The disulphide-bonded nature of procollagen and the role of the extension peptides in the assembly of the molecule.

1. The molecular weights of chick tendon and cartilage procollagens, and their constituent polypeptides, were determined by gel filtration and gel electrophoresis. The values obtained are in good agreement and indicate that the mol.wts. of the secreted procollagens (types I and II) and their individual pro-alpha-chains are of the order of 405 000-445 000 and 137 000-145 000 respectively.2. Digestion of tendon procollagen with human rheumatoid synovial collagenase gave products consistent with the presence of large non-helical peptide extensions at both N-and C-termini. Electrophoretic analysis gave apparent mol.wts. of 17 500 and 36 000 for the respective N- and C-terminal extensions of pro-alpha1(I)-and pro-alpha2-chains, and inter-chain disulphide bonds were restricted to the C-terminal location. 3. During the biosynthesis of procollagen by tendon and cartilage cells a close correlation was observed between the extent of inter-chain disulphide bonding and the proportion of procollagen polypeptides having a triple-helical conformation. These processes appeared to commence in the rough endoplasmic reticulum and be completed in the smooth endoplasmic reticulum, but the rate at which they occur in cartilage cells is markedly slower than that found in tendon cells. 4. When the intracellular [14C]procollagen polypeptides present in the rough-endoplasmic-reticulum fractions of tendon and cartilage cells were analysed under non-reducing conditions on agarose/polyacrylamide composite gels, no significant pools of dimeric intermediates were detected. 5. In both cell types, inter-chain disulphide-bond formation occurred even when hydroxylation, and hence triple-helix formation, was inhibited. The presence of pro-alpha1- and pro-alpha2-components in a ratio of 2:1 in the disulphide-linked unhydroxylated procollagen isolated from tendon cells demonstrated that correct chain association occurs in the absence of hydroxylation. This observation is consistent with a model for the assembly of pro-gamma112-chains in which the recognition and selection of pro-alpha1-and pro-alpha2-chains in a 2:1 ratio are directed by the non-helical C-terminal extension peptides of tendon procollagen.     

Studies on the glycosylation of hydroxylysine residues during collagen biosynthesis and the subcellular localization of collagen galactosyltransferase and collagen glucosyltransferase in tendon and cartilage cells

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [(14)C]lysine were assayed for hydroxy[(14)C]lysine and hydroxy[(14)C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[(14)C]lysine and glucosylgalactosylhydroxy[(14)C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [(14)C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. (14)C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5'-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.     

Hydroxylation of lysine and glycosylation of hydroxylysine during collagen biosynthesis in isolated chick-embryo cartilage cells.

Hydroxylation of lysine and glycosylation of hydroxylysine during collagen biosynthesis in isolated chick-embryo cartilage cells were studied by using continuous labelling and pulse-chase labelling experiments with [14C]lysine. Control experiments with [14C]proline indicated that in continuous labelling the hydroxylation of [14C]proline became linear with time after about 4 min and the secretion of collagen after about 35 min, as reported previously. In similar experiments with [14C]lysine the hydroxylation of [14C]lysine and the glycosylations of hydroxy[14C]lysine became linear at about 4 min, suggesting that these reactions were initiated while the polypeptide chains were growing on the ribosomes. Pulse-chase labelling experiments with [14C]lysine indicated that after a 5 min pulse-label the hydroxylation of [14C]lysine and the glycosylations of hydroxyl[14C]lysine continued during the chase period for about 20 min. The data suggest that these reactions are continued after the release of complete polypeptide chains into the cisternae of the endoplasmic reticulum, whereas the reactions are probably not continued after the formation of the triple helix and the movement of the molecules into the Golgi vacuoles.     

The biosynthesis of basement-membrane collagen by isolated rat glomeruli.

A technique is described for the rapid isolation of highly purified preparations of viable glomeruli from rat kidney cortex. The synthesis of protein as judged by the incorporation of [14C]proline into non-diffusible material was shown to be linear for up to 6 h. The synthesis of collagen, measured as non-diffusible 4-hydroxy[14C]proline, was also linear over this period but represented only a small proportion of total protein synthesis. Similar studies conducted in vivo confirmed that collagen synthesis accounted for less than 5% of total protein synthesis in glomeruli. When isolated glomeruli were incubated with [14C]proline, it was found that approximately 16% of the hydroxyproline present in the collagenous component occurred as the 3-isomer. When glomeruli were incubated with [14C]lysine over 90% of the hydroxy[14C]lysine synthesised was glycosylated and most of the glycosylated hydroxy[14C]lysine was present as glucosyl-galactosyl-hydroxy[14C]lysine. The size of the basement membrane collagen synthesised by the isolated glomeruli was estimated by treating the 14C-labelled protein with mercaptoethanol and sodium dodecyl sulphate and then chromatographing the 14C-labelled protein on an agarose column equilibrated and eluted with buffer containing 0.1% (w/v) sodium dodecyl sulphate. The initial form of [14C]collagen synthesised was found to consist of polypeptide chains which had molecular weights of approximately 140 000 and which were shown to be distinctly larger than the polypeptide chains from embryonic chick tendon procollagen. Also when glomeruli were labelled with [14C]proline for 2 h and chased with unlabelled proline for 4 h there was a time-dependent conversion of the initially synthesised collagen moiety to collagen polypeptide chains which co-chromatograph with tendon pro-alpha chains (molecular weight approx. 120 000).     

The route of secretion of procollagen. The influence of alphaalpha''-bipyridyl, colchicine and antimycin A on the secretory process in embryonic-chick tendon and cartilage cells.

I. Embryonic-chick tendon cells were pulse-labelled for 4 min with [14C]proline and the 14C-labelled polypeptides were chased with unlabelled proline for up to 30 min. Isolation of subcellular fractions during the chase period and their subsequent analysis for bacterial collagenase-susceptible 14C-labelled peptides demonstrated the transfer of procollagen polypeptides from rough to smooth microsomal fractions and thence to the extracellular medium. Parallel analyses of Golgi-enriched fractions indicated the involvement of this organelle in the secretory pathway of procollagen. Sodium dodecylsulphate/polyacrylamide-gel electrophoresis of the 14C-labelled polypeptides present in the Golgi-enriched fractions demonstrated that the procollagen polypeptides were all present as disulphide-linked pro-gamma components. 2. When similar kinetic studies of the intracellular transport of procollagen were conducted with embryonic-chick cartilage cells almost identical results were obtained, but the rate of translocation of cartilage procollagen was significantly slower than that observed for tendon procollagen. 3. When hydroxylation of procollagen polypeptides was inhibited by alphaalpha'-bipyridyl, the nascent polypeptides accumulated in the rough microsomal fraction. 4. When cells were pulse-labelled for 4min with [14C)proline and the label was chased in the presence of colchicine, secretion of procollagen was inhibited and an intracellular accumulation of procollagen 14C-labelled polypeptides was observed in the Golgi-enriched fractions. 5. The energy-dependence of the intracellular transport of procollagen was demonstrated in experiments in which antimycin A was found to inhibit the transfer of procollagen polypeptides from rough to smooth endoplasmic reticulum. 6. It is concluded that procollagen follows the classical route of secretion taken by other extracellular proteins.     

The coordinate synthesis and cotranslational assembly of type I procollagen

Nascent polysome-associated type I procollagen pro-alpha-chains isolated from chick embryo tendon fibroblasts were examined for their proteinase resistance. The distribution of chain sizes and their proteinase resistance were also determined following chain elongation in an in vitro readout system in the absence of chain initiation factors. Chains were labeled with [14C]proline in the cells and with [3H]proline in the readout system. Differences in the ratios of 14C to 3H in the double-labeled nascent chains before and after chymotryptic digestion, determined by slicing and counting polyacrylamide gels after electrophoresis, permitted analysis of the relative stabilities of in vivo and in vitro elongated portions of the chains. In confirmation of earlier work, the polysome-bound nascent procollagen contained chymotrypsin, chymotrypsin plus trypsin, and pepsin-resistant alpha-chain size components. The readout system data showed that the full length chains produced in the cell were more resistant to digestion than the fully elongated readout-completed chains. The protease resistance of the chains was taken to indicate the registration of the chains prior to the induction of helix formation during the isolation procedure. These data support the model in which chain selection and folding are facilitated by the organization of the attachment of the ribosomes to the endoplasmic reticulum surface.     

