Lysosomal function in the degradation of defective collagen in cultured lung fibroblasts

Human fibroblasts when induced to make nonhelical , defective collagen have mechanisms for degrading up to 30% of their newly synthesized collagen intracellularly prior to secretion. To determine if at least a portion of the degradation of defective collagen occurs by lysosomes, extracts of cultured HFL-1 fibroblasts were examined for proteinases capable of degrading denatured type I [3H]procollagen. The majority of the proteolytic activity against denatured [3H]-procollagen had a pH optimum of 3.5-4; it was stimulated by dithiothreitol and inhibited 95% by leupeptin, 10% by pepstatin, and 98% by leupeptin and pepstatin together. Extracts of purified lysosomes from the fibroblasts were active in degrading denatured [3H]procollagen and were completely inhibited by leupeptin and pepstatin. To demonstrate directly that human lung fibroblasts can translocate a portion of their defective collagen to lysosomes, cultured cells were incubated with cis-4-hydroxyproline and labeled with [14C]proline to cause the cells to make nonhelical [14C]procollagen. About 3% of the total intracellular hydroxy[14C]proline was found in lysosomes. If, however, the cells were also treated with NH4Cl, an inhibitor of lysosomal function, 18% of the intracellular hydroxy[14C]proline was found in lysosomes. These results demonstrate that cultured human lung fibroblasts induced to make defective collagen are capable of shunting a portion of such collagen to their lysosomes for intracellular degradation.     

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.     

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.     

The synthesis and secretion of cartilage procollagen.

1. Isolation of free and membrane-bound ribosomes from embryonic chick sternal-cartilage cells labelled for 4min with [14C]proline and their subsequent analysis for hydroxy[14C]proline indicated that cartilage procollagen biosynthesis occurs on bound ribosomes. 2. Nascent procollagen polypeptides on bound ribosomes isolated from cells labelled with [14C]lysine were found to contain hydroxy[14C]lysine indicating that hydroxylation of lysine commences while the growing chains are still attached to the ribosomes. 3. Analysis of bound ribosomes labelled with either [14C]proline or [14C]lysine on sucrose density gradients indicated that cartilage procollagen is synthesized on large polyribosomes in the range 250-400S. 4. Microsomal preparations isolated from cells pulse-labelled for 4 min with [14C]proline were used to determine the direction of release of nascent procollagen polypeptides. Puromycin induced the vectorial release of nascent procollagen polypeptides into the microsomal vesicles suggesting that the first step in the secretion of procollagen polypeptides is their transfer from the ribosomes through the membrane of the endoplasmic reticulum into the cisternal space. 5. The procollagen polypeptides secreted by cartilage cells were shown to be linked by inter-chain disulphide bonds. 6. Examination of the state of aggregation of pro-alpha chains in subcellular fractions isolated from cartilage cells labelled with [14C]proline for various periods of time have provided data on the timing and location of inter-chain disulphide-bond formation. This process commences in the rough endoplasmic reticulum after the release of completed pro-alpha chains from membrane-bound ribosomes. Pro-alpha chains isolated from fractions of smooth endoplasmic reticulum were virtually all present as disulphide-bonded aggregates, suggesting that either disulphide bonding is completed in this cellular compartment, or that procollagen needs to be in a disulphide-bonded form to be transferred to this region of the endoplasmic reticulum. 7. Comparison of these results with previously published data on disulphide bonding in tendon cells suggest that the rate of inter-chain disulphide-bond formation is significantly slower in cartilage cells.     

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.     

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.     

Effects of preformed proline and proline amino acid precursors (including glutamine) on collagen synthesis in human fibroblast cultures

A technique of derivatizing proline and 4-hydroxyproline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to measure the radioactivities, concentrations and specific activities of proline and hydroxyproline. The technique was used to study the conditions of procollagen synthesis in cultured human foreskin fibroblasts. Procollagen synthesis appeared to be independent of the proline concentration in the medium, in the presence of glutamine, when monitored by the assay of non-dialyzable hydroxyproline, but not when monitored by [14C]proline incorporation. In the absence of unlabelled proline added to labelled proline in the medium, the specific activity of the secreted procollagen did not reach a plateau over a 24-h period. When the medium was supplemented with glutamine, glutamic acid, or aspartic acid, both the radioactivity and concentration of intracellular free proline decreased. Pyrrolidone-2-carboxylic acid and ornithine both induced a slight increase in concentration of the intracellular free proline. Glutamine competed with [14C]proline for incorporation into prolyl-tRNA and procollagen, independently of free intracellular proline, and it stimulated the biosynthesis of procollagen (expressed as non-dialyzable hydroxyproline) by a factor of 2.3.     

