Copolymerization of normal type I collagen with three mutated type I collagens containing substitutions of cysteine at different glycine positions in the alpha 1 (I) chain.

Previous observations with type I collagen from a proband with lethal osteogenesis imperfecta demonstrated that type I collagen containing a substitution of cysteine for glycine alpha 1-748 copolymerized with normal type I collagen (Kadler, K. E., Torre-Blanco, A., Adachi, E., Vogel, B. E., Hojima, Y., and Prockop, D. J. (1991) Biochemistry 30, 5081-5088). Here, three preparations containing normal type I procollagen and type I procollagen with a substitution of cysteine for glycine alpha 1-175, glycine alpha 1-691, or glycine alpha 1-988 were purified from cultured skin fibroblasts from probands with osteogenesis imperfecta. The procollagens were then used as substrates in a system for assaying the self-assembly of type I collagen into fibrils. The cysteine-substituted collagens in all three preparations were incorporated into fibrils. The cysteine alpha 1-175 and cysteine alpha 1-691 collagens were shown to increase the lag time and decrease the propagation rate constant for fibril assembly. All three preparations containing cysteine-substituted collagens formed fibrils with diameters that were two to four times the diameter of fibrils formed under the same conditions by normal type I collagen. Also, the three preparations containing cysteine substituted collagens had higher solubilities than normal type I collagen. The results, therefore, demonstrated that the three cysteine-substituted collagens copolymerized with normal type I collagen. The effects of the mutated collagens on fibril assembly can be understood in terms of a recently proposed model of fibril growth from symmetrical tips by assuming that the mutated monomers partially inhibit tip growth but not lateral growth of the fibrils. Of special interest was the observation that the Cys alpha 1-175 collagen from a proband with a non-lethal variant of osteogenesis imperfecta had quantitatively less effect on several parameters of fibril assembly at 37 degrees C than cysteine-substituted collagens from three probands with lethal variants of the disease.     

Assembly of collagen fibrils de novo by cleavage of the type I pC-collagen with procollagen C-proteinase. Assay of critical concentration demonstrates that collagen self-assembly is a classical example of an entropy-driven process

Type I procollagen was purified from the medium of cultured human fibroblasts incubated with 14C-labeled amino acids, the NH2-terminal propeptides were cleaved with procollagen N-proteinase, and the resulting pC-collagen was isolated by gel filtration chromatography. pC-collagen did not assemble into fibrils or large aggregates even at concentrations of 0.5 mg.ml-1 at 34 degrees C in a physiological buffer. However, cleavage of pC-collagen to collagen with purified C-proteinase (Hojima, Y., (1985) J. Biol. Chem. 260, 15996-16003) generated fibrils that were visible by eye and that were large enough to be separated from solution by centrifugation at 13,000 x g for 4 min. With high concentrations of enzyme, the pC-collagen was completely cleaved in 1 h, and turbidity was near maximal in 3 h, but collagen continued to be incorporated in fibrils for over 10 h. Because the pC-collagen was uniformly labeled with 14C-aminoacids, the concentration of soluble collagen and, therefore, the critical concentration of polymerization were determined directly. The critical concentration was independent of the initial pC-collagen concentration and of the rate of cleavage. The critical concentration decreased with temperature between 29 and 41 degrees C and was 0.12 +/- 0.06 (S.E.) microgram.ml-1 at 41 degrees C. The thermodynamic parameters of assembly were essentially independent of temperature in the range 29 to 41 degrees C. The process was endothermic with a delta H value of +56 kcal.mol-1, but entropy driven with a delta S value of +220 cal.K-1.mol-1. The Gibbs energy change for polymerization was -13 kcal.mol-1 at 37 degrees C. The data demonstrate, for the first time, that type I collagen fibril formation de novo is a classical example of an entropy-driven self-assembly process similar to the polymerization of actin, flagella, and tobacco mosaic virus protein.     

Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases

A new system was developed for studying the assembly of collagen fibrils in vitro. A partially purified enzyme preparation containing both procollagen N-proteinase and c-proteinase (EC 3.4.24.00) activities was used to initiate fibril formation by removal of the N- and C-propeptides from type I procollagen in a physiological buffer at 35-37 degrees C. The kinetics of fibril formation were similar to those observed for fibril formation with tissue-extracted collagen in the same buffer system, except that the lag phase was longer. The longer lag phase was in part accounted for by the time required to convert procollagen to collagen. Similar results were obtained when an intermediate containing the C-propeptide but not the N-propeptide was used as a substrate. Therefore, removal of the c-propeptide appeared to be the critical step for fibril formation under the conditions used here. The fibrils formed by enzymic cleavage of procollagen or pCcollagen appeared microscopically to be more tightly packed than fibrils formed directly from collagen under the same conditions. This impression was confirmed by the observation that the fibrils formed by cleavage of procollagen were stable to temperatures 1.5-2 degrees C higher than fibers formed from extracted collagen under the same conditions. When smaller amounts of procollagen proteinase were used, the rate of cleavage of procollagen to collagen was markedly reduced. The fibrils which formed under these conditions were up to 3 micrometers in diameter. Some appeared to contain branch points.     

Temperature-induced post-translational over-modification of type I procollagen. Effects of over-modification of the protein on the rate of cleavage by procollagen N-proteinase and on self-assembly of collagen into fibrils.

Previous observations suggested that incubating fibroblasts at elevated temperature caused over-modification of type I procollagen by post-translational enzymes because of a delay in folding of the collagen triple helix. Here, human skin fibroblasts were incubated at 40.5 instead of 37 degrees C, and the type I procollagen secreted into the medium was isolated. Analysis of the protein indicated that there was an increase of about 5 residues of hydroxylysine/alpha chain and about 1 residue of glycosylated hydroxylysine/alpha chain. Assays with procollagen N-proteinase indicated that the N-propeptide of the over-modified collagen was cleaved at a decreased rate, apparently because the over-modification altered the conformation-dependent cleavage site for the enzyme. Assays in a system for assembly of collagen into fibrils demonstrated that the over-modified protein had a higher critical concentration for self-assembly. Also, the fibrils formed from the over-modified collagen at 31 and 29 degrees C had smaller diameters than fibrils formed from normal type I collagen. The results provide direct evidence for earlier suggestions that post-translational over-modification of a fibrillar collagen can alter the morphology of the fibrils formed. The results also indicate that some of the biological consequences of the mutations in type I procollagen causing heritable disorders must be ascribed to the effects of post-translational over-modifications that frequently occur as secondary consequences of changes in the primary structure of the protein.     

Mechanism of in vitro collagen fibril assembly. Kinetic and morphological studies

The kinetics of in vitro fibril assembly of Type I collagen preparations that contain different amounts of covalently cross-linked oligomers was studied with turbidimetry. Fibril formation showed a lag phase with no solution turbidity and a growth phase with a sigmoidal increase in the solution turbidity. The length of the lag phase was inversely related to both the total collagen concentration and the amount of covalently cross-linked oligomers in the solution. Double logarithmic plots of t1/4, the amount of time it takes for 1/4 of the collagen to assemble into fibrils, versus the total collagen concentration were linear but the slope decreased from -0.84 to -2.3 with decreasing amounts of covalently cross-linked oligomers in the samples. Electron microscopy showed the formation of unbanded microfibrils with diameters in the range of 3-15 nm early in the lag phase and larger diameter banded fibrils coexisting with the microfibrils near the end of the lag phase. Centrifugation of the solution at the lag phase prolonged the lag time, presumably by removal of microfibrils, but subsequent growth of the fibrils was unaffected. The results suggest a cooperative nucleation-growth mechanism for the in vitro assembly of collagen fibrils which is consistent with the results of an equilibrium study of the fibril assembly reaction we reported earlier (Na, G. C., Butz, L. J., Bailey, D. G., and Carroll, R. J. (1986) Biochemistry 25, 958-966).     

FTIRS in H2O demonstrates that collagen monomers undergo a conformational transition prior to thermal self-assembly in vitro

The assembly of type I collagen molecules into native fibrils can be accomplished in vitro in solutions at physiological ionic strength and pH by raising the temperature above 30 degrees C. The thermal self-assembly reaction exhibits a distinct lag phase. This lag phase has been proposed to be evidence for a conformational transition in the monomer. Fourier transform infrared spectroscopy (FTIRS) is a very sensitive probe of the H-bonded states within the triple helix. The carbonyl group spectrum (amide I, 1700-1600 cm-1) has been investigated in collagen/H2O solutions at 1 mg/mL under self-assembly conditions from 4 to 34 degrees C and, in the same range, at a higher ionic strength where self-assembly does not occur. The deconvoluted spectra show three very clear bands at approximately 1660, 1644, and 1630 cm-1. These bands vary in both frequency maxima and relative intensity over the temperature range examined. Spectra were also obtained in the amide II and III regions. Spectral changes were evident in the 22-26 degrees C range, under fibril-forming conditions, which lead to the hypothesis that the triple helix of the semiflexible collagen molecule is actually perfected during the lag phase, facilitating nucleation and intermolecular interaction. Further spectral changes after fibrils do form show that the molecules are once again distorted as they are bent to fit within the fibrils.     

Collagen fibrils in vitro grow from pointed tips in the C- to N-terminal direction.

Growth of collagen fibrils was examined in a system in which collagen monomers are generated by specific enzymic cleavage of type IpCcollagen with procollagen C-proteinase. Fibrils formed at 37 degrees C had highly tapered and symmetrical pointed tips. The pattern of cross-striations in the pointed tips indicated that all the molecules were oriented so that the N-termini were directed towards the tip. At 29 degrees C and 32 degrees C, the fibrils formed were thicker. One end of fibrils formed at 29 degrees C was blunt, and the other was pointed. Growth of the fibrils was exclusively from pointed tips. Occasionally a spear-like projection appeared at a blunted end. The spear-like projection then became a new pointed tip for growth in the opposite direction. The results suggested a model for fibril growth with at least three distinct binding sites for monomers. In the model, the pointed tip is the site with the highest affinity for the binding of monomers and most probably defines the critical concentration for fibril assembly. The main shaft of the fibril is a site with very low affinity for binding. The blunted end defines a low-affinity binding site where monomers can bind in opposite orientation to produce growth from a new pointed end.     

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.     

Thermodynamic and structural characteristics of collagen fibrils formed in vitro at different temperatures and concentrations

Analysis of the results of calorimetric study of reconstituted collagen (type I) fibrils, in particular, the half-width of the temperature transition, shows that the collagen packing density in the fibrils and the size of cooperative blocks therein depend on the assembly temperature and on the initial collagen concentration. The least dense fibrils are formed at subphysiological temperatures (25° or 30°C) and low concentration (0.3 mg/ml). The extent of ordering does not change upon doubling the concentration but increases upon quadrupling it. At physiological temperature (35°C) the fibrils are densely packed regardless of collagen concentration. The enthalpy of fibril assembly is minimal at 35°C, 1.2 mg/ml, and ionic strength of 0.17 M. The influence of temperature on particular steps of fibrillogenesis and the role of water in these processes are discussed.     

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.     

Monomer and oligomer of type I collagen: molecular properties and fibril assembly

Type I collagen purified from calf skin was further separated into monomeric and oligomeric fractions and characterized with gel electrophoresis and measurement of solution viscosity. The thermal stabilities of the triple-helical structure of the collagen molecules of these preparations and the fibrils assembled therefrom were determined with differential UV spectroscopy and scanning microcalorimetry. The monomeric collagen was reduced with NaBH4-, and the kinetics and equilibrium of the reversible fibril assembly-disassembly were examined in detail. Fibril assembly and disassembly of the collagen induced by slow scans of temperature showed hysteresis. The assembly curve was very sharp whereas the disassembly curve was gradual. Equilibrium centrifugation showed the collagen disassembled from the fibrils to be predominantly monomers. However, unlike the unassembled collagen, the collagen disassembled from fibrils by cooling showed no lag phase in subsequent cycles of fibril assembly. The thermodynamic parameters of fibril growth were derived from a fibril disassembly curve. Fibril growth was weaker for the NaBH4-reduced monomeric collagen than the native crude collagen, perhaps due to the removal of oligomers and the changes in the molecular structure brought by the reduction. The results corroborated the strongly cooperative mechanism for the fibril assembly proposed in the preceding paper.     

Turbidimetric and morphological studies of type I collagen fibre self assembly in vitro and the influence of fibronectin

Collagen undergoes several stages of self assembly including turbidimetric lag, growth and plateau steps. The later stages of type I collagen self assembly were studied by turbidity—time measurements, low angle laser light scattering and by determination of the birefringence retardation of collagen fibres formed in vitro. These studies were conducted in the presence and absence of fibronectin to evaluate the effect of fibronectin on the kinetics and extent of type I collagen fibrillogenesis. The results of these studies indicate that the collagen fibres observed at the end of the lag phase appear to be identical to fibres seen in the growth phase of turbidity—time curves based on fibre diameter and birefringence retardation measurements. Birefringence retardation measurements suggest that the diffracting unit may be the collagen fibril and that the volume fraction of fibrils in fibres is about 0.95 using a model developed for a series of parallel ellipsoids. Morphological observations suggest that the distribution of fibre diameters formed in vitro during the growth phase is narrow and appears to be independent of time with only the mass of collagen in fibres increasing during the growth phase. During the growth phase, layers of parallel fibres are formed with alternating layers appearing almost orthogonal. In the presence of fibronectin the mechanism of fibre formation appeared unchanged. It was concluded that fibronectin appeared to modify the kinetics of self assembly by preventing collisions between collagen molecules.     