Hydroxylation of peptide-bound proline and lysine before and after chain completion of the polypeptide chains of procollagen

The synthesis of procollagen hydroxyproline and hydroxylysine was examined in matrix-free cells which were isolated from embryonic tendon by controlled enzymic digestion and then incubated in suspension. After the cells were labeled with [¹⁴C]proline for 2 min, or about one-third the synthesis time for a Pro-α chain, [¹⁴C]hydroxyproline was found in short peptides considerably smaller than the Pro-α chains of procollagen. The results, therefore, confirmed previous reports indicating that the hydroxylation of proline can begin on nascent chains. In similar experiments in which the cells were labeled with [¹⁴C]lysine, [¹⁴C]hydroxylysine was found in short, newly synthesized peptides, providing the first evidence that the hydroxylation of lysine can also begin on nascent peptides. However, further experiments demonstrated that the synthesis of hydroxyproline and hydroxylysine continues until some time after assembly of the polypeptide chains is completed.     

Immunochemical and immunocytochemical studies of the C-propeptide of type II procollagen in chondrocytes of the growth plate.

The C-propeptide of type II procollagen has previously been implicated in cartilage calcification. To further characterize this propeptide, we have investigated its molecular status and intracellular distribution in bovine fetal growth plate chondrocytes, particularly within the calcifying zone, using cell isolation, Western blotting, and localization with immunofluorescence and immunogold techniques. We found that in all cells freshly isolated by collagenase digestion the C-propeptide was a component of type II pro-alpha chains. No free C-propeptide was detected intracellularly. In situ localization of the C-propeptide by immunostaining employing immunofluorescence revealed the presence of procollagen in most growth plate cells, staining being most intense in hypertrophic cells. In the latter, large dilations of the rough endoplasmic reticulum were observed. These were not found in proliferating cells and had an approximate diameter of 5 microns. With immunogold localization these, together with Golgi-derived secretory granules, stained for the C-propeptide. These combined results suggest that in all cells of the growth plate the C-propeptide is a constituent part of type II collagen pro-alpha chains, and that it is usually segregated in the rough endoplasmic reticulum at a time when, according to other studies, collagen synthesis ceases in the lower hypertrophic zone and calcification of the extracellular matrix ensues. This suggests that the intracellular translocation of type II collagen pro-alpha chains may change in hypertrophic cells at this time.     

Accumulation of procollagen in the degenerative articular cartilage of dogs with osteoarthritis.

Collagen metabolism was studied in degenerative articular cartilage of dogs with spontaneous, early onset osteoarthritis. A fraction of collagen which represented about 1.5% of the total was extracted from cartilage samples with dilute phosphate buffer (pH 7.4) containing 0.2% sodium dodecyl sulfate. Agarose gel filtration in the presence of sodium dodecyl sulfate revealed that extracts of degenerative cartilage had about 24% procollagen whereas extracts of normal samples had only 3%. The isolated procollagen fraction was rechromatographed on agarose columns in the presence of mercaptoethanol. This resulted in the identification of a collagen species which migrated between marker beta and alpha collagen chains. The molecular weight of this collagen was estimated to be 150,000. Based on incorporation of [14C]proline, its ratio of hydroxy[14C]proline to total 14C was 0.32. Procollagen was not found after limited pepsin digestion (pH 3, 4 degrees C, 16 h) of degenerative cartilage samples. Since the total collagen content (microgram hydroxyproline/mg cartilage), hydroxy-[14C]proline/mg cartilage, specific radioactivity of hydroxyproline in the extractable collagen fraction were similar for normal and degenerative cartilage we propose that procollagen accumulated in the degenerative cartilage due to a partial defect in conversion of procollagen to collagen.     

Accumulation of procollagen in the degenerative articular cartilage of dogs with osteoarthritis

Collagen metabolism was studied in degenerative articular cartilage of dogs with spontaneous, early onset osteoarthritis. A fraction of collagen which represented about 1.5.% of the total was extracted from cartilage samples with dilute phosphate buffer (pH 7.4) containing 0.2% sodium dodecyl sulfate. Agarose gel filtration in the presence of sodium dodecul sulfate revealed that extracts of degenerative cartilage had about 24% procollagen whereas extracts of normal samples had only 3%. The isolated procollagen fraction was rechromatographed on agarose columns in the presence of mercaptoethanol. This resulted in the identification of a collagen species which migrated between marker β and α collagen chains. The molecular weight of this collagen was estimated to be 150000. Based on incorporation of [¹⁴C]proline, its ratio of hydroxy[¹⁴C]proline to total ¹⁴C was 0.32. Procollagen was not found after limited pepsin digestion (pH 3,4°C, 16 h) of degenerative cartilage samples.Since the total collagen content (μg hydroxyproline/mg cartilage), hydroxy[¹⁴C]proline/mg cartilage, specific radioactivity of hydroxy[¹⁴C]proline (cpm/μg), in the whole cartilage, and the specific radioactivity of hydroxyproline in the extractable collagen fraction were similar for normal and degenerative cartilage we propose that procollagen accumulated in the degenerative cartilage due to a partial defect in conversion of procollagen to collagen.     

