The promotion of collagen polymerization by lanthanide and calcium ions.

Ca2+ (1-5 mM) and lanthanide (20-250 microM) ions enhance the rate of polymerization of purified calf skin collagen (1.5 mg/ml) at pH 7.0 in the presence of 30mM-Tris/HCl and 0.2 M-NaCl. Both the nucleation phase and the growth phase of polymerization are accelerated. The activation energy of the growth phase, 239.3 +/- 24.3 kJ/mol (57.2 +/- 5.8 kcal/mol), is decreased to 145.6 +/- 9.6 kJ/mol (34.8 +/- 2.3 kcal/mol) by 5 mM-Ca2+ and to 75.3 +/- 4.6 kJ/mol (18.0 +/- 1.1 kcal/mol) by 25 microM-Sm3+. In contrast, the activation energy of the nucleation phase, 191.6 +/- 23.4 kJ/mol (45.8 +/- 5.6 kcal/mol), is only slightly decreased by Ca2+ or Sm3+. Collagen fibrils formed in the presence of Sm3+ are thinner than control fibrils, and more thermoresistant.     

The mechanism of nucleation for in vitro collagen fibril formation.

The formation of collagen fibrils from soluble monomers and aggregates by thermal gelation at neutral pH can be divided into two distinct stages: a nucleation phase and a growth phase. Turbidity studies of the kinetics of the precipitation reaction show that the lag-phase time or nucleation reaction time, t′_l, is markedly temperature dependent while the growth reaction time is temperature independent. The activation energy of the nucleation reaction is essentially constant over the temperature range studied. In monitoring the nucleation-phase reaction by various physicochemical techniques, including viscosity, sedimentation equilibrium, and light scattering, no evidence for the formation of aggregates was observed. Enrichment of the initial collagen solution with aggregates accelerates nucleation, but de novo nuclei formation is still required even in highly aggregated collagen preparations. Removal of pepsin and pronase susceptible peptides lengthens the nucleation reaction time and increases the sensitivity of the rate of nuclei formation to changes in ionic strength. Electron microscope studies show the fibrils formed from the protease-treated collagen to be less well organized. With pepsin-treated collagen, subfibrils and obliquely striated fibrils are seen, showing that while microfibrils are formed interactions between them are modulated by the enzyme susceptible peptides in the same way that these regions modulate nuclei assembly. It appears that pepsin and pronase susceptible peptide regions of collagen play a more prominent role in the in vitro assembly of collagen molecules to form D-stagger nuclei and fibrils than do ionic interactions between helical molecular regions. A mechanism of nucleation of collagen fibrillogenesis is discussed.     

The effects of dipalmitoyl phosphatidyl choline on the precipitation of native fibrils and segment-long-spacing aggregates from collagen solution

The effect of dipalmitoyl phosphatidyl choline (DPPC), the major phospholipid component of pulmonary surfactant, on the precipitation of collagen in the form of native fibrils and segment-long-spacing (SLS) aggregates was studied in vitro. The effects of DPPC on both phases of collagen fibrillogenesis were analyzed spectrophotometrically, and alterations in the morphology of precipitated fibrils and SLS aggregates were ascertained by transmission electron microscopy (TEM). Low concentrations of DPPC inhibited the growth phase of fibrillogenesis, while higher concentrations were required to inhibit nucleation. Both the meshwork density and mean width of precipitated fibrils were altered by DPPC, as was the size of SLS aggregates. Segment-long-spacing aggregates prepared from pepsin-treated collagen were inhibited to a greater degree than SLS aggregates prepared from untreated collagen, indicating that the pepsin-susceptible residues of the telopeptide extensions of tropocollagen molecules stabilize SLS aggregates against the effects of DPPC. Based on these results and the inhibition of the growth phase at lower concentrations than those which inhibited the nucleation phase of fibrillogenesis, it was concluded that the primary mechanism of DPPC inhibition is electrostatic interference between the positively charged phospholipid molecules and the net positive charge of collagen. It is proposed that pathological conditions involving the pulmonary epithelium may allow interaction between surfactant and collagen, which could further weaken the interstitial connective tissue.     

Type VII collagen is a major structural component of anchoring fibrils

Anchoring fibrils are specialized fibrous structures found in the subbasal lamina underlying epithelia of several external tissues. Based upon their sensitivity to collagenase and the similarity in banding pattern to artificially created segment-long spacing crystallites (SLS) of collagens, several authors have suggested that anchoring fibrils are lateral aggregates of collagenous macromolecules. We recently reported the similarity in length and banding pattern of anchoring fibrils to type VII collagen SLS crystallites. We now report the construction and characterization of a murine monoclonal antibody specific for type VII collagen. The epitope identified by this antibody has been mapped to the carboxyl terminus of the major helical domain of this molecule. The presence of type VII collagen as detected by indirect immunofluorescence in a variety of tissues corresponds exactly with ultrastructural observations of anchoring fibrils. Ultrastructural immunolocalization of type VII collagen using a 5-nm colloidal gold-conjugated second antibody demonstrates metal deposition upon anchoring fibrils at both ends of these structures, as predicted by the location of the epitope on type VII collagen. Type VII collagen is synthesized by primary cultures of amniotic epithelial cells. It is also produced by KB cells (an epidermoid carcinoma cell line) and WISH (a transformed amniotic cell line).     

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.     