The C-terminal extrahelical peptide of type I collagen and its role in fibrillogenesis in vitro

The formation in vitro of fibrils from type I acid-soluble calf skin collagen has been studied before and after removal of the extrahelical peptides with carboxypeptidase and with pepsin. Turbidimetric studies show that the mechanism of fibril growth in undigested collagen is similar to that in pepsin-digested collagen; following carboxypeptidase digestion, however, a different growth mechanism was apparent. The two mechanisms have been further characterized by electron microscopy. In the course of formation of fibrils from undigested collagen, “early fibrils” (short D-periodic fibrils that have both ends visible) occurred in the lag phase under the precipitating conditions employed here. After pepsin or carboxypeptidase digestion of the collagen no “early fibrils” were seen. In carboxypeptidase-digested collagen, lateral assembly was inhibited; after pepsin digestion, linear assembly was inhibited. Complete removal of the extrahelical peptides prevented fibril formation under the conditions used here. Electron-optical examination of segment-long-spacing (SLS) dimers established a more complete removal of the C-terminal peptide after carboxypeptidase digestion than after pepsin digestion. Analyses of staining patterns of SLS dimers and fibrils from undigested and digested samples showed that the C-terminal peptide in SLS crystallites and fibrils formed from undigested collagen is in a condensed conformation. A proposed conformation, in which condensation occurs predominantly in a hydrophobic region at the proximal end of the C-terminal peptide, is discussed in terms of a dual role for the C-terminal peptide in fibrillogenesis. One role, shared with the N-terminal peptide, is to participate in interactions between the 4D-staggered molecules leading to the formation of linear aggregates; the other is to participate in interactions between these linear aggregates giving rise to D-periodic aggregates and lateral (as well as linear) growth.     

Thermodynamic characteristics of collagen fibrils reconstructed in vitro at different temperatures and concentrations

The results of a calorimetric study of type I collagen fibrillogenesis were analyzed. The dependence of the half-width of the temperature transition of a collagen solution on the concentration and temperature of collagen formation was studied. It was demonstrated that, by varying temperature and collagen concentration, one can regulate the density of packing and dimensions of cooperative fibril blocks. At temperatures below the physiological level (25 degrees C and 30 degrees C), and a relatively low concentration of collagen (0.3 mg/ml), fibrils with the lowest density of packing are formed. The degree of order does not change as the collagen concentration increases twofold but grows as the concentration increases fourfold. It was shown that, at the physiological temperature (35 degrees C), fibrils with a dense packing of molecules are formed at all collagen concentrations studied. The value of fibril formation enthalpy is minimal at a temperature of 35 degrees C, pH 7.2, an ionic strength of 0.17 M and a concentration of 1.2 mg/ml. Based on the results obtained, a conclusion was made that the packing density of fibrils formed at physiological temperature does not depend on collagen concentration over the concentration range of 0.3 - 1.2 mg/ml.     

In vitro culture decreases the expression of TGF(beta), Hsp47 and type I procollagen and increases the expression of CTGF in avian tendon explants

Weight-bearing tendons in many species, including humans, chickens and horses, are prone to failure, in many cases without a discernible cause. The normal function of the tendon depends on the proper assembly of fibrils of type I collagen, the main structural component of the tendon. We studied the effect of in vitro culture, temperature (37 degrees C vs. 43 degrees C) and wounding on the expression of mRNAs for several collagen regulators, transforming growth factor beta (TGF(beta)), heat shock protein 47 (Hsp47) and connective tissue growth factor (CTGF), in chicken embryonic gastrocnemius tendon explants. The expression of mRNAs for TGF(beta) and Hsp47, a chaperone of collagen assembly, remained strong during the first day of in vitro culture, but then it decreased, slightly more at higher temperature. Additional injury in selected tendons had no significant effect on the levels of TGF(beta) and Hsp47 mRNAs. Likewise, the level of immunostained type I procollagen also decreased with the length of culture. The expression of CTGF gradually increased from 0 at the time of tendon removal with the duration of culture to strong after three days of culture when the expression of TGF(beta) and Hsp47 was low. We conclude that in vitro culture over the period of several days rather than an increase in temperature or additional wounding decreases the expression of TGF(beta), Hsp47 and type I procollagen and increases the expression of CTGF.     

