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. 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.     

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.     

Electron microscopic and biochemical characterization of collagen in blattarian insects

The occurrence of collagen in the cockroach Leucophaea maderae has been demonstrated by electron optical and biochemical techniques. Electron micrographs of tissues of this and a related species (Blaberus craniifer) are presented and they show that collagenous-type fibers occur in the stroma of nonneural as well as neural organs of these insects. Hydroxyproline and hydroxylysine, amino acids considered to be "markers" for collagen, have been shown to be present in proteins extracted from material rich in neuroglandular tissue (corpus cardiacum plus corpus allatum). Trimmed carcasses of Leucophaea maderae have been shown to contain a protein or proteins soluble in hot trichloroacetic acid, with compositional characteristics similar to those of collagens in general, including diagnostic proportions of glycine, proline, hydroxyproline, and hydroxylysine. This collagen is not soluble in dilute acetic acid or in concentrated solutions of guanidinium chloride. It is measurably digested by bacterial collagenase.     

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.     

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.     

Proline and hydroxyproline metabolism: implications for animal and human nutrition

Proline plays important roles in protein synthesis and structure, metabolism (particularly the synthesis of arginine, polyamines, and glutamate via pyrroline-5-carboxylate), and nutrition, as well as wound healing, antioxidative reactions, and immune responses. On a per-gram basis, proline plus hydroxyproline are most abundant in collagen and milk proteins, and requirements of proline for whole-body protein synthesis are the greatest among all amino acids. Therefore, physiological needs for proline are particularly high during the life cycle. While most mammals (including humans and pigs) can synthesize proline from arginine and glutamine/glutamate, rates of endogenous synthesis are inadequate for neonates, birds, and fish. Thus, work with young pigs (a widely used animal model for studying infant nutrition) has shown that supplementing 0.0, 0.35, 0.7, 1.05, 1.4, and 2.1% proline to a proline-free chemically defined diet containing 0.48% arginine and 2% glutamate dose dependently improved daily growth rate and feed efficiency while reducing concentrations of urea in plasma. Additionally, maximal growth performance of chickens depended on at least 0.8% proline in the diet. Likewise, dietary supplementation with 0.07, 0.14, and 0.28% hydroxyproline (a metabolite of proline) to a plant protein-based diet enhanced weight gains of salmon. Based on its regulatory roles in cellular biochemistry, proline can be considered as a functional amino acid for mammalian, avian, and aquatic species. Further research is warranted to develop effective strategies of dietary supplementation with proline or hydroxyproline to benefit health, growth, and development of animals and humans.     

Effects of Dietary Supplementation with Fish Scales-Derived Collagen Peptides on Skin Parameters and Condition: A Randomized,Placebo-Controlled,Double-Blind Study

Fish scales-derived collagen peptides (CPs) are characterized by their specific amino acid composition with a high concentration of glycine, proline and hydroxyproline. These amino acids have been known to exert beneficial effects on human skin. The aim of the present study was to examine the effects of collagen peptides obtained from fish scales on changes in periorbital wrinkles, facial skin hydration, and skin elasticity in healthy women aged 30–60 years. In the present randomized, placebo-controlled, double-blind trial, 71 subjects consumed a 20 mL beverage containing 3000 mg of CPs or placebo once per day over 12 weeks. Significant decreases in periorbital wrinkles (p?<?0.05) were observed in the treatment group after 12 weeks of CPs ingestion compared to the control group. This study also demonstrated a consistent trend of enhanced facial skin moisture (p?<?0.001) and skin elasticity (p?<?0.001) by dietary intake of CPs without any side effects or adverse events. These findings indicate that fish-derived CPs hold great promise as a natural supplement with cutaneous anti-aging properties.     

Insoluble collagen of methylcholanthrene induced sarcoma

The insoluble collagen from methylcholanthrene induced sarcoma was isolated and characterized. It contains more glycine, hydroxyproline and acidic amino acids than normal connective tissue collagen. An anionic character of tumour collagen was stated (pI 6.1). No typical collagen subunits in this protein were found. The tumour collagen is strongly bound to acidic glycoprotein containing a significant amount of hydroxylysine. Such an insoluble complex is resistant to the dispersing action of EDTA. It dissociates during heating in concentrated urea.     

Structural aspects of hydroxyproline-containing proteins

The occurrence of hydroxyproline (Hyp) in collagen, C1q and acetylcholineesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the beta-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific beta-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to C1q and acetylcholineesterase both of which share the general structural and functional properties of collagen in their "tail" regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of C1q and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.     

Characterization of Acid- and Pepsin-Soluble Collagens from the Cuticle of <Emphasis Type="Italic">Perinereis nuntia</Emphasis> (Savigny)

To extend the practical applications of collagen, alternatives to mammalian sources are needed. In this study, acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from the cuticle of Perinereis nuntia (Savigny), and their physicochemical features and structures were examined. The yields of ASC and PSC were 3.89% and 6.74%, respectively. The glycine contents of both collagens accounted for approximately one-third of the total amino acid residues, and the sum totals of proline and hydroxyproline in ASC and PSC were 212 and 214 residues/1000 residues, respectively. However, the proline hydroxylation rates of ASC and PSC were 84.0% and 83.6%, respectively. The maximum absorption peaks of both ASC and PSC were detected at 233 nm. Zeta potential studies indicated that ASC and PSC have a net zero charge at pH 4.89 and 4.95, respectively. Fourier-transform infrared spectroscopy, circular dichroism, and X-ray diffraction confirmed the triple helical structure of the collagen. The denaturation temperatures (T_d) of ASC and PSC were 36.5 °C and 33 °C, respectively. Moreover, the collagens appeared to be loose, fibrous, and porous by scanning electron microscopy. These results suggested that collagen from the cuticle of Perinereis nuntia (Savigny) has potential commercial applications in the food, nutraceutical, and pharmaceutical industries.     

Collagen structure: the Madras triple helix and the current scenario

This year marks the 50th anniversary of the coiled-coil triple helical structure of collagen, first proposed by Ramachandran's group from Madras. The structure is unique among the protein secondary structures in that it requires a very specific tripeptide sequence repeat, with glycine being mandatory at every third position and readily accommodates the imino acids proline/hydroxyproline, at the other two positions. The original structure was postulated to be stabilized by two interchain hydrogen bonds, per tripeptide. Subsequent modeling studies suggested that the triple helix is stabilized by one direct inter chain hydrogen bond as well as water mediated hydrogen bonds. The hydroxyproline residues were also implicated to play an important role in stabilizing the collagen fibres. Several high resolution crystal structures of oligopeptides related to collagen have been determined in the last ten years. Stability of synthetic mimics of collagen has also been extensively studied. These have confirmed the essential correctness of the coiled-coil triple helical structure of collagen, as well as the role of water and hydroxyproline residues, but also indicated additional sequence-dependent features. This review discusses some of these recent results and their implications for collagen fiber formation.     

On the Chemical Composition and Developmental Role of the Mesoglea of Hydra

Two aspects of the chemistry and function of the mesoglea ofhydra were studied, (i) Chemical composition: Its componentneutral sugars and amino acids were analyzed to determine ifthis structure contains collagen, (ii) Role in morphogenesis:Hydra were exposed to the lathyrogen 3-aminopropionitrile, aninhibitor of collagen crosslinking, to discover if new cross-linkedmesoglea is required for normal regeneration to occur. Large amounts of the neutral sugars glucose and galactose andsmall amounts of fucose and rhamnose were found, as was theamino sugar glucosamine. Evidence that the major protein componentof the mesoglea is collagen was revealed by the detection ofhydroxylysine, hydroxyproline, and proline, and of large amountsof glycine. The glucose and galactose are joined as a dimerby an alkali-stable bond to the polypeptide backbone of thecollagen. The lathyrogen 3-aminopropionitrile, known to block the assemblyof newly synthesized collagen into fibers, causes abnormal headregeneration in hydra. The drug has its maximum effect between24 and 48 hr after the previous head is removed.     

Effect of amino acids on the growth ofAcetobacter suboxydans

Summary Published disagreements between amino acid growth requirements and the reported ability of extracts to synthesize amino acids prompted a nutritional study onAcetobacter suboxydans ATCC strain 621. A chemically defined medium containing no amino acids supported cellular growth. Growth was stimulated by the addition of either glutamate, glutamine, -ketoglutarate, proline, histidine, and to a lesser extent by the addition of either glycine, hydroxyproline, or mesaconate.     

Collagen binding specificity of the discoidin domain receptors: Binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1

The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I-III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.     

Application of high-pressure liquid chromatography to studies of collagen production by isolated cells in culture

Techniques for assessing collagen production by cells in culture are usually based on evaluation of uptake of radiolabeled proline into collagen. Although simple in theory, this approach is often flawed because of uncertainties concerning the specific activity of labeled proline in the precursor pool for collagen synthesis. An alternative approach is to assess collagen production directly by measuring hydroxyproline in proteins secreted by cultured cells, although this has been difficult, due to the insensitivity of the methods available. Here we apply high-pressure liquid chromatography using reverse-phase elution of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole derivatives of hydroxyproline to measure collagen production by fibroblasts. The method is easy to perform and allows quantitation of hydroxyproline down to 5 pmol, making it applicable to fibroblasts in 12-well culture plates. Collagen production was shown to be constant over a period of 24 h, with a mean rate of 391 +/- 18 (SE n = 14) ng collagen/10(6) cells/h. Similar values were obtained using thin-layer chromatography and an enzyme-linked immunosorbent assay for type I collagen, but these techniques were judged to be less convenient and required additional assumptions compared with the technique described here in full.     

Amino acid synthesis from glucose-U-14C in Glossina morsitans

Amino acid synthesis from glucose-U-¹⁴C was investigated in 2 day post-emergent and pregnant females of Glossina morsitans. This insect can synthesize alanine, aspartic acid, cystine, glutamic acid, glycine, proline, and serine from glucose. Arginine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, threonine, and valine showed no radioactivity and hence may be classified as nutritionally indispensable amino acids. Although tyrosine and hydroxyproline were not synthesized from glucose, they are at least partially dispensable nutrients for this insect because their synthesis from phenylalanine has been demonstrated. After the labelled glucose injection the highest radioactivity was recovered in the proline fraction. This is probably related to its rôle as an important energy reserve for flight. The radioactive amino acids recovered from females and from their offspring following glucose-U-¹⁴C injection were similar to those recovered from younger females. Radioactivity was also detected in the expired CO₂ and the excreta. The amino acids alanine, arginine, cystine, glycine, histidine, leucine/isoleucine, lysine, methionine, proline, and valine were identified in the excreta, of which arginine and histidine were in the largest amounts. Only excreted alanine, glycine, and proline showed radioactivity.     

Characterization of pepsin-solubilized bovine heart-valve collagen.

Collagens extracted from heart valves by using limited pepsin digestion were fractionated by differential salt precipitation. Collagen types were identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, amino acid analysis and cleavage with CNBr. Heart-valve collagen was heterogeneous in nature, consisting of a mixture of type-I and type-III collagens. The identity of type-III collagen was established on the basis of (a) insolubility in 1.7 M-NaC1 at neutral pH, (b) behaviour of this collagen fraction on gel electrophoresis under reducing and non-reducing conditions, (c) amino acid analysis showing a hydroxyproline/proline ratio greater than 1, and (d) profile of CNBr peptides on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showing a peak characteristic for type-III collagen containing peptides alpha1(III)CB8 and alpha1(III)CB3. In addition to types-I and -III collagen, a collagen polypeptide not previously described in heart valves was identified. This polypeptide represented approx. 30% of the collagen fraction precipitated at 4.0 M-NaCl, it migrated between beta- and alpha1-collagen chains on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and its electrophoretic behaviour was not affected by disulphide-bond reduction. All collagen fractions from the heart valves contained increased amounts of hydroxylysine when compared with type-I and -III collagens from other tissues. The presence of beta- and gamma-chains and higher aggregates in pepsin-solubilized collagen indicated that these collagens were highly cross-linked and suggested that some of these cross-links involved the triple-helical regions of the molecule. It is likely that the higher hydroxylysine content of heart-valve collagen is responsible for the high degree of intermolecular cross-linking and may be the result of an adaptive mechanism for the specialized function of these tissues.