The effect of bovine tendon glycoprotein on the formation of fibrils from collagen solutions.

The formation of collagen fibrils under physiological conditions of ionic strength, pH and temperature was markedly affected by the presence of small amounts of bovine tendon glycoprotein. The absorbance of the gels at 400 nm was decreased, and they took longer to form. Over the range of concentration tested, the negative specific absorbance, -delta Asp., and the specific retardation, Rsp., both increased with the glycoprotein to collagen ratio. When added during the nucleation phase, glycoprotein was still able to exert its effect almost fully, and so must act to inhibit the later stages of fibril formation. Several pieces of evidence showed that glycoprotein acts via a weak binding to the collagen molecule. Electron microscopy established that fibrils formed in the presence of glycoprotein had a normal cross-striation pattern, but were significantly thinner than fibrils formed in control gets. The results suggest that glycoprotein could act in tissues to help regulate the diameter of collagen fibrils.     

Lanthanide-mediated DNA hydrolysis

Lanthanide ions are remarkably effective catalysts for the hydrolytic cleavage of phosphate ester bonds, including the robust bonds of DNA. This makes Ln(III) and Ce(IV) ions attractive candidates for developing selective and efficient artificial nucleases, which could have many biochemical and clinical applications. Both small-molecule-based and biopolymer-based lanthanide complexes are being pursued.     

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.     

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.     

Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro

During bone and dentin mineralization, the crystal nucleation and growth processes are considered to be matrix regulated. Osteoblasts and odontoblasts synthesize a polymeric collagenous matrix, which forms a template for apatite initiation and elongation. Coordinated and controlled reaction between type I collagen and bone/dentin-specific noncollagenous proteins are necessary for well defined biogenic crystal formation. However, the process by which collagen surfaces become mineralized is not understood. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein expressed during the initial stages of mineralized matrix formation in bone and dentin. Here we show that DMP1 bound specifically to type I collagen, with the binding region located at the N-telopeptide region of type I collagen. Peptide mapping identified two acidic clusters in DMP1 responsible for interacting with type I collagen. The collagen binding property of these domains was further confirmed by site-directed mutagenesis. Transmission electron microscopy analyses have localized DMP1 in the gap region of the collagen fibrils. Fibrillogenesis assays further demonstrated that DMP1 accelerated the assembly of the collagen fibrils in vitro and also increased the diameter of the reconstituted collagen fibrils. In vitro mineralization studies in the presence of calcium and phosphate ions demonstrated apatite deposition only at the collagen-bound DMP1 sites. Thus specific binding of DMP1 and possibly other noncollagenous proteins on the collagen fibril might be a key step in collagen matrix organization and mineralization.     

Growth of Collagen Fibril Seeds from Embryonic Tendon: Fractured Fibril Ends Nucleate New Tip Growth

Collagen fibrils are the principal tensile element of vertebrate tissues where they occur in the extracellular matrix as spatially organised arrays. A major challenge is to understand how the mechanisms of nucleation, growth and remodelling yield fibrils of tissue-specific diameter and length. Here we have developed a seeding system whereby collagen fibrils were isolated from avian embryonic tendon and added to purified collagen solution, in order to characterise fibril surface nucleation and growth mechanisms. Fragmentation of tendon in liquid nitrogen followed by Dounce homogenisation generated fibril length fragments. Most (> 94%) of the fractured ends of fibrils, which show an abrupt square profile, were found to act as nucleation sites for further growth by molecular accretion. The mechanism of this nucleation and growth process was investigated by transmission electron microscopy, atomic force microscopy and scanning transmission electron microscopy mass mapping. Typically, a single growth spur occurred on the N-terminal end of seed fibrils whilst twin spurs frequently formed on the C-terminal end before merging into a single tip projection. The surface nucleation and growth process generated a smoothly tapered tip that achieved maximum diameter when the axial extension reached ∼ 13 μm. Lateral growth also occurred along the entire length of all seed fibrils that contained tip projections. The data support a model of collagen fibril growth in which the broken ends of fibrils are nucleation sites for propagation in opposite axial directions. The observed fibril growth behaviour has direct relevance to tendon matrix remodelling and repair processes that might involve rupture of collagen fibrils.     

Immunoelectron microscopic studies of type X collagen in endochondral ossification

Immunofluorescence and immunoelectron microscopy were used in conjunction with a monoclonal antibody to investigate the localization of type X collagen in the proximal tibial growth plate of 7-d-old chicks. This molecule was detected throughout the hypertrophic zone first appearing when chondrocytes exhibited hypertrophy: it was absent from the proliferative zone. Type X collagen was primarily associated with type II collagen fibrils as demonstrated by immunogold staining. Type X collagen was not concentrated in the focal calcification sites nor was it associated with matrix vesicles. These observations suggest that type X collagen may play a role other than that directly related to the nucleation of calcification.     

Inhibition, by lanthanides, of neutral proteinases secreted by human, rheumatoid synovium

Fragments of human, rheumatoid synovium were maintained on organ culture for three days under serum-less conditions. Their conditioned media contained collagenolytic, gelatinolytic and caseinolytic activities, which were susceptible to inhibition by lanthanide ions. Of the four lanthanides tested, Sm3+ proved the best inhibitor of gelatinase and caseinase, while La3+ inhibited collagenase the most strongly. Inhibition of collagenase by La3+ was uncompetitive. A direct binