Chick Embryo Tendon Research Articles

Identification of collagen α1(I) trimer in embryonic chick tendons and calvaria

Collagen with a molecular composition [ α 1(I)] 3 has been identified in acetic acid extracts from lathyritic chick embryo tendons and calvaria. These molecules characteristically have greater solubility than Type I collagen at neutral pH and contain increased amounts of hydroxylysine residues. It is suggested that these molecules represent a separate gene product of embryonic cells which may be important in the process of maturation and development.

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.

Tetramers and Monomers of Prolyl Hydroxylase in Isolated Chick-Embryo Tendon Cells. The Association of Inactive Monomers to Active Tetramers and a Preliminary Characterization of the Intracellular Monomer-Size Protein

In freshly isolated chick-embryo tendon cells about 65% of the prolyl hydroxylase protein is present in the form of active enzyme tetramers, and about 35% in a form corresponding in molecular weight to the enzyme monomers when studied by gel filtration. After incubation of these cells with 0.45 mM dithiothreitol for 30 min, the enzyme activity, the enzyme tetramers as a percentage of total enzyme protein, and the formation of hydroxyl [14C]proline in the cells after a pulse with [14C]proline, all decreased to about 0–5% of their original values. When the cells were incubated for an additional 5 h without dithiothreitol, all these parameters again increased to about 50–60% of their original values, the increases in enzyme activity and percentage of enzyme tetramers being similar irrespective of whether cycloheximide was present during the reversal period. When the cells were incubated for 1 h with a mixture of radioactively-labelled amino acids, the specific activity of the enzyme tetramers was about one fifth of that of the monomer-size protein. During a chase period of up to 6 h, a small increase occurred in the specific activity of the tetramers and a decrease in that of the monomer-size protein, while the specific activity of the total enzyme protein remained essentially unchanged. When the enzyme protein was first labelled for 1 h and then dissociated with dithiothreitol, re-formation of radioactively-labelled tetramers took place, and the specific activity of these tetramers was markedly higher than that of the tetramers originally present. Preliminary characterization of the radioactively-labelled intracellular monomer-size enzyme protein by polyacrylamide gel electro-phoresis in the presence of sodium dodecylsulphate and 2-mercaptoethanol indicated that it consisted of two main polypeptide chains, the molecular weights of which were about the same as those of the enzyme monomers. The data suggest that the monomer-size protein in the cell represents, at least in part, precursors of the enzyme tetramers, and that it can be associated to active tetramers after its ribosomal biosynthesis. It is also suggested that the half-life of prolyl hydroxylase protein in these cells is relatively long, being probably more than one day.

Effect of cortisol acetate on collagen biosynthesis and on the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase in chick-embryo tendon cells

Collagen synthesis and the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase were studied in isolated chick-embryo tendon cells after the administration of cortisol acetate to the chick embryos. When the steroid was injected 1 day before isolation of the tendon cells, collagen synthesis was decreased, even though the enzyme activities were not changed. When cortisol acetate was given as repeated injections over a period of 4 days, both collagen synthesis and the enzyme activities decreased. The hydroxylase activities decreased even more than the two collagen glycosyltransferase activities, both in isolated cells and in whole chick embryos. The amount of prolyl hydroxylase protein diminished to the same extent as the enzyme activity, indicating that cortisol acetate inhibits enzyme synthesis. The inhibitory effect of cortisol acetate on collagen synthesis and on the enzyme activities was partially reversible in 3 days. Total protein synthesis was completely restored within this time. Only massive doses of cortisol acetate inhibited collagen synthesis in vitro. Additional experiments indicated that cortisol acetate did not decrease the rate of the enzyme reactions when added directly to the enzyme incubation mixtures. The results suggest that cortisol acetate decreases collagen synthesis both by its direct effect on collagen polypeptide-chain synthesis and by decreasing the activities of enzymes involved in post-translational modifications.

Purification and characterization of a peptide from the carboxy-terminal region of chick tendon procollagen type I.

