Comparative analysis of the structure and thermal stability of sea urchin peristome and rat tail tendon collagen

We have purified collagen from two distinct sources; the vertebrate, rat tail tendon and an invertebrate, sea urchin adult tissue, the peristome. The collagenous nature of the purification products was confirmed by amino acid compositional analysis. Both preparations had high contents of glycine and proline residues and hydroxyproline was also present. The total pyrrolidine (proline+hydroxyproline) content decreased from 17.9 mole% in rat tail collagen to 12.9 mole% in peristome collagen. Distinctly different circular dichroic spectra were measured for these collagens. Analyses of spectra, measured as a function of temperature, revealed distinct thermal denaturation profiles. The melting temperature for rat tail collagen was 38.5 degrees C, while the corresponding value for peristome collagen was significantly lower at 27 degrees C. A similar thermal denaturation profile was obtained for rat tail collagen in digestion experiments using a 41-kDa gelatinase activity, isolated from sea urchin eggs. These results identify structural differences between a typical, vertebrate type I fibrillar collagen and an echinoderm collagen which serves as a constituent of a mutable connective tissue. These differences may relate to the functional roles played by collagen in these distinctly different tissues.     

Hydroxyproline content determines the denaturation temperature of chick tendon collagen

A series of nine procollagen samples in which the hydroxyproline content varied from <1% to 44% of the total imino acids was prepared by incubating embryonic chick tendon cells with varying concentrations of α,α′-dipyridyl, an inhibitor of proline hydroxylase. The thermal stability of these procollagen preparations was then investigated by using pepsin digestion at different temperatures as an enzymatic probe of conformation. Using this technique, the denaturation temperature of the procollagen was found to be directly proportional to the hydroxyproline content. A denaturation temperature of 23.5 °C was found for the unhydroxylated procollagen and 37.9 °C for fully hydroxylated procollagen. These results suggest that hydroxyproline is crucial to the thermal stability of the collagen triple helix. They also imply that unhydroxylated molecules are not triple helical within the cell at 37 °C and that triple helix formation may be necessary for normal secretion.     

Influence of bleomycin on the metabolism of dermal collagen in albino rats

The metabolism of collagen in male rats by treatment with bleomycin was studied following the injection of [3H]proline and the determination of specific and total activity of [3H]hydroxyproline in skin collagen fractions and urine. In the case of the bleomycin-treated animals, there was found to be an increase in the neutral salt soluble collagen content with no change in insoluble collagen content as compared to the control group. The specific and total radioactivity of [3H]hydroxyproline in soluble and insoluble collagen fractions was also increased. Examination of [3H]hydroxyproline activity in soluble and insoluble collagen showed that the conversion of soluble to insoluble collagen was improved by the bleomycin-treated group. It was found that this was accompanied by a decrease in urinary excretion of total hydroxyproline and [3H]hydroxyproline during the first 12 hr after the administration of [3H]proline. Therefore, the results of the present investigation clearly indicate that the maturation of soluble to insoluble collagen is promoted and accompanied by a decrease in the catabolism of soluble collagen in the bleomycin-treated animals. In addition, administration of bleomycin increased the synthesis of collagen.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs. Evidence for the formation and degradation of a partially hydroxylated collagen