Translation of embryonic-chick tendon procollagen messenger ribonucleic acid in two cell-free protein-synthesizing systems

Embryonic-chick tendon poly(A)-containing RNA was translated in the wheat-germ and mRNA-dependent rabbit reticulocyte-lysate systems. The ability of each system to synthesize polypeptides similar to pro-alpha chains of collagen was tested on the bases of electrophoretic mobility and susceptibility to highly purified bacterial collagenase. Very small amounts of polypeptides in the size range of pro-alpha chains were synthesized in the wheat-germ system, whereas efficient synthesis of two polypeptides similar to pro-alpha1 and pro-alpha2 chains was achieved in the reticulocyte lysate. The collagenous nature of the major high-molecular-weight products synthesized was demonstrated by their susceptibility to collagenase and ability to act as a substrate for purified collagen proline hydroxylase. Determinations of the relative amounts of these translation products suggest that the 2:1 ratio of pro-alpha1 and pro-alpha2 chains found in type I procollagen is reflected in proportional amounts of translatable mRNA for pro-alpha1 and pro-alpha2 chains. Comparisons of the electrophoretic mobilities of hydroxylated and unhydroxylated reticulocyte-lysate translation products were made with appropriate standards of hydroxylated and unhydroxylated procollagen polypeptides. The results suggest that, in common with a number of secreted proteins, procollagen is synthesized as pre-pro molecules consistent with the ;Signal Hypothesis'.     

Elastin biosynthesis in chick-embryo arteries. Studies on the intracellular site of synthesis of tropoelastin.

Electrophoretic analyses of the products of cell-free translation of elastin mRNA isolated from 17-day chick-embryo thoracic arteries have demonstrated that the elastin mRNA codes for polypeptides that are slightly larger than the cellular tropoelastin polypeptides synthesized and secreted by matrix-free artery cells. Pulse-chase experiments with cells labelled with [3H]proline established that newly synthesized tropoelastin polypeptides were associated solely with membrane-bound particulate fractions. Cell-free translation of membrane-bound and free polyribosomes isolated from artery cells revealed that the tropoelastin mRNA was associated predominantly with the membrane-bound fraction. When rough-microsomal fractions, isolated from cells labelled with [3H]proline for 10 min, were treated with proteinases in the presence and in the absence of detergent, the nascent tropoelastin polypeptides were shown to be susceptible to proteolysis only when the integrity of the membranes was destroyed by detergent treatment. In similar experiments tropoelastin polypeptides synthesized by membrane-bound polyribosomes in the nuclease-treated reticulocyte lysate were also resistant to the proteolytic-enzyme treatment. The results suggest that tropoelastin polypeptides are synthesized on membrane-bound polyribosomes and discharged into the lumen of the endoplasmic reticulum with co-translational removal of a signal peptide.     

Hydroxylation of prolyl residues in type II procollagen in vitro and in cellulo. Lack of preferential hydroxylation of specific regions of the protein

[14C]Proline-labeled protocollagen, the unhydroxylated form of procollagen, was isolated from cartilage cells incubated with alpha, alpha'-dipyridyl. For examination of the initial steps in the hydroxylation of the protein, it was incubated in vitro with prolyl hydroxylase so that an average of 1.3-2.7 prolyl residues per chain was hydroxylated. The partially hydroxylated alpha chain were cleaved with cyanogen bromide, and the fragments were separated by polyacrylamide gel electrophoresis or column chromatography. The cyanogen bromide fragments were hydroxylated to the same degree. The results indicated, therefore, that in the initial hydroxylation of alpha chains in vitro, there was no preferential hydroxylation of any specific regions of the protein. In a second series of experiments, cartilage cells were incubated with [14C]proline and alpha, alpha'-dipyridyl so that prolyl hydroxylase in the cells was extensively, but not completely, inhibited. Partially hydroxylated alpha chains were isolated, and cyanogen bromide fragments of the alpha chains from the cells were assayed for hydroxy[14C]proline. The alpha chains contained an average of two residues of hydroxyproline per chain, and the cyanogen bromide fragments were hydroxylated to about the same degree. The results indicated, therefore, that when prolyl hydroxylase activity in cells is low relative to the rate at which pro alpha chains are synthesized, hydroxylation of prolyl residues occurs as it does in vitro, and there is no preferential hydroxylation of a specific region of the protein.     