A disulfide-bonded peptide with a molecular weight of about 100 000 was isolated from the medium of cultured chick embryo tendons. It was shown to be a trimer with two types of subunits in a 2:1 ratio, and tryptic fingerprinting and immunological evidence indicated that it was derived from the carboxy-terminal-precursor-specific region of procollagen. Amino acid analysis after reduction and alkylation indicated that the trimer contains about 30 residues of half-cystine involved in intrachain as well as interchain disulfide bonding. The interchain bonds could be reduced and alkylated under nondenaturing conditions. Carbohydrate analysis showed that each of the three peptide chains in the trimer contains about two residues of N-acetylglucosamine and about ten residues of mannose. This suggests the presence of one or two oligosaccharide units per chain.

Incorporation of proline analogs into procollagen: Assay for replacement of imino acids by cis-4-hydroxy- l-proline and cis-4-fluoro- l-proline

Previously, several proline analogs have shown to be incorporated into protein and, in particular, into procollagen polypeptides. Here a new technique was used to determine the extent to which two proline analogs, cis-4-hydroxy- l-proline and cis-4-fluoro- l-proline, replaced proline and hydroxyproline in newly synthesized pro-α and pro-γ chains of procollagen. Matrix-free chick embryo tendon cells, when incubated with 1.53 m m, cis-4-hydroxy- l-proline, synthesized collagenous polypeptides in which from 13 to 19% of the total imino acid residues were replaced with the analog. Incubation of cells with 1.50 m m, cis-4-fluoro- l-proline resulted in the synthesis of polypeptides in which 27% of the imino acid residues were replaced by the analog. With lower concentrations, proportionally less of the analog was incorporated into protein. The observations here extend previous indications that proline analogs in relatively low concentrations may have a specific effect on the synthesis of collagen.

Effect of betamethasone-17-valerate on synthesis of collagen and prolyl hydroxylase activity in chick embryo tendon cells

The effect of betamethasone-17-valerate on the biosynthesis of collagen was studied in matrix-free chick embryo tendon cells in vitro and the activity of prolyl hydroxylase was assayed in the cells after incubation with the steroid in vitro and after injection of the steroid on to chorioallantoic membrane of embryonated eggs. Tendon cells synthesized collagen at a rapid rate and the synthesis was essentially linear for up to about 7 hr when studied by labelling with [ 14C]proline. The secretion of collagen [ 14C]hydroxyproline was also almost linear for up to 7 hr. Betamethasone-17-valerate decreased the total incorporation of [ 14C]proline and the decrease in the synthesis of [ 14C]hydroxyproline was larger than the decrease in the total incorporation, indicating that the collagen synthesis was affected more than other protein synthesis. Betamethasone-17-valerate had no effect on the activity of prolyl hydroxylase, when the cells were incubated with this steroid in vitro or, when the activity of the purified enzyme was assayed in the presence of this steroid. When the steroid was injected on to chorioallantoic membrane of embryonated eggs, the activity of prolyl hydroxylase decreased markedly in the tendon cells, indicating that in the system studied the amount of prolyl hydroxylase is decreased or that the enzyme is inactivated by this steroid in vivo.

Prolyl 3‐Hydroxylase: Partial Characterization of the Enzyme from Rat Kidney Cortex

The formation of 3-hydroxyproline was studied with crude rat kidney cortex extract as a source of enzyme and chick embryo tendon protocollagen and procollagen or cartilage protocollagen as a substrate. Synthesis of 3-hydroxyproline was observed with all these substrates and the formation of 3-hydroxyproline ranged up to seven residues per pro-alpha-chain. The highest rate of 3-hydroxylation took place at 20 degrees C and the reaction required Fe2+, O2,2-oxoglutarate and ascorbate. The formation of 3-hydroxyproline was affected by chain length and the conformation of the substrate, in that longer polypeptide chains proved better substrates, while the native triple-helical conformation of protocollagen or procollagen completely prevented the reaction. Formation of 3-hydroxyproline with tendon procollagen as a substrate was not inhibited by antiserum to prolyl 4-hydroxylase or by poly(L-proline) when these substances were used in concentrations which clearly inhibited 4-hydroxyproline formation with tendon protocollagen as a substrate. Furthermore, pure prolyl 4-hydroxylase did not synthesize any 3-hydroxyproline under conditions in which the crude rat kidney cortex enzyme would readily do so. The data thus strongly suggest that prolyl 3-hydroxylase and prolyl 4-hydroxylase are separate enzymes.

Effect of L-azetidine-2-carboxylic acid on glycosylations of collagen in chick-embryo tendon cells.

The glycosylations of hydroxylysine during collagen biosynthesis in isolated chick-embryo tendon cells were studied by using pulse-chase labelling experiments with [14C]-lysine. The hydroxylation of lysine and the glycosylations of hydroxylysine continued after a 5 min pulse label for up to about 10 min during the chase period. These data differ from those obtained previously in isolated chick-embryo cartilage cells, in which, after a similar 5 min pulse label, these reactions continued during the chase period for up to about 20 min. The collagen synthesized by the isolated chick-embryo tendon cells differed markedly from the type I collagen of adult tissues in its degree of hydroxylation of lysine residues and glycosylations of hydroxylysine residues. When the isolated tendon cells were incubated in the presence of L-azetidine-2-carboxylic acid, the degree of glycosylations of hydroxylysine during the first 10 min of the chase period was identical with that in cells incubated without thcarboxylic acid for at least 60 min, whereas no additional glycosylations took place in the control cells after the 10 min time-point. As a consequence, the collagen synthesized in the presence of this compound contained more carbohydrate than did the collagen synthesized by the control cells. Additional experiments indicated that azetidine-2-carboxylic acid did not increase the collagen glycosyltransferase activities in the tendon cells or the rate of glycosylation reactions when added directly to the enzyme incubation mixture. Control experiments with colchicine indicated that the delay in the rate of collagen secretion, which was observed in the presence of azetidine-2-carboxylic acid, did not in itself affect the degree of glycosylations of collagen. The results thus suggest that the increased glycosylations were due to inhibition of the collagen triple-helix formation, which is known to occur in the presence of azetidine-2-carboxylic acid.

Segment-long-spacing aggregates and isolation of COOH-terminal peptides from type I procollagen.

Type I procollagen secreted by matrix-free cells from chick embryo tendons was purified by DEAE-cellulose chromatography. Electron microscopy of segment-long-spacing aggregates of the procollagen demonstrated the presence of both NH2-terminal and COOH-terminal extensions not found in collagen. The procollagen was digested with bacterial collagenase and the COOH-terminal fragments were isolated by gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Analysis of tryptic peptides demonstrated that the COOH-terminal extensions on the pro alpha 1 and pro alpha 2 chains had different primary structures.

Characterization of procollagen synthesized by matrix-free cells isolated from chick embryo tendons

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.

Removal of amino-terminal and carboxy-terminal extension peptides from procollagen during synthesis of chick embryo tendon collagen

Matrix-free chick embryo tendon cells were incubated with [ 14C]proline for 60 minutes and protein synthesis was stopped by the addition of cycloheximide. Newly synthesized collagen precursors recovered in the incubation medium were mostly intact procollagen molecules which contain both amino-terminal and carboxy-terminal extensions. If the cells were further incubated for 2 hours in the presence of cycloheximide, most of the procollagen was converted to precursor molecules which were devoid of amino-terminal extensions. Removal of the carboxy-terminal extensions from procollagen was not observed. Similar experiments with intact tendons demonstrated that procollagen synthesized by the intact tissues in vitro was readily converted to an intermediate form devoid of amino-terminal extensions and then to collagen. The results suggest that the removal of the amino-terminal and carboxy-terminal extensions from procollagen is catalyzed by two separate enzymic activities.

Primary and secondary effects of ascorbate on procollagen synthesis and protein synthesis by primary cultures of tendon fibroblasts

The effects of ascorbic acid on collagen biosynthesis were studied in primary cultures of fibroblasts from chick embryo tendons. Addition of ascorbate to the cultures increased the rate of synthesis of procollagen hydroxyproline, but the effect was not explained by activation of prolyl hydroxylase as has been seen in other cell cultures. Instead the increase in the rate of hydroxyproline synthesis appeared to be the result of some direct cofactor effect of the vitamin. In the presence of ascorbate, most of the newly synthesized procollagen was hydroxylated and became triple helical. In the absence of ascorbate, the overall degree of hydroxylation in newly synthesized procollagen was reduced, but a small fraction of newly synthesized procollagen was near-maximally hydroxylated and became triple helical. When cultures were exposed to ascorbic acid for more than 6 h, there was an increase in rate of protein synthesis, rate of procollagen synthesis, and fraction of membrane-bound ribosomes. The increases in these parameters in the presence of ascorbate appeared to be a secondary effect produced by the accumulation of stable triple-helical procollagen in the culture system.

Carbohydrate moieties of procollagen: incorporation of isotopically labeled mannose and glucosamine into propeptides of procollagen secreted by matrix-free chick embryo tendon cells.

Cells obtained from chick embryo tendons incorporate isotopically labeled glucosamine and mannose into the pro-alpha1 and pro-alpha2 chains of procollagen as judged by sodium dodecyl sulfate-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The label was further localized to the propeptides of pro-alpha1 and pro-alpha2 by its chromatographic behavior after digestion with bacterial collagenase or alpha-chymotrypsin. Carbohydrate analysis of isolated pro-alpha chains showed the presence of labeled galactosamine in addition to mannose and glucosamine. Resistance to mild alkaline hydrolysis suggested that greater than 90% of the oligosaccharide units are not linked to the propeptide backbone by either serine or threonine.

Ascorbate increases the synthesis of procollagen hydroxyproline by cultured fibroblasts from chick embryo tendons without activation or prolyl hydroxylase

An improved procedure was developed to extract prolyl hydroxylase from tendon cells of chick embryos with detergent, and improved assays were developed for both the activity of the enzyme and the amount of enzyme protein. Freshly isolated tendon cells were found to contain approx. 100 μg of enzyme protein per 10 8 cells and 40–50% of the enzyme protein was active. When the cells were cultured, they were found to contain the same amount of enzyme protein by only 15–20% of the enzyme protein was active. Gel filtration of cell extracts indicated that the active form of prolyl hydroxylase in freshly isolated tendon cells and in cultured tendon cells had the same apparent size and the same activity per μg of immunoreactive protein as enzyme which was shown to be a tetramer. The inactive form was found to have about the same apparent size as subunits of the enzyme. When freshly isolated cells were incubated for 2 h in the presence of 40 μg per ml of ascorbate, there was a slight increase in the rate of hydroxyproline synthesis. In cultured cells, ascorbate at a concentration of 40 μg per ml caused a 2-fold increase in the rate of hydroxyproline synthesis within 30 min. However, ascorbate did not increase the activity of prolyl hydroxylase in extracts from either cell system. Therefore it appears that the influence of ascorbate on synthesis of procollagen hydroxyproline by the cells studied here must be ascribed to a cofactor effect on the hydroxylation reaction similar to that observed with purified enzyme, and it does not involve “activation” of inactive enzyme protein to active enzyme as has been observed in cultures of L-929 and 3T6 mouse fibroblasts.

Further characterization of embryonic tendon fibroblasts and the use of immunoferritin techniques to study collagen biosynthesis.

Morphological studies were carried out on fibroblasts from chick embryo tendons, cells which have been used in a number of recent studies on collagen biosynthesis. The cells were relatively rich in endoplasmic reticulum and contained a well-developed Golgi complex comprised of small vesicles, stacked membranes, and large vacuoles. Techniques were then devised for preparing cell fragments which were penetrated by ferritin-antibody conjuates but which retained the essential morphological features of the cells. Finally, the new procedures were employed to develop further information as to how collagen is synthesized. As reported elsewhere, preliminary studies with ferritin-labeled antibodies showed that prolyl hydroxylase was found in the endoplasmic reticulum of freshly isolated fibroblasts and that procollagen is found in both the cisternae of the endoplasmic reticulum and the large Golgi vacuoles. In the experiments described here, the cells were manipulated so that amino acids continued to be incorporated into polypeptide chains but assembly of the molecule was not completed because hydroxylation of prolyl and lysyl residues was prevented. The results indicated that these manipulations produced no change in the distribution of prolyl hydroxylase. Examination of the cells with ferritin conjugated to antibodies which reacted with protocollagen, the unhydroxylated form of procollagen, demonstrated that protocollagen was retained in the cisternae of the endoplasmic reticulum during inhibition of the prolyl and lysyl hydroxylases. Assays for prolyl hydroxylase with an immunologic technique demonstrated that although the enzyme is found within the endoplasmic reticulum, it is not secreted along with procollagen. The observations provided further evidence for a special role for prolyl hydroxylase in the control of collagen biosynthesis.