1. After the administration of l-[G-(3)H]proline to guinea pigs deprived of ascorbic acid for increasing periods of time, the specific radioactivities of proline and hydroxyproline in skin collagen and aortic elastin were determined at various time-intervals after administration of the labelled compound with a view to studying the formation and degradation of collagen and elastin both deficient in hydroxyproline. 2. As judged from the incorporation of radioactivity into elastin proline, elastin synthesis was not decreased in the ascorbic acid-deficient animals. There was however, a rapid decline in the specific radioactivity of elastin hydroxyproline. The proline/hydroxyproline specific-radioactivity ratio was approx. 1.5:1 after 6 days and 20:1 after 12 days of ascorbic acid deprivation, in contrast with the ratio of 1:1 in controls. The results suggested that the effect of ascorbic acid deficiency on elastin biosynthesis could be regarded as simply an elimination of hydroxylation of elastin proline with the formation and retention of a polymer increasingly deficient in hydroxyproline. 3. Collagen proline and hydroxyproline specific radioactivities were derived from material that was soluble in hot trichloroacetic acid, non-diffusible and collagenase-degradable. In contrast with elastin, there was a rapid decline in the specific radioactivity of proline as well as hydroxyproline in collagen from the ascorbic acid-deficient animals. However, the proline/hydroxyproline specific-radioactivity ratio in all samples from scorbutic animals was consistently slightly above 1:1. The results suggest the appearance in place of collagen, but in rapidly diminishing amounts, of a partially hydroxylated collagen in which the degree of hydroxylation may be decreased only by approx. 10%. 4. Incorporation of radioactivity into the diffusible hydroxyproline in skin remained relatively high despite the rapid decline in the incorporation of radioactivity into collagen. This observation is interpreted as indicative of an increasing degree of degradation of partially hydroxylated collagen to diffusible peptides. An alternative explanation might be that partially hydroxylated peptides are released to an increasing extent from ribosomes before they attain a length at least sufficient to render them non-diffusible. In either case it implies the accumulation in scurvy of low-molecular-weight peptides enriched in proline and deficient in hydroxyproline and could explain the failure to accumulate a high-molecular-weight collagen deficient in hydroxyproline. 5. It is thought, however, that, in addition, an inhibition of ribosomal amino acid incorporation leading to decreased synthesis of partially hydroxylated collagen may also occur, perhaps secondarily to impaired hydroxylation.     

微等离子束对豚鼠皮肤超微结构分析和羟脯氨酸测定

目的观察微等离子束对豚鼠皮肤胶原组织作用效应的组织学和超微结构变化及羟脯氨酸含量测定,探讨微等离子束的作用机理。方法选择15只豚鼠,每只豚鼠背部划分为实验侧和空白对照侧2个等分区域,给予60W/10 kJ微等离子束照射,于作用后即刻、1周后和1月后分别切取实验侧及空白对照部位皮肤行组织病理维多利亚-立春红染色,透射电镜分析和羟脯氨酸检测试剂盒进行含量测定。结果 60 W/10 kJ即刻表现为表皮局灶性出现点阵状改变,部分表皮出现汽化缺失或者坏死变性,真皮浅层胶原组织出现点阵化表现和明显均质化;特殊染色显示微等离子束主要影响真皮胶原纤维,形成局灶性胶原纤维凝集和变性。1周后皮肤浅层胶原组织结构逐渐致密,排列有序,有少量组织细胞。1月后皮肤浅层胶原组织明显增厚,胶原纤维增粗并排列致密,弹力纤维呈局灶性增粗。透射电镜显示微等离子束作用后表皮细胞较完整,细胞间结构正常,但真皮胶原丧失正常结构,细胞结构消失,大量细胞凋亡明显,1月后仍可见少量细胞凋亡的表现但胶原结构逐渐恢复,浅层胶原纤维排列明显致密。羟脯氨酸测定显示微等离子束作用1周后羟脯氨酸含量要高于作用之前,但是差异性不具有统计学意义（P〉0.05）;1月后羟脯氨酸含量要明显高于作用前,差异性具有统计学意义（P〈0.05）。结论微等离子束对豚鼠皮肤胶原组织作用有明显的刺激效应,其主要靶组织为真皮胶原组织,可以明显促进皮肤新生胶原的增生。     

Quantitative study of tissue collagen metabolism

A procedure for the quantification of various parameters of metabolism of collagen in fibrotic mouse liver has been developed. The method involves derivatization of hydroxyproline, a marker of collagen, with dansyl chloride, high-performance liquid chromatography of the derivative on an octadecyl C-18 column, and its detection by fluorescence. This assay improves upon existing procedures in several respects: It extends the analysis so that not only the collagen content of the tissue but also the metabolism of collagen is determined at levels found intracellularly. It is sensitive enough to quantify 0.1-10 nmol of hydroxyproline, and it includes three major amino acids (hydroxyproline, glycine, and proline) of collagen and two assay controls; it generates information on both the purity and quantity of collagen in each assay. The determination of specific activity of intracellular free [14C]proline, which is the precursor of protein-bound hydroxyproline, defines the specific activity of [14C]hydroxyproline of collagen converted from precursor residues of [14C]proline by the action of prolyl hydroxylase. The specific activity of [14C]hydroxyproline can be used for the evaluation of collagen synthesis and secretion and intracellular and extracellular degradation of the newly synthesized and secreted collagen in the tissue. The determination of specific activities of [14C]hydroxyproline and [14C]proline and of the ratio of [14C]hydroxyproline to [14C]proline of newly secreted collagen provides information concerning the extent of hydroxylation of [14C]proline residues of newly synthesized collagen.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs

1. After the administration of labelled proline to guinea pigs deprived of ascorbic acid for 15 days, the dorsal skin was examined 5 days later in an attempt to detect the presence of hydroxyproline-deficient collagen (protocollagen). The extent of incorporation of proline into skin collagens indicated a severe impairment of collagen synthesis. 2. A comparison of proline and hydroxyproline specific radioactivities in diffusible peptides obtained by treatment with collagenase of either purified skin collagens or direct hot-trichloroacetic acid extracts of skin failed to indicate the presence of protocollagen. Possible reasons for this are discussed. 3. The incorporation results did not indicate an inability of normal collagen, i.e. collagen hydroxylated to the normal degree, to cross-link in scurvy. 4. Incorporation of labelled proline into aortic elastin isolated from the same animals did not indicate a decrease in elastin biosynthesis in ascorbic acid deficiency, beyond that attributable to the inanition accompanying the vitamin deficiency. The proline/hydroxyproline specific-radioactivity ratio in elastin from scorbutic guinea pigs was about 6:1 in contrast with the 1:1 ratio in control groups. It is concluded that the formation of elastin hydroxyproline was ascorbate-dependent and that a hydroxyproline-deficient elastin is formed and retained in scurvy. The formation of desmosines was unimpaired in scorbutic animals. 5. Studies with chick embryos confirmed the formation of elastin hydroxyproline from free proline. Incorporation of free hydroxyproline into elastin hydroxyproline was negligible. 6. Digestion of solubilized samples with collagenase indicated that the hydroxyproline in guinea-pig aortic elastin preparations was not derived from contamination by collagen. It is suggested that most if not all of the hydroxyproline in the guinea pig elastin preparations investigated can be considered an integral part of the elastin molecule.     

Hepatic collagen production in the rat is unaffected by methotrexate

Methotrexate (MTX) has been implicated in the pathogenesis of hepatic fibrosis. However, no information exists regarding the effects of MTX on hepatic collagen metabolism. Therefore, we studied the role of MTX in hepatic collagen production in vivo in rats receiving an 8-week course of varying doses of MTX. Twenty-four hours prior to sacrifice animals received an injection of [5-3H]proline. Collagen was extracted with hot trichloroacetic acid and the proteinbound [3H]hydroxyproline was used as a measure of de novo collagen production. The hepatic collagen content was essentially the same in the control and treatment groups in spite of evidence of hepatotoxicity. Similarly, no significant differences were present among the control and MTX-treated groups in the de novo absolute collagen production. In summary, we found no evidence of increased hepatic fibrogenesis in small groups of animals after 8 weeks of treatment with MTX. Data clearly supporting the claim that MTX itself is responsible for hepatic fibrosis are lacking.     

Investigation of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline content in tissues from the Arabian camel (Camelus dromedarius)

This study was conducted to determine the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline (Hyp) in tissues from the Arabian camel (Camelus dromedarius). Results indicated that there were significant differences in the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen Hyp in various tissues (P < 0.01). Camel kidney showed a significantly high concentration of total, free, peptide-bound and protein-bound Hyp and collagen content as compared to other tissues examined (P < 0.01). Kidney also showed a significantly high concentration of soluble collagen Hyp as compared to other tissues examined (P < 0.01). However, the concentration of insoluble collagen Hyp was significantly high in liver when compared to other tissues (P < 0.01). These variations may result from differences in the collagen structure and/or composition in this species.     

High-performance liquid chromatographic quantitation of collagen biosynthesis in explant cultures

The capacity of lung explant cultures to synthesize collagen can be estimated by determining the content of [³H]hydroxyproline in protein following incubation with [³H]proline. The technique requires acid hydrolysis followed by quantitative separation of hydroxyproline from proline for scintillation counting and is often restricted to methods that can accommodate large samples because of relatively low specific radioactivity. A method which is useful for such samples, providing rapid separation of nonderivatized amino acids by ion-exchange HPLC, is described here. The HPLC system employs an HPX-87C cation-exchange column in 10 mm calcium acetate, pH 5.5, at 85°C. Under isocratic conditions hydroxyproline is completely resolved from proline with quantitative recovery of the ³H cpm applied to the column. Large amounts of material, equivalent to at least 150 mg wet wt of lung, can be applied without affecting resolution or recovery, and samples can be injected at intervals as short as 40 min. This method was used to study collagen biosynthesis in a model of pulmonary fibrosis induced in rabbits by the tumor-promoting agent, phorbol myristate acetate (PMA), and provides information concerning total protein synthesis as well as production of collagen. The data show a doubling in the rate of collagen production in lung explants prepared from animals treated with PMA compared with explants from control animals.     

