Possible role and mechanism of action of dissolved calcium in the degradation of bone collagen by lysosomal cathepsins and collagenase.

Equilibrium experiments with bone powder, at pH values ranging from 6.3 to 3.5, show a linear relation between log([Ca2+]/[Ca2+]0) (where [Ca2+]0 = 1 M-Ca2+) and pH, indicating that [Ca2+] could reach levels of 25 mM at pH 5 and 90 mM at pH 4. These elevated Ca2+ concentrations stimulated the lysis of insoluble bone collagen in vitro by purified lysosomes and by mouse bone collagenase, whose activities were additive at acid pH. At neutral pH, the addition of 10-100 mM-CaCl2 did not influence the susceptibility of acid-soluble skin collagen in solution towards bone collagenase, but increased it markedly towards collagen in the fibrillar form. Increasing the [Ca2+] did not influence the susceptibility of collagen to trypsin. Elevated [Ca2+] and a co-operation between lysosomal cysteine proteinases and matrix collagenase could thus participate in the osteoclastic breakdown of bone collagen.     

Cathepsin B1. A lysosomal enzyme that degrades native collagen

1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released (14)C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human alpha(2)-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5-5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an alpha-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.     

Quantitative and metabolic changes of hepatic collagens in rats after carbon tetrachloride poisoning

1. The collagen hydroxyproline in rat liver was composed of 3.5% neutral-soluble collagen, 4.9% acid-soluble collagen and 91.6% insoluble collagen. In labelling studies with [(14)C]proline in vitro, the specific radioactivities of neutral-soluble, acid-soluble and insoluble collagens in rat liver were found to be 233000, 69000 and 830d.p.m./mumol of hydroxyproline respectively after 1h. 2. During subacute carbon tetrachloride poisoning the hepatic content of insoluble collagen markedly increased, whereas those of soluble collagens did not change. During recovery from subacute poisoning hepatic contents of soluble collagens were markedly decreased. 3. After 8 weeks of carbon tetrachloride poisoning the specific radioactivities of hepatic soluble collagens increased, while that of insoluble collagen decreased. During recovery from subacute poisoning, the specific radioactivities of soluble collagens decreased to the normal range and that of insoluble collagen further decreased. 4. Hepatic collagenolytic activity solubilizing insoluble collagen, which differs from mammalian collagenase, decreased under the conditions of the subacute poisoning and also during recovery from subacute poisoning.     

Influence of thyroid hormones on collagen content in tissues of guinea pigs.

Collagen fractions content and level of collagen catabolites in body fluids were determined in normal, hypo- and hyperthyroid guinea pigs. An increase of urinary excretion of hydroxyproline and hydroxylysine as well as concentration of these amino acids in blood serum was found in hyperthyroidism, and a decrease was shown in hyperthyroid guinea pigs. Hyperthyroidism stimulated an increase of neutral-salt-soluble and acid-soluble collagen in skin and liver, and a decrease of insoluble collagen in skin as well as increase of all collagen fractions in bone samples. Hypothyroidism induced a decrease of all collagen fractions in skin and liver, and an increase of acid-soluble and insoluble collagen in bone samples.     

The formation and thermal stability of in vitro assembled fibrils from acid-soluble and pepsin-treated collagens.

The role of the non-helical regions of the collagen molecule in fibrillogenesis has been investigated by comparing the kinetics of fibril formation of pepsin-treated acid-soluble collagen, acid-soluble collagen and mixtures of the two and by comparison of the thermal stabilities of the fibrils formed. The acid-soluble collagen was found to aggregate more rapidly than the pepsin-treated collagen under physiological conditions of pH and ionic strength. Variations in ionic strength, at physiological pH, were found to have differing effects on the aggregation of these two forms of soluble collagen. Fibrils formed from the pepsinized-collagen had a lower thermal stability tha n those formed from the intact collagen. The behavior observed with mixtures of acid-soluble and pepsin-treated collagens was found to be quantitatively consistent with the pepsinized collagen being able to utilize the nuclei formed by the acid-soluble collagen for subsequent growth. However, the use of the acid-soluble nuclei by the pepsinized collagen for growth did not enhance its rate of precipitation during the growth phase, nor did it enhance the thermal stability of the fibrils formed from the pepsinized collagen.     

Interaction between collagen and adenosine 5'-triphosphate.

A quantitative study was made of the adsorption of adenosine 5′-triphosphate (ATP) on collagen by following the change in the absorbance at 258 nm of ATP in the soaking solution. The amount of ATP adsorbed decreased exponentially with the increase of pH up to pH 8 and fell off more rapidly at higher pH values. At a given pH, when the concentration of ATP was increased, the amount of ATP adsorbed increased following the pattern of a Langmuir isotherm. The adsorption was independent of the cation present. The adsorption of adenosine 5′-diphosphate was essentially the same as that for ATP. For tendons deposited with calcium phosphate, the amount of ATP adsorbed decreased compared to natural tendons. The adsorption of ATP on collagen fibers inhibited the contraction caused by calcium chloride, calcium bromide, and lithium bromide. In solution, ATP had very little effect on the denaturation of acid-soluble collagen caused by calcium chloride.     

Synthesis and degradation rates of collagens in vivo in whole skin of rats, studied with 1802 labelling.

Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.     

Synthesis and degradation of collagens in skin of healthy and protein-malnourished rats in vivo, studied by 18O2 labelling.

To explore the effects of growth retardation, caused by restricted protein intake, on collagen turnover in the whole skin, Sprague-Dawley rats (n = 20) were labelled with 18O2 and fed on either an adequate (18%) or a low (3%) lactalbumin diet. Skin biopsies were obtained at intervals during the following 6 months. Independent groups of animals (n = 186) were used to determine the size of the 0.5 M-acetic acid-soluble and -insoluble collagen pools in the entire skin of healthy and malnourished rats. Collagen was estimated by measurement of hydroxyproline. Soluble-collagen synthesis rates were equivalent to 99 +/- 8 mumol of hydroxyproline/day in healthy animals and 11 +/- 2 mumol/day in malnourished rats. Insoluble-collagen synthesis rates were 32 and 5 mumol of hydroxyproline/day in the healthy and protein-depleted rats respectively. The degradation of soluble collagen amounted to 37 +/- 8 and 6 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Efflux of collagen from the soluble collagen, defined as the sum of the rate of soluble collagen that is degraded plus that which matures into insoluble collagen, was 70 +/- 8 and 11 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Insoluble collagen was not degraded in either group. The fraction of soluble collagen leaving the pool that was converted into insoluble collagen was 0.46 in both diet groups. It is concluded that the turnover of soluble collagen is markedly decreased with malnutrition, but degradation and conversion into insoluble collagen account for the same proportions of efflux from the soluble-collagen pool as in rapidly growing rats.     

The self-assembly of collagen molecules

The aggregation of native acid-soluble collagen (N-ASC) and of pronase-treated acid soluble collagen (P-ASC) was examined in solution under conditions which varied from those of minimum collagen-collagen interaction to those leading to incipient fiber formation. Molecular weights and weight distributions were determined in the analytical ultracentrifuge using the Yphantis high speed sedimentation equilibrium and Aarchiblad approach-to-equilibrim techniques. The aggregation was pH and ionic strength dependent in each case. Under conditions of minimum aggregation (low pH, low ionic strength), N-ASC showed the presence of permant aggregates. At higher pH and ionic strength, a higher fraction of aggregate was formed but these were of the same charcter and molecular weight as the permanent aggregates. The aggregates were of a single molecular size, with a weight of 1.5 × 10⁶ daltons, compared with a monomer collagen weight of 3.1 × 10⁵ daltons. The P-ASC formed aggregates also but to a much lower extent and the maximum aggregate size corresponded to dimers in molecular weight. These data show the major importance of molecular end-regions in collagen aggregation to form native type fibers and, by virtue of the discrete size of the N-ASC aggregates, support the microfibrillar hypothesis for the assembly of collagen fibrills.     

Interaction of collagen molecules from the aspect of fibril formation: acid-soluble, alkali-treated, and MMP1-digested fragments of type I collagen.

Collagen type I extracted with acid or digested with pepsin forms fibrils under physiological conditions, but this ability is lost when the collagen is treated with alkaline solution or digested with matrix metalloproteinase 1 (MMP1). When acid-soluble collagen was incubated with alkali-treated collagen, the fibril formation of acid-soluble collagen was inhibited. At 37 degrees C, at which alkali-treated collagen is denatured, the lag time was prolonged but the growth rate of fibrils was not affected. At 30 degrees C, at which the triple helical conformation of alkali-treated collagen is retained, the lag time was prolonged and the growth rate reduced. Heat-denatured alkali-treated collagen and MMP1-digested fragments have no inhibitory effect on the fibril formation of acid-soluble collagen. This means that the triple helical conformation and the molecular length are important factors in the interaction of collagen molecules and that alkali-treated collagen acts as a competitive inhibitor for fibril formation of collagen. We found that alkali-treated collagen and MMP1-digested fragments form fibrils that lack the D periodic banding pattern and twisted morphology under acidic conditions at the appropriate ionic strength. We also calculated the relative strengths of hydrophobic and electrostatic interactions between collagen molecules. When the hydrophobic interaction between linear collagen molecules was considered, we found a pattern of periodic maximization of the interactive force including the D period. On the other hand, the electrostatic interaction did not show the periodic pattern, but the overall interaction score affected fibril formation.     

Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.     

Liquid crystallinity in collagen solutions and magnetic orientation of collagen fibrils

Studies of the optical birefringence of solutions of acid-soluble collagen from rat-tail tendon at 22°C in the pH range 1.0–6.0 show that collagen exhibits an isotropic to mesophase transition only between pH 2.4 and 3.0 at 10% weight concentration. Such liquid crystalline order is probably essential for the orientation of collagen in a magnetic field. When solutions of neutral salt-soluble collagen were precipitated at pH 7.0 by warming to 37°C (“heat gelling”) in a magnetic field of ca. 20 kG, the resulting fibrils wee oriented perpendicular to the direction of the field. Heat gelling is shown to be a useful technique for maintaining the orientation induced in precursor solutions even after the sample is removed from the magnetic field.     

The effect of semicarbazide on the nature and stability of collagen fibrils

Semicarbazide affected both the final width and stability of fibrils reconstituted from solutions of acid-soluble collagen. Fibril width was increased after semicarbazide treatment at pH2.6 and 4.3, whereas after treatment at pH8.9 it decreased. Fibril stability was decreased after semicarbazide treatment at all values of pH and temperature, indicating the inhibition of intermolecular cross-linking. A direct binding of semicarbazide to the alphabeta-unsaturated aldehyde groups in the intramolecular cross-link was demonstrated.     

The action of cathepsin B and collagenolytic cathepsin in the degradation of collagen.

Cathepsin B and collagenolytic cathepsin were obtained from bovine spleen and human placenta and identified as thiol proteinases. Both enzymes degraded insoluble fibrous collagen maximally at pH 3.5 and soluble monomeric collagen near pH 4.5. The response to activators and inhibitors was similar for both enzymes. Collagenolytic cathepsin was unable to degrade the synthetic substrates of cathepsin B and was also shown to differ in its physico-chemical properties. Minor differences were noted in the action of these cathepsins on insoluble fibrous collagen from different tissues. It was concluded that the rate and extent of the dissolution of fibrous collagen was determined by the number and location of the interchain cross-links, the amount of the associated non-collagenous components and the type of solvent ions, but not by the collagen phenotype.     

The induction of cross-links in soluble rat skin collagen in vitro

1. Starch-gel electrophoresis was used to investigate the subunit composition of salt-soluble and acid-soluble rat skin collagen. 2. Cross-linkage of collagen subunits in vitro was performed (a) when in fibrillar form and (b) when in solution. In the former case the increase in number of cross-links appeared to be predominantly intermolecular and in the latter case predominantly intramolecular.     

Changes induced by ozone and ultraviolet light in type I collagen. Bovine Achilles tendon collagen versus rat tail tendon collagen

High-molecular-mass aggregates were made soluble from insoluble collagens of bovine Achilles tendon and rat tail tendon by limited thermal hydrolysis. These polymeric collagen aggregates were cross-linked by 390-nm-fluorescent 3-hydroxy-pyridinium residues (excited at 325 nm) in the former tendon and by unknown non-fluorescent residues in the latter. With the solubilized insoluble-collagens from both tendons, as well as with acid-soluble collagen from rat tail tendon, other 350-385-nm fluorescence intensities (excited at 300 nm) were found to be higher in monomeric chains than in dimeric and polymeric chains. Low levels of ozone inhibited fibril formation of acid-soluble collagen particularly from young rat tail tendon, reacting with tyrosine residues and the 350-385-nm fluorophores. Aldehyde groups, involved in cross-linking, were not effectively modified by ozone. beta-Components (alpha-chain dimers) were not efficiently dissociated even by higher doses of ozone compared to gamma-components (alpha-chain trimers). Polymeric chain aggregates from bovine Achilles tendon collagen, whose 3-hydroxy-pyridinium cross-links are cleaved by ozone, were more readily dissociated by ozone than those from rat tail tendon collagen. Ultraviolet (300-nm) light, which destroyed the 350-385-nm fluorophores, inhibited fibril formation less effectively than ultraviolet (275-nm) light, which is absorbed by tyrosine residues, and did not dissociate collagen polymers from rat tail tendon. On the other hand, ultraviolet (320-nm) light, absorbed by 3-hydroxy-pyridinium cross-links which were rapidly photolyzed, partially dissociated polymeric collagen aggregates from bovine Achilles tendon after subsequent heating.     