Antibodies to Chick‐Tendon Procollagen

Antisera were prepared by immunizing a rabbit with procollagen synthesized and secreted by cells from chick embryo tendons. Specific antibodies were then purified from the antisera by using an immunoadsorbent which contained the isolated NH2‐terminal extensions of procollagen. The purified antibodies were shown to react specifically with intact procollagen as well as with procollagen in which the interchain disulfide bonds were ruptured by reduction under non‐denaturing conditions. The antibodies also reacted with the pro‐α1 chain but not the pro‐α2 chain isolated from the procollagen. There was no reaction after the intrachain bonds in the pro‐α chains of the procollagen were reduced under denaturing conditions and alkylated.In the course of characterizing the antibodies it was shown that the NH2‐terminal extensions on the two pro‐α1 chains and the one pro‐α2 chain of type I procollagen are non‐identical. Also, it was shown that the serum of chick embryos contains an antigen which reacts with the specific antibodies to procollagen.

Effects of Temperature on Conformation, Hydroxylation, and Secretion of Chick Tendon Procollagen

Abstract When freshly isolated chick embryo tendon fibroblasts were incubated at 37° with [14C]proline and 0.5 mm α,α'-dipyridyl, a ferrous iron chelator which inhibits prolyl hydroxylase, the labeled unhydroxylated procollagen molecules accumulated intracellularly. When pepsin digestion at 15° was used as an enzymatic probe of conformation, these molecules were shown to be non-triple helical, indicating that hydroxylation of proline is necessary for triple helix formation at normal body temperatures. When cells which had accumulated unhydroxylated procollagen at 37° were then incubated at 25°, large amounts of unhydroxylated procollagen were secreted into the medium. Although these secreted molecules were sensitive to pepsin they were shown to be largely triple helical when prolyl hydroxylase was used as another probe of collagen conformation. The retained molecules were shown to be largely non-triple helical by this same test. This result suggests that triple helix formation may be necessary for normal secretion of procollagen by these cells. When cells which had accumulated unhydroxylated procollagen at 37° were then incubated at different temperatures between 20° and 37° in the presence of excess ferrous iron to reverse the inhibition of prolyl hydroxylase, the accumulated molecules were secreted and the extent of hydroxyproline formation increased as the temperature during the reversal period increased. At 20° less than 1% of the [14C]proline in the secreted molecules was hydroxylated while at 37° this value increased to 33%. This result implies that under some conditions the body temperature of an organism may in part regulate the hydroxyproline content of the collagen molecules.

Ferritin-conjugated antibodies used for labeling of organelles involved in the cellular synthesis and transport of procollagen.

An improved procedure was used to conjugate ferritin to antibodies specific for the NH(2)-terminal extensions on the precursor form of collagen known as procollagen. The ferritin-antibody conjugates were then used to determine the localization of procollagen in fibroblasts isolated from chick-embryo tendons. Procollagen was found in the cisternae of the endoplasmic reticulum, indicating that the protein passes into this compartment early in its biosynthesis. Specific labeling of large Golgi vacuoles was also observed, suggesting that this compartment is also involved in the secretory process. When cells were incubated with colchicine so that the secretion of procollagen was delayed, there was an increase of large, smooth-surfaced vacuoles in the cells and these vacuoles were labeled with the ferritin-antibody conjugate.

Time lag in the secretion of collagen by matrix-free tendon cells and inhibition of the secretory process by colchicine and vinblastine

Matrix-free cells were isolated from chick embryo tendons and incubated with [ 14Cproline in order to follow the time course for the synthesis and secretion of [ 14C]-collagen. The results indicated a lag of 2–3 min between the time at which the incorporation of [ 14C]proline and the synthesis of peptide-bound [ 14C]hydroxyproline reached steady-state levels. A lag of about 18 min was observed between the initiation of the synthesis of collagen [ 14C]hydroxyproline and the secretion of [ 14C]collagen from the cells into the incubation medium. Colchicine and vinblastine were found to inhibit the rate of secretion of [ 14C]-collagen by about 70% and with appropriate concentrations of these agents the amount of intracellular [ 14C]collagen increased about 2-fold in 2 h. The results suggested that the secretion of collagen depends at least in part on the intensity of microtubules. Addition of dibutyryl cyclic AMP to the incubation medium produced a small increase in the fraction of total [ 14C]collagen which was secreted into the medium by the cells in 1 h.