Biosynthesis of basement membrane collagen by rabbit corneal endothelium in vitro.

The synthesis of collagen has been demonstrated in endothelial cells of Descemet's membrane isolated from rabbit cornea. Incorporation of [14C]proline and [14C]lysine into nondialyzable protein was measured in the medium and cell fraction after incubating Descemet's membrane for up to 5 hours. In the [14C]collagen synthesized by the endothelium, 15% of the hydroxy[14C]proline was present as the 3-isomer. About 98% of the hydroxy[14C]lysine in the 14C-labeled-protein found in the medium was glycosylated; 95% of the glycosylated hydroxy[14C]lysine was in the form of the disaccharide glucosyl-galactosyl-hydroxy[14C]lysine. Time course experiments with [14C]proline indicated that there was a delay of about 60 min before significant amounts of [14C]collagen were secreted into the medium. The initial polypeptides of [14C]collagen synthesized by the corneal endothelium had an apparent molecular weight of 155,000. The chemical and physical properties of the [14C]collagen synthesized by rabbit corneal endothelium are consistent with those of basement membrane collagen synthesized by other cell types.     

Synthesis of procollagen by matrix-free cells from embryonic-chick arteries

Cells were isolated from the major arteries of 17-day chick embryos by digestion of the tissue with collagenase and trypsin. The cells, when examined immediately after isolation, exhibited a high degree of viability and they were shown to synthesize and secrete procollagen at a high and constant rate for several hours when incubated in suspension in modified Krebs medium. Continuous labelling of the cells with [(14)C]proline demonstrated a lag of about 30min between the time at which the synthesis of non-diffusible peptide-bound hydroxy[(14)C]proline became linear and the time at which its secretion into the medium became linear. This lag time compares with that of 18min observed for freshly isolated matrix-free cells from embryonic-chick tendon, which synthesize and secrete the same type of collagen. Gel-filtration chromatography and polyacrylamide-gel electrophoresis indicated that the collagenous polypeptides secreted into the medium were in the precursor form, known as procollagen, and that the constituent pro-alpha-chains were linked by interchain disulphide bonds and were also in a triple-helical conformation. Characterization of the secreted procollagen by gel-filtration chromatography, polyacrylamide-gel electrophoresis, DEAE-agarose chromatography, and polyacrylamide-gel electrophoresis of peptides obtained by CNBr cleavage, indicated that the predominant form was type-I procollagen. This work extends the range of freshly isolated matrix-free cell systems, which have been characterized for use in studies on the biosynthesis and secretion of procollagen, and it indicates differences in the rates of secretion of procollagen in different cell types secreting the same type of procollagen.     

Biosynthesis and in vitro translation of type IV procollagens

The present paper describes how epithelial cells, cultured from bovine anterior lens capsule explants, synthesize and secrete procollagen type IV polypeptide chains alpha 1(IV) and alpha 2(IV). Metabolic labeling of these cells with [14C]proline for different time intervals and subsequent analysis by SDS/polyacrylamide gel electrophoresis revealed the presence of two polypeptide chains with apparent molecular masses of 180 kDa and 170 kDa. The procollagens were bacterial-collagenase-sensitive and were specifically immunoprecipitated by antibodies raised against the 7S domain of type IV collagen. Type IV procollagen poly(A)-rich RNA was isolated from cultured lens capsule cells and translated in a reticulocyte lysate cell-free system. Two polypeptides with apparent molecular masses of 152 kDa and 145 kDa were identified as procollagen type IV unmodified chains by gel electrophoresis, collagenase digestion and specific immunoprecipitation. During experiments in which cells were labeled in the presence of alpha, alpha'-bipyridyl, type IV procollagen appeared as one major band comigrating with a 145 kDa polypeptide on SDS-gel electrophoresis.     

