybridizations were used to quantitate procollagen mRNAs during formation of granulation tissue. The period of rapid collagen synthesis was characterized by high levels of procollagen mRNAs, which were reduced when collagen production returned to a low basal level. The rate of collagen synthesis and the levels of procollagen mRNAs during the period of rapid reduction in collagen production did not, however, parallel with each other. This suggests that translational control mechanisms are important during this time in preventing overproduction of collagen. In silicotic lungs, the early stages of fibroblast activation follow a similar path but appear faster. At a later stage, however, the RNA levels increase again and permit collagen synthesis to continue at a high rate, resulting in massive collagen accumulation.     

Cortisol decreases the concentration of translatable type-I procollagen mRNA species in the developing chick-embryo calvaria.

The calvarial mRNA species of chick embryos were translated in the rabbit reticulocyte-lysate cell-free translation system. The amount of procollagen type-I mRNA species was determined by digestion with bacterial collagenase and by fluorography of the cell-free translation products. Administration of cortisol resulted in a specific decrease in the cellular concentration of translatable procollagen type-I mRNA species in the calvaria of developing chick embryos. There was a lag period of up to 12 h before the response, which was dose-dependent. The data suggest that the decrease in amounts of procollagen mRNA species is the main reason for the lower amount of tissue collagen after topical or systemic administration of glucocorticoids, although other factors may contribute to the response.     

Bleomycin treatment of chick fibroblasts causes an increase of polysomal type I procollagen mRNAs. Reversal of the bleomycin effect by dexamethasone

Bleomycin treatment of primary chick skin fibroblasts and chick lung fibroblasts resulted in a selective dose-dependent increase of cell layer procollagen synthesis. Solid support hybridization of total cellular RNA to 32P-labeled pro-alpha 1(I) and pro-alpha 2(I) cDNAs did not indicate an increase of total cellular procollagen type I mRNAs in bleomycin-treated cells. However, bleomycin treatment of chick skin fibroblasts causes a redistribution of procollagen type I mRNAs within the nuclear, cytoplasmic, and polysomal subcellular fractions. Both the nuclear and cytoplasmic procollagen type I mRNAs are significantly decreased in concentration after bleomycin administration. In contrast, the polysomal procollagen type I mRNAs are significantly increased in both chick skin and lung fibroblasts treated with bleomycin. Administration of dexamethasone to bleomycin-treated fibroblasts resulted in a reversal of the bleomycin-induced increase in cell layer procollagen synthesis. The increased amounts of polysomal procollagen type I mRNAs in bleomycin-treated cells were also reduced by subsequent administration of dexamethasone. These data indicate that bleomycin treatment of chick skin and chick lung fibroblasts results in a specific increase in procollagen synthesis in the cell layer which is mediated by elevated levels of polysomal type I procollagen mRNAs via a repartitioning of these mRNAs within the fibroblast. Furthermore, dexamethasone reverses the bleomycin-induced elevations of both cell layer procollagen synthesis and polysomal type I procollagen mRNAs.     

New pepsin-solubilized low molecular weight collagenous component possibly unique to periodontal ligament

Limited pepsin digestion of bovine periodontal ligament releases genetic types I, III, and V collagen and a high cystine containing low molecular weight collagenous component. Salt fractionation and molecular sieve chromatography allowed the isolation of the latter as an apparently pure homogeneous moiety which had an approximate molecular mass of 30 000 daltons. Reduction with mercaptoethanol yielded a single 10 000-dalton band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This led us to conclude that the newly isolated low molecular weight collagen fragment consists of three similar molecular weight chains. Unreduced collagen-like glycoprotein (CGP) [Jander, R., Troyer, D., & Rauterberg, J. (1984) Biochemistry 23, 3675-3681] after extraction from tissues with collagen denaturing solvents yields the GP140 glycoprotein upon reduction and does not release any collagen fragment below 90 000 daltons upon mild or vigorous pepsin digestion. The GP140 glycoprotein [Heller-Harrison, R. A., & Carter, W. G. (1984) J. Biol. Chem. 259, 6858-6864] isolated by extraction under reducing and collagen denaturing solvent conditions did not yield a collagen fragment below 40 000 daltons after pepsin treatment. It was clearly shown that both CGP and GP140 yield type VI collagen fragments in the above-cited reports. Since this report demonstrates that the Mr 30 000 collagen fragment is only released by pepsin treatment of nondenaturing solvent treated periodontal ligament and that only very small peptides are found in denaturing solvent treated tissue after pepsin digestion, it is concluded that the newly isolated Mr 30 000 collagen fragment reported here is not derived from type VI collagen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interchain disulfide bonds at the COOH-terminal end of procollagen synthesized by matrix-free cells from chick embryonic tendon and cartilage.

