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.     

Pleomorphism in type I collagen fibrils produced by persistence of the procollagen N-propeptide

The assembly of type I collagen and type I pN-collagen was studied in vitro using a system for generating these molecules enzymatically from their immediate biosynthetic precursors. Collagen generated by C-proteinase digestion of pC-collagen formed D-periodically banded fibrils that were essentially cylindrical (i.e. circular in cross-section). In contrast, pN-collagen generated by C-proteinase digestion of procollagen formed thin, sheet-like structures that were axially D-periodic in longitudinal section, of varying lateral widths (up to several microns) and uniform in thickness (approximately 8 nm). Mixtures of collagen and pN-collagen assembled to form a variety of pleomorphic fibrils. With increasing pN-collagen content, fibril cross-sections were progressively distorted from circular to lobulated to thin and branched structures. Some of these structures were similar to fibrils observed in certain heritable disorders of connective tissue where N-terminal procollagen processing is defective. The observations are considered in terms of the hypothesis that the N-propeptides are preferentially located on the surface of a growing assembly. The implications for normal diameter control of collagen fibrils in vivo are discussed.     

SLRP interaction can protect collagen fibrils from cleavage by collagenases

Decorin, fibromodulin and lumican are small leucine-rich repeat proteoglycans (SLRPs) which interact with the surface of collagen fibrils. Together with other molecules they form a coat on the fibril surface which could impede the access to collagenolytic proteinases. To address this hypothesis, fibrils of type I or type II collagen were formed in vitro and treated with either collagenase-1 (MMP1) or collagenase-3 (MMP13). The fibrils were either treated directly or following incubation in the presence of the recombinant SLRPs. The susceptibility of the uncoated and SLRP-coated fibrils to collagenase cleavage was assessed by SDS/PAGE. Interaction with either recombinant decorin, fibromodulin or lumican results in decreased collagenase cleavage of both fibril types. Thus SLRP interaction can help protect collagen fibrils from cleavage by collagenases.     

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.     

Localization of pN-type IIA procollagen on adult bovine vitreous collagen fibrils.

Type II procollagen is synthesized in long (type IIA) and short (type IIB) forms because of alternative splicing of mRNA; the long form containing an additional cysteine-rich domain in the amino-propeptide. An antiserum (IIA) that recognizes this domain was used for immunolocalization studies on adult bovine vitreous at light and electron microscopic levels and for Western blot analyses. The immunolocalization studies revealed labelling by the IIA antiserum of the vitreous collagen fibrils. This labelling was removed by prior extraction of the fibrils with 6 M guanidine hydrochloride (GuHCl) and the extract was shown to contain pN-type IIA procollagen. Adult vitreous collagen fibrils are coated with pN-type IIA procollagen, a finding with potential implications for vitreous collagen fibril structure and function.     

Active negative control of collagen fibrillogenesis in vivo. Intracellular cleavage of the type I procollagen propeptides in tendon fibroblasts without intracellular fibrils

It is established fact that type I collagen spontaneously self-assembles in vitro in the absence of cells or other macromolecules. Whether or not this is the situation in vivo was unknown. Recent evidence shows that intracellular cleavage of procollagen (the soluble precursor of collagen) to collagen can occur in embryonic tendon cells in vivo, and when this occurs, intracellular collagen fibrils are observed. A cause-and-effect relationship between intracellular collagen and intracellular fibrils was not established. Here we show that intracellular cleavage of procollagen to collagen occurs in postnatal murine tendon cells in situ. Pulse-chase analyses showed cleavage of procollagen to collagen via its two propeptide-retained intermediates. Furthermore, immunoelectron microscopy, using an antibody that recognizes the triple helical domain of collagen, shows collagen molecules in large-diameter transport compartments close to the plasma membrane. However, neither intracellular fibrils nor fibripositors (collagen fibril-containing plasma membrane protrusions) were observed. The results show that intracellular collagen occurs in murine tendon in the absence of intracellular fibrillogenesis and fibripositor formation. Furthermore, the results show that murine postnatal tendon cells have a high capacity to prevent intracellular collagen fibrillogenesis.     

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.     