Effects of dietary xylitol on collagen content and glycosylation in healthy and diabetic rats

The effects of three-month dietary xylitol supplementation on the amounts and hexose contents of acid-soluble collagen as well as on the amounts and fluorescence of collagenase-soluble collagen were studied in healthy and streptozotocin-diabetic male rats. Collagen was extracted from skin samples. In the healthy rats, supplementation with xylitol (10%) increased the hydroxyproline content of the acid-soluble fraction and skin thickness. In diabetic rats receiving and not receiving xylitol, the acid-soluble collagen fraction was markedly lower than in healthy rats. However, its amount was significantly elevated when xylitol had been added to the diet. Supplementation with xylitol caused no changes in the amounts of collagenase-soluble fraction in either healthy or diabetic rats. Supplementation with xylitol (10%) significantly decreased the hexose content of acid-soluble collagen and the fluorescence of the collagenase-soluble fraction in both healthy and diabetic rats. The results indicate that dietary xylitol affects collagen synthesis and collagen glycosylation.     

Alkali-treated collagen retained the triple helical conformation and the ligand activity for the cell adhesion via alpha2beta1 integrin

Alkaline treatment is a good method for extracting collagen with high recovery even from an aged animal specimen. However, the properties of collagen treated under alkaline conditions have not been well established yet. By the treatment with a solution of 3% sodium hydroxide and 1.9% monomethylamine, the isoelectric point of type I collagen was lowered from 9.3 to 4.8 because of the conversions of Asn and Gln to Asp and Glu. With the acidification of the pI, the denaturation temperature of the collagen was decreased from 42 to 35 degrees C after 20 d treatment, but the collagen-specific triple helical conformation was maintained. Human keratinocytes and fibroblasts adhered to the alkali-treated collagen via the collagen receptor integrin alpha2beta1. This indicates that the alkali-treated collagen maintained its property as a biological adherent molecule. Unlike acid-soluble collagen, alkali-treated collagen lost the ability to form fibrils at neutral pH under physiological conditions. This ability was lost even after 4 h of alkaline treatment, when the denaturation temperature of the collagen did not change. On the other hand, the alkali-treated collagen formed a fibrous precipitate with a uniform diameter of 50-70 nm under acidic conditions at 30 degrees C.     

Observations on the different substrate behavior of tropocollagen molecules in solution and intermolecularly cross-linked tropocollagen within insoluble polymeric collagen fibrils.

Bacterial collagenase was used to compare the extent of digestion of tropocollagen monomers in solution and in reconstituted fibrils with that of tropocollagen molecules intermolecularly cross-linked within insoluble polymeric collagen fibrils obtained from mature tendons at given time-intervals. The extent of digestion of tropocollagen monomers in solution was directly proportional to the enzyme concentration (a range of enzyme substrate molar ratios 1:200 to 1:10 was used). The extent of digestion of polymeric collagen was followed by measuring the solubilization of fluorescent peptides from fluorescent-labelled insoluble polymeric collagen fibrils. The extent of digestion of tropocollagen within polymeric collagen was linear over a very small range of enzyme concentrations, when the enzyme/substrate ratio in the reaction mixture was less than 1:400 on a molecular basis. The behavior of tropocollagen in the form of reconstituted collagen fibrils, which had been matured at 37 degrees C for 8 weeks, was intermediate between the behaviour of solutions of tropocollagen and insoluble polymeric collagen fibrils. The significance of the results is discussed in terms of the structure of polymeric collagen fibrils and the protection against enzymic attack provided by tropocollagen molecules on the circumference of the fibril. The results suggest that assays of collagenase activities based on tropocollagen as substrate cannot be directly related to the ability of these enzymes to degrade mature insoluble collagen fibrils.     

Biosynthesis of skin collagens in normal and diabetic mice.

Synthesis of collagens in vitro was studied on minced mouse skins incubated with [3H]-proline in organ-culture conditions. A comparative study was carried out on genetically diabetic mice (KK strain) and control mice (Swiss strain). After incubation, neutral-salt-soluble and acid-soluble collagens were extracted. The insoluble dermis was digested by pepsin and type I and type III collagens separated by differential precipitation in neutral salt solutions. Type I and Type III collagens were characterized by ion-exchange and molecular-sieve chromatography, amino acid analysis and by the characterization of CNBr peptides. In diabetic-mouse skin, the relative proportion of type III collagen was significantly higher than in control-mouse skin. The incorporation of radioactively labelled proline into hydroxyproline of type III collagen was significantly faster in diabetic-mouse skin than in control-mouse skin.No significant modifications in the total collagen content of the skin or of their rates of synthesis were observed between the two strains. Alteration in the ratio of type III to type I collagen in the diabetic-mouse skin can be interpreted as a sign of alteration of the regulation of collagen biosynthesis and may be related to the structural alterations observed in the diabetic intercellular matrix.