Matrix-free cells from tendons and cartilage of chick embryos were incubated in suspension, and the procollagens secreted by the cells were isolated in the presence of protease inhibitors. Tendon procollagen was shown to contain both NH_2? and COOH-terminal extensions and interchain disulfide bonds were located in the COOH-terminal region. A disulfide-linked fragment previously isolated after digestion of the molecule with bacterial collagenase was shown to originate from the COOH-terminal end. Cartilage procollagen was also shown to contain interchain disulfide bonds in the COOH-terminal region.     

Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane

Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.     

Partial purification and characterization of a neutral protease which cleaves the N-terminal propeptides from procollagen

A rapid assay procedure was developed for cleavage of the N-terminal propeptides of procollagen. With the assay a neutral procollagen N-protease was purified about 300-fold from chick embryo tendon extract. The enzyme had an apparent molecular weight of 260 000 and a pH optimum of 7.4. Ca2+ was required for enzymic activity but this requirement was partially replaced by Mg2+ or Mn2+. The enzyme was bound to concanavalin A-agarose and therefore was presumably a glycoprotein. The N-propeptides released from type I procollagen were of about 23 000 and 11 000 daltons as estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The partially purified enzyme was also found to cleave type II procollagen and the N-propeptide obtained was about 18 000 daltons. Heat denaturation of either type I or type II procollagen decreased the rate at which the proteins were cleaved by the N-protease.     

Decreased extracellular matrix production in scurvy involves a humoral factor other than ascorbate

Our recent studies suggested that decreased collagen synthesis in bone and cartilage of scorbutic guinea pigs was not related to ascorbate-dependent proline hydroxylation. The decrease paralleled scurvy-induced weight loss and reduced proteoglycan synthesis. Those results led us to propose that the effects of ascorbate deficiency on extracellular matrix synthesis were caused by changes in humoral factors similar to those that occur in fasting. Here we present evidence for this proposal. Exposure of chick embryo chondrocytes to scorbutic guinea pig serum, in the presence of ascorbate, led to effects on extracellular matrix synthesis similar to those seen in scorbutic animals. The rates of collagen and proteoglycan synthesis were reduced to approximately 30-50% of the levels in cells cultured in normal guinea pig serum plus ascorbate, but proline hydroxylation and procollagen secretion were unaffected. Similar results were obtained with serum from fasted guinea pigs supplemented in vivo with ascorbate. The growth rate of the chondrocytes was not significantly affected by scorbutic guinea pig serum.     

Characterization of the tissue form of type V collagen from chick bone

Type V collagen was prepared from acetic acid extracts of lathyritic chick bone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracted material demonstrated two collagenous bands of slower mobility than pepsin-extracted alpha 1(V) and alpha 2(V) chains. Cyanogen bromide peptide maps of these protein bands identified them as forms of alpha 1(V) and alpha 2(V). Segment long spacing (SLS) crystallite banding patterns of the acid-extracted Type V were identical within the triple-helical domain to the SLS banding patterns of pepsin-extracted Type V collagen, supporting the identification of this material. A globular domain at one end of the triple helix of the acid-extracted Type V was visualized by both rotary shadowing and negative staining of SLS crystallites. The molecular weights of the globular terminal peptides were 18,000 and 29,000, respectively, for alpha 1(V) and alpha 2(V), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after bacterial collagenase digestion of the isolated alpha chains. The results presented here indicate that fully processed Type V collagen in chick bone exists as a higher molecular weight form than that from pepsin extracts and retains a globular domain at one end of the triple helix. This is in contrast to the interstitial collagens in which only very small non-triple-helical domains (telopeptides) are retained in the fully processed molecules. In vitro aggregation studies demonstrated the intact fully processed form of Type V collagen forms uniform small-diameter fibrous structures. These results suggest that Type V collagen may be present in fibrous structures within tissues.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo.

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.