Stabilization of collagen fibrils by hydroxyproline

The substitution of hydroxyproline for proline in position Y of the repeating Gly-X-Y tripeptide sequence of collagen-like poly(tripeptide)s (i.e., in the position in which Hyp occurs naturally) is predicted to enhance the stability of aggregates of triple helices, while the substitution of Hyp in position X (where no Hyp occurs naturally) is predicted to decrease the stability of aggregates. Earlier conformational energy computations have indicated that two triple helices composed of poly(Gly-Pro-Pro) polypeptide chains pack preferentially with a nearly parallel orientation of the helix axes [Nemethy, G., & Scheraga, H.A. (1984) Biopolymers 23, 2781-2799]. Conformational energy computations reported here indicate that the same packing arrangement is preferred for the packing of two poly(Gly-Pro-Hyp) triple helices. The OH groups of the Hyp residues can be accommodated in the space between the two packed triple helices without any steric hindrance. They actually contribute about 1.9 kcal/mol per Gly-Pro-Hyp tripeptide to the packing energy, as a result of the formation of weak hydrogen bonds and other favorable noncovalent interatomic interactions. On the other hand, the substitution of Hyp in position X weakens the packing by about 1.7 kcal/mol per Gly-Hyp-Pro tripeptide. Numerous published experimental studies have established that Hyp in position Y stabilizes an isolated triple helix relative to dissociated random coils, while Hyp in position X has the opposite effect. We propose that Hyp in position Y also enhances the stability of the assembly of collagen into microfibrils while, in position X, it decreases this stability.     

Thermodynamic studies of the assembly in vitro of native collagen fibrils

Measurements of the solubility of calf-skin tropocollagen in neutral phosphate buffers in the temperature range 20-37 degrees C show that native collagen fibril formation is an endothermic process made thermodynamically favourable by a large positive entropy of precipitation associated with structural changes in the surrounding solvent. The effect of inorganic ions and small solute molecules on precipitation seems to be correlated with their structural effects on liquid water. Heterogeneity in the precipitation properties of the collagen solutions may be related to changes in the configurational entropy of the macromolecules due to intramolecular cross-linking.     

Reconstituted collagen fibrils. Fibrillar and molecular stability of the collagen upon maturation in vitro.

During the maturation in vitro of reconstituted collagen fibrils prepared from rat skin, the mechanical and thermal stability of collagen increased and the pepsin-solubility decreased. At the same time a larger fraction of the pepsin-soluble collagen attained a lower molecular thermal stability that resulted in a biphasic thermal transition of the soluble collagen. Type-I collagen, with a similar biphasic thermal transition, was isolated from acid-insoluble rat skin collagen.     

Crystalline regions in collagen fibrils

A new image processing technique, content-dependent anisotropic spatial frequency filtering, has been developed to visualize the location and orientation of crystalline regions in collagen fibril cross-sections. The results show that most crystalline regions are oriented with their approximately 4 nm periodicity directed radially from the fibril centre. This periodicity corresponds to the separation between rows of molecular ends in the quasi-hexagonal molecular packing scheme. The extent of crystallinity increases with radius and frequently the lattice is either continuously distorted or interrupted by sharp discontinuities.     

Oblique banding pattern in collagen fibrils reconstituted in vitro after trypsin treatment.

Collagen fibres from rat tail tendon suspended in small pieces in a solution (pH 7.8) containing 0.5 M CaCl2 were treated with purified bovine trypsin at 20 degrees C for 20 h. After the enzyme treatment collagen from this solution was precipitated out and reconstituted in vitro into native-type fibrils. The banding pattern in these reconstituted fibrils was found to be oblique. This is comparable to that observed recently in fibrils reconstituted from cartilage collagen. On the other hand, normal transverse banding pattern was observed in the fibrils reconstituted in vitro from collagen solution of rat tail tendon which was not pre-treated with trypsin. No significant change was, however, observed in the segment long spacing fibrils precipitated from the enzyme-treated collagen solution. It is possible that the enzyme might affect the mode of organization of tropocollagen molecules during in vitro fibrillogenesis into native-type fibrils either by interacting with the "telopeptide" regions or with the non-collagenous components associated with the native protein and this could probably result into the formation of fibrils with oblique banding pattern.