Arginine catabolism in the cyanobacterium Synechocystis sp. Strain PCC 6803 involves the urea cycle and arginase pathway

Cells of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 supplemented with micromolar concentrations of L-[(14)C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of L-[(14)C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [(14)C]ornithine preceded that of [(14)C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with L-[(14)C]ornithine, suggesting that the reaction of arginase, rendering ornithine and urea, is the main initial step in arginine catabolism. Ornithine followed two metabolic pathways: (i) conversion into citrulline, catalyzed by ornithine carbamoyltransferase, and then, with incorporation of aspartate, conversion into argininosuccinate, in a sort of urea cycle, and (ii) a sort of arginase pathway rendering glutamate (and glutamine) via Delta(1)pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an argF (ornithine carbamoyltransferase) insertional mutant was impaired in the production of [(14)C]citrulline from [(14)C]arginine; (ii) a proC (Delta(1)pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [(14)C]proline, [(14)C]glutamate, and [(14)C]glutamine from [(14)C]arginine or [(14)C]ornithine; and (iii) a putA (proline oxidase) insertional mutant did not produce [(14)C]glutamate from L-[(14)C]arginine, L-[(14)C]ornithine, or L-[(14)C]proline. Mutation of two open reading frames (sll0228 and sll1077) putatively encoding proteins homologous to arginase indicated, however, that none of these proteins was responsible for the arginase activity detected in this cyanobacterium, and mutation of argD (N-acetylornithine aminotransferase) suggested that this transaminase is not important in the production of Delta(1)pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [(14)C]arginine catabolism also provide a rationale for understanding how nitrogen is made available to the cell after mobilization of cyanophycin [multi-L-arginyl-poly(L-aspartic acid)], a reserve material unique to cyanobacteria.     

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.     

A study of the biosynthesis of collagenous and non-collagenous proteins and of the proline/hydroxyproline ratios of collagens isolated from embryonic tissues

1. After incubation of chick-embryo skin slices with [(14)C]proline for 2hr. the specific activities of [(14)C]proline and [(14)C]hydroxyproline in soluble and insoluble collagens and [(14)C]proline in non-collagenous proteins were determined as well as the total amounts of both imino acids in these proteins. On the basis of these results it was demonstrated that soluble collagens having a high proline/hydroxyproline ratio are contaminated with non-collagenous proteins. 2. It was found that, in the presence of a mixture of amino acids in the incubation medium, the rate of synthesis of soluble collagen is significantly decreased. 3. The metabolic activity of collagenous proteins is related to their solubility, but that of non-collagenous proteins is not.     

Fibroblast procollagen production rates in vitro based on [3H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [3H]proline and measuring the subsequent formation of [3H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [3H]proline, and [3H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [14C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45-55]. Procollagen production rates were readily determined by this method using 5 to 20 microCi [3H]proline and approximately 10(6) cells. It was found that 3H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/microgram DNA . h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.     

Further studies on the effect of the collagen triple-helix formation on the hydroxylation of lysine and the glycosylations of hydroxylysine in chick-embryo tendon and cartilage cells

The hydroxylation of lysine and glycosylations of hydroxylysine were studied in isolated chick-embryo tendon and cartilage cells under conditions in which collagen triple-helix formation was either inhibited or accelerated. The former situation was obtained by incubating the tendon cells with 0.6mm-dithiothreitol, thus decreasing their proline hydroxylase activity by about 99%. After labelling with [(14)C]proline, the formation of hydroxy[(14)C]proline was found to have declined by about 95%. Since the hydroxylation of a relatively large number of proline residues is required for triple-helix formation at 37 degrees C, the pro-alpha-chains synthesized under these conditions apparently cannot form triple-helical molecules. Labelling experiments with [(14)C]lysine indicated that the degree of hydroxylation of the lysine residues in the collagen synthesized was slightly increased and the degree of the glycosylations of the hydroxylysine residues more than doubled, the largest increase being in the content of glucosylgalactosylhydroxylysine. Recovery of chick-embryo cartilage cells from temporary anoxia was used to obtain accelerated triple-helix formation. A marked decrease was found in the extent of hydroxylation of the lysine residues in the collagen synthesized under these conditions, and an even larger decrease occurred in the glycosylations of the hydroxylysine residues. The results support the previous suggestion that the triple-helix formation of the pro-alpha-chains prevents further hydroxylation of lysine residues and glycosylations of hydroxylysine residues during collagen biosynthesis.     

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.     