Hydroxyproline content and location in relation to collagen thermal stability.

A new analysis has been made on studies of the influence of imino acid content on the changes of collagen thermal stability (t_m). It is shown that, for the interstitial vertebrate collagens, there is a strict regularity in the changes of t_m depending on hydroxyproline content. No correlation is observed between t_m and proline content. Also, no correlation between t_m and hydroxyproline content is observed for invertebrate and basement membrane collagens. On the basis of the reported data, the dependence of t_m on hydroxyproline content is considered to be not a correlation between t_m and the total content of hydroxyproline, but only as the correlation between t_m and the content of hydroxyproline occurring at the third position in the sequence (Gly-R₂-R₃)_n. The results agree with the idea that the influence exerted by proline and hydroxyproline on the stabilization of the triple helix of collagen is different.     

Effect of intravenously infused dexamethasone on collagen metabolism in skin of merino sheep.

The effects of an 8-day intravenous infusion of dexamethasone (7.6 mg kg-0.75 body weight) on collagen biosynthesis and wool growth in skin were examined in four Merino wethers. Plasma dexamethasone concentrations reached their peaks during the first 24 h infusion, which were followed by relatively stable levels (c. 1 X 10(-7) M) for the next 4--5 days. Minor increases in plasma dexamethasone levels were recorded during the final 2 days of treatment. Dexamethasone concentrations quickly fell below the level of detection once infusion ceased. Marked decreases in the wet weight, thickness and protein content of skin were observed at the end of infusion. DNA content was reduced to 42.4 +/- 4.9% s.e.m. during the first 2 days of treatment, but in the next 4 days of infusion gradually increased to 85.0 +/0 12.5% of controls. The level of collagen (expressed as hydroxyproline content of its acid hydrolysate) was elevated throughout the infusion and then gradually declined in 3 weeks to about 60% of controls. The biosynthesis of collagen measured by the rate of [14C]hydroxyproline formation and the activity of proline, 2-oxoglutarate dioxygenase (EC 1.14.11.2. formerly prolyl hydroxylase) was reduced during the first half of treatment to a greater extent than the rate of [14C]proline incorporation into proteins. Wool growth was reduced by 80.4 +/- 11.6% in the post-infusion period which allowed three sheep out of four to be defleeced. Inhibition of collagen biosynthesis in sheep skin was due initially to a decrease in the activity of proline, 2-oxoglutarate dioxygenase and later to the reduced rate of proline incorporation into proteins. It was also evident that changes in biosynthetic rate of collagen were not reflected in the total level of skin collagen. The extent of wool growth depression in individual animals paralleled the changes in DNA content and the extent of collagen biosynthesis inhibition.     

Silicon deprivation decreases collagen formation in wounds and bone,and ornithine transaminase enzyme activity in liver

We have shown that silicon (Si) deprivation decreases the collagen concentration in bone of 9-wk-old rats. Finding that Si deprivation also affects collagen at different stages in bone development, collagen-forming enzymes, or collagen deposition in other tissues would have implications that Si is important for both wound healing and bone formation. Therefore, 42 rats in experiment 1 and 24 rats in experiment 2 were fed a basal diet containing 2 or 2.6 μg Si/g, respectively, based on ground corn and casein, and supplemented with either 0 or 10 μg Si/g as sodium metasilicate. At 3 wk, the femur was removed from 18 of the 42 rats in experiment 1 for hydroxyproline analysis. A polyvinyl sponge was implanted beneath the skin of the upper back of each of the 24 remaining rats. Sixteen hours before termination and 2 wk after the sponge had been implanted, each rat was given an oral dose of ¹⁴C-proline (1.8 μCi/100 g body wt). The total amount of hydroxyproline was significantly lower in the tibia and sponges taken from Si-deficient animals than Si-supplemented rats. The disintegrations per minute of ¹⁴C-proline were significantly higher in sponge extracts from Si-deficient rats than Si-supplemented rats. Additional evidence of aberrations in proline metabolism with Si deprivation was that liver ornithine aminotransferase was significantly decreased in Si-deprived animals in experiment 2. Findings of an increased accumulation of ¹⁴C-proline and decreased total hydroxyproline in implanted sponges and decreased activity of a key enzyme in proline synthesis (liver ornithine aminotransferase) in Si-deprived animals indicates an aberration in the formation of collagen from proline in sites other than bone that is corrected by Si. This suggests that Si is a nutrient of concern in wound healing as well as bone formation. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area is an equal opportunity/affirmative action employer, and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Stabilization of collagen structure: Dependence of collagen denaturation enthalpy on the imino acid content

An analysis of the available data on the enthalpy (ΔH_r) of denaturation (melting) of collagens with different imino acid content in solution and in the aggregated state has shown that ΔH_r in solution increases with increasing denaturation temperature, whereas in the aggregated state there is an inverse dependence. ΔH_r in solution correlates with the hydroxyproline content but not with that of proline. No correlation between the change of ΔH_r and the imino acid content is observed for the aggregated state.     

Oestradiol inhibits collagen breakdown in the involuting rat uterus

1. The earlier observation (Woessner, 1969) of oestradiol inhibition of collagen breakdown is confirmed and extended. Administration of 100mug of oestradiol-17beta/day to parturient rats strongly inhibits the loss of collagen from the involuting uterus. Three experiments show that this effect is due to an inhibition of collagen degradation rather than to a stimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[(14)C]-proline by the administration of [(14)C]proline near the end of pregnancy. By 3 days post partum, control uteri lost 83% of their collagen and 90% of their hydroxy[(14)C]proline. Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxyproline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorporation of [(14)C]proline into uterine collagen hydroxyproline in vivo was not affected by oestradiol treatment. 4. Urinary excretion of hydroxyproline was increased in post-partum control rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucylglycyl- l-prolyl-d-arginine (a substrate for clostridial collagenase) increased in activity in the post-partum uterus and was unaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagen extensively at pH3.2. This digestion was unaffected by the oestradiol treatment. 7. Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenates contained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline. The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradiol treatment.     

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.     

Alterations in myocardial collagen content affect rat papillary muscle function

We investigated the influence of myocardial collagen volume fraction (CVF, %) and hydroxyproline concentration (microg/mg) on rat papillary muscle function. Collagen excess was obtained in 10 rats with unilateral renal ischemia for 5 wk followed by 3-wk treatment with ramipril (20 mg. kg(-1). day(-1)) (RHTR rats; CVF = 3.83 +/- 0. 80, hydroxyproline = 3.79 +/- 0.50). Collagen degradation was induced by double infusion of oxidized glutathione (GSSG rats; CVF = 2.45 +/- 0.52, hydroxyproline = 2.85 +/- 0.18). Nine untreated rats were used as controls (CFV = 3.04 +/- 0.58, hydroxyproline = 3.21 +/- 0.30). Active stiffness (AS; g. cm(-2). %L(max)(-1)) and myocyte cross-sectional area (MA; micrometer(2)) were increased in the GSSG rats compared with controls [AS 5.86 vs. 3.96 (P < 0.05); MA 363 +/- 59 vs. 305 +/- 28 (P < 0.05)]. In GSSG and RHTR groups the passive tension-length curves were shifted downwards, indicating decreased passive stiffness, and upwards, indicating increased passive stiffness, respectively. Decreased collagen content induced by GSSG is related to myocyte hypertrophy, decreased passive stiffness, and increased AS, and increased collagen concentration causes myocardial diastolic dysfunction with no effect on systolic function.     

Evidence for the role of 4-hydroxyproline localized in the third position of the triplet (Gly-X-Y) in adaptational changes of thermostability of a collagen molecule and collagen fibrils

An analysis of the available data on the thermostability and imino acid content of various collagens has shown that the change of the denaturation temperature (t_m) of the collagen triple helix, as well as the temperature of hydrothermic shrinkage (t_s) of collagen fibrils, depends on the number of hydroxyproline residues localized in the third position of the collagen triplet. This change does not depend on the content of proline and 3- and 4-hydroxyproline localized in the second position of the triplet. Empiric equations have been obtained connecting t_m and t_s with the content of 4-hydroxyproline. The results of the analysis are in good agreement with one of the collagen structure models recently proposed by the Ramachandran school.