Mechanism for the regulation of post-translational modifications of procollagens synthesized by matrix-free cells from chick embryos

Three possible mechanisms are considered to account for the variations of post-translational modifications in different collagen types. 1) The cells have different amounts of post-translational modifying enzymes, 2) the rate of prolylhydroxylation of different procollagen types is varied, and 3) the rate of chain association of pro-alpha chains of different collagen types is modulated. In an attempt to examine the three possibilities, we have determined the activities of prolyl hydroxylase and lysyl hydroxylase, and we have examined the kinetics of the secretion of procollagens and the kinetics of pro-gamma chain formation of different procollagen types in matrix-free cells isolated from tissues of 17-day-old chick embryos. Type II collagen synthesized by cartilage cells contains more hydroxylysine than type I collagen synthesized by tendon and cornea cells. It was found, however, that cartilage cells contain significantly less lysyl hydroxylase than tendon and cornea cells. In contrast, we found only a small difference in the amount of prolyl hydroxylase in tendon, cornea, and cartilage cells. The secretion of type I procollagen by tendon and cornea cells can be described by two first order processes. In contrast, the secretion of type II procollagen by cartilage cells, type IV procollagen by lens cells, and type V procollagen by cornea cells can be described by single first order processes. Examination of the formation of pro-gamma components of procollagen types I and II revealed that it occurs via intermediate dimers of two pro-alpha chains. The formation or pro-gamma(I) chains in tendon and cornea cells is about three times faster than the formation of pro-gamma(II) chains in cartilage cells. These results are consistent with the hypothesis that the rate of association of pro-alpha chains regulates the synthesis of procollagens with different degrees of post-translational modifications.     

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.     

Termination of procollagen chain synthesis by puromycin. Evidence that assembly and secretion require a COOH-terminal extension.

Embryonic chick fibroblasts were incubated with [14C]proline and puromycin in the low concentrations of 1 to 3 mug/ml. The molecular weight of the synthesized procollagen chains, as measured by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, was progressively reduced by increasing concentrations of puromycin in this range. For example, at 3 mug/ml the great majority of the [14C]proline was contained in procollagen chains having an average molecular weight of about 95,000 instead of the control value of 125,000. Associated with this decrease in molecular weight there was a marked decrease in the incorporation of cysteine although [14C]proline incorporation was relatively unaffedted. Disulfide bond formation was drastically inhibited as was triple helix formation as measured by resistance of the procollagen to pepsin digestion. Although the shortened procollagen chains were of normal hydroxyproline content, they nevertheless were secreted much more slowly than normal procollagen. Based upon these findings, we postulate that: (a) low concentrations of puromycin terminate procollagen chains before a COOH-terminal extension is completed, (b) these COOH-terminal extensions are required for normal assembly of the three individual procollagen chains and for triple helix formation, and (c) only assembled, triple helical procollagen molecules are selected for normal secretion.     

Synthesis and processing of a type I procollagen containing shortened pro-alpha 1(I) chains by fibroblasts from a patient with osteogenesis imperfecta

Skin fibroblasts from a patient with a lethal form of osteogenesis imprefecta were found to synthesize equal amounts of normal pro-alpha 1(I) chains and pro-alpha 1(I) chains which are about 10% shorter because of a deletion of about 100 amino acids in the middle of the alpha chain domain. The pro-alpha 1(I) chains were incorporated into three different kinds of trimers: a normal type I trimer with normal length pro-alpha 1(I) chains; a type Is trimer with one shortened pro-alpha 1(I) chain and two normal length chains; and a type Iss trimer containing two shortened pro-alpha 1(I) chains and one normal length pro-alpha 2(I) chain. As judged by resistance to digestion by chymotrypsin and trypsin, the type Is and Iss trimers denatured at a temperature at least 3 degrees C lower than normal type I procollagen. Procollagen containing the shortened pro-alpha 1(I) chains was slowly secreted by the cells but was degraded by extracellular proteinases within 6 h of chase into the medium. The results indicated that the presence of the shortened pro-alpha 1(I) chains in procollagen trimers produces a delay in rate of helix formation, overmodification of the polypeptides by post-translational enzymes, a decrease in the thermal stability of the trimers, and increased susceptibility of the protein to endogenous proteinases. Additionally, the fibroblasts of this patient synthesized and secreted a type III-like species of procollagen with unusual chromatographic properties.