Metabolism of [14C]citrulline in the perfused sheep and goat udder

1. A lactating-sheep mammary gland was perfused for 12h in the presence of l-[2-(14)C]-citrulline and received adequate quantities of glucose, acetate and amino acids. Two lactating-goat udders were similarly perfused in the presence of either l-[carbamoyl-(14)C,-2-(14)C]citrulline or l-[carbamoyl-(14)C,1-(14)C]citrulline and l-[4-(3)H]arginine. 2. In these experiments, [(14)C]citrulline was substantially oxidized to CO(2) and converted into arginine and proline of casein. 3. The specific radioactivities of arginine, ornithine and proline of the plasma increased after passage through the udders, demonstrating that [(14)C]citrulline is metabolized by the mammary gland. 4. The presence of two unknown radioactive metabolites of [(14)C]citrulline was detected in the perfusate. These substances were not found after incubation in vitro of oxygenated blood in the presence of the radioactive precursor. 5. From these experiments, it is concluded that citrulline is metabolized in mammary tissue by way of arginine to urea, ornithine and proline.     

Diabetes stimulates procollagen degradation in rat tendon in vitro

To identify the mechanisms responsible for the paucity of recently synthesized collagen in connective tissues during diabetes, in vitro procollagen metabolism was studied in non-diabetic (control) and diabetic rats. Achilles tendons from the two groups were incubated for 1-8 h (35 degrees C) in medium containing [14C]proline and the radiolabeled collagen in the tissue, and that released into the media, were examined by SDS-polyacrylamide gel electrophoresis and fluorography. The bulk of the radiolabeled collagen in tendon from the diabetics was recovered as degradation products; these, but also procollagen and collagen components, were prominent in the control tissues. Moreover, the collagenous components synthesized by the diabetic rat tendons were more readily digested in vitro by trypsin than those produced by control tissues. We conclude that diabetes reduces collagen accretion in connective tissues in part due to increased intracellular degradation of procollagen.     

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).     

Fibroblast procollagen production rates in vitro based on [H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [³H]proline and measuring the subsequent formation of [³H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [³H]proline, and [³H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [¹⁴C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45–55]. Procollagen production rates were readily determined by this method using 5 to 20 μCi [³H]proline and approximately 10⁶ cells. It was found that ³H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/μg DNA · h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.     

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.     

A disorder of collagen biosynthesis in patients with cerebral artery aneurysm

12 patients with subarachnoid hemorraghe due to rupture of a cerebral aneurysm were examined clinically for symptoms and signs of a connective tissue disorder and biochemically for details of the biosynthesis of collagen. No uniform clinical pattern of any connective tissue disorder was seen in these patients, although selected signs were observed. Skin fibroblast cultures were then established. The rate of procollagen production in two cell lines was reduced by 40% and 50%, respectively, and the intracellular accumulation of hydroxyl[¹⁴C]proline (as a percentage of total hydroxy[¹⁴C]proline) was increased by 70% in each relative to eight control cell lines. No difference was found in the degree of intracellular degradation of procollagen. After pulse-labelling, however, the radioactive procollagen was secreted into the medium in 1 h in the control cells, but required at least 3 h in the two aneursym patient cell lines. The results, thus, suggest that delayed secretion of procollagen rather than increased intracellular degradation led to the reduction in the rate of procollagen synthesis in these two fibroblast lines from patients with cerebral artery aneurysm.     

A post-translational modification, unrelated to hydroxylation, in the collagenous domain of nonhelical pro-alpha 2(I) procollagen chains secreted by chemically transformed hamster fibroblasts.

Transformed Syrian hamster embryo (NQT-SHE) fibroblasts do not synthesize the pro-alpha 1 subunit of type I procollagen, but secrete two modified forms of the pro-alpha 2(I) subunit that migrate more slowly than the normal chain during gel electrophoresis (Peterkofsky, B., and Prather, W. (1986) J. Biol. Chem. 261, 16818-16826). By electrophoretic analysis of cyanogen bromide and V8 protease-derived peptides from the collagenous domains of intra- and extracellular pro-alpha 2(I) chains, we find that the modification occurs almost exclusively in secreted molecules, is located in the region spanned by the cyanogen bromide peptide CB3,5, and persists when hydroxylation is inhibited. Thus, modification is due to a post-translational reaction other than hydroxylation. The modified chains appear to be secreted in the denatured state since: 1) helical structures formed at 4 degrees C under acidic conditions were unstable under neutral conditions at 37 degrees C; 2) conditions that destabilize the type I procollagen helix and thus inhibit its secretion, i.e. inhibition of proline hydroxylation or incorporation of the proline analog cis-hydroxyproline, did not affect secretion of the modified chains. The time courses for secretion of nonhelical modified chains from NQT-SHE and of hydroxylated helical procollagen I from control cells, as a proportion of total collagen synthesized, were similar. Although cis-hydroxyproline did not inhibit the secretion of the modified chains, it induced their rapid intracellular degradation.