Self-assembly of collagen type I molecules without telopeptides

The effect of temperature on the kinetics of formation of fibrils from rat tail collagen molecules devoid of telopeptides was studied. It was shown that the rats of fibril formation at 30 and 35 degrees C increases five- and eightfold, respectively, as compared with that at 25 degrees C. It was found that enthalpy of fibril denaturation at 30 degrees C is maximal for the collagen both with intact telopeptides and devoid of telopeptides. It was found that essential for the fibrilogenesis of type I collagen devoid of telopeptides are temperatures of 30 and 35 degrees C.     

Formation of collagen type I fibrils in vitro

The assembly of collagen fibrils as a function of temperature and collagen concentration was studied. It was shown that temperature increases from 25 to 35 degrees C, the degree of ordering of collagen fibrils increases 1.5-fold at collagen concentration above 1 mg/ml and 2-fold at low collagen concentration. A maximum ordering of fibril structure occurs under conditions close to physiological (T approximately 35 degrees C and collagen concentration 1.2 mg/ml). As temperature is elevated from 30 to 35 degrees C, the packing of collagen molecules in fibrils becomes more ordered: the values of enthalpy and entropy of the transition of fibrils from the native to a disordered state decrease at all collagen concentrations used. At high collagen concentration, the dimensions of cooperative blocks in fibrils formed at 25 and 30 degrees C coincide with those of cooperative blocks of monomeric collagen in solution. Upon increasing the temperature to 35 degrees C, the dimensions of cooperative blocks increase.     

Binding of soluble type I collagen to fibroblasts: effects of thermal activation of ligand, ligand concentration, pinocytosis, and cytoskeletal modifiers

Efficient binding of native, soluble 125I-labeled type I rat collagen to mouse 3T3 fibroblast monolayers requires prior warming of the ligand to 35-37 degrees C for 10-30 min. Decreased binding at high ligand concentrations is ascribed to ligand-ligand interactions rather than to negative cooperativity. Addition of bacterial collagenase to monolayers labeled with the 125I-ligand releases a constant fraction (80%) of the bound ligand over a 2-h interval at 37 degrees C, indicating that little of the ligand becomes inaccessible by pinocytosis. Colchicine (10(-7) M) and vinblastine (5 X 10(-8) M) do not inhibit binding by morphologically intact monolayers. Cytochalasins and concanavalin A show dose-related inhibition of binding by intact monolayers that is due to a reduction in the number of available binding sites rather than to a change in binding site affinity. The collagen binding site on the fibroblast surface is proposed as an organizing center for the assembly of periodic type I collagen fibrils.     

Conformational stability of type I collagen triple helix: evidence for temporary and local relaxation of the protein conformation using a proteolytic probe

Native collagen polypeptides exist in a unique triple helical conformation resistant to most proteinases. In this study, the stability of type I collagen triple helix, employing a mixture of trypsin and alpha-chymotrypsin as a proteolytic probe, was examined. The degradation of type I [3H]collagen was monitored as 3H-labeled peptides soluble in trichloroacetic acid (TCA) or by sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis. In one set of experiments, collagen substrates were preincubated at various temperatures for up to 8 h, followed by a 15-min proteolytic treatment at the same temperature. At 43 degrees C, most of the collagen was degraded, while the fraction of the substrate degraded at 40, 38, and 35 degrees C was 53, 41 and 19%, respectively. This fraction was independent of the preincubation time which varied from 10 to 480 min. Thus, at any given temperature, a constant fraction of the collagen substrate was susceptible to proteolysis. Measurement of the midpoint temperature (Tm) of the helix to coil transformation for type I collagen, at neutral pH employing an increasing temperature gradient and brief proteolysis at the individual temperatures, indicated a value of 38.8 degrees C. However, determination of the Tm by employing proteolytic digestions at a constant temperature (30 degrees C) using conditions under which the nonhelical peptides are readily digested to TCA-soluble peptides while native collagen resists such proteolysis, indicated a value of 42.7 degrees C. In further studies, collagen was subjected to continuous proteolysis for up to 24 h. A large fraction of collagen was digested at 30 or 34 degrees C, temperatures well below the Tm of the helix to coil transformation. SDS-polyacrylamide gel electrophoresis of the degradation products obtained at these temperatures revealed multiple cleavage fragments. Finally, temperature double-jump experiments indicated that the destabilization of the triple helix is reversible provided that the Tm of the substrate is not exceeded. The results provide evidence for reversible and local relaxation of the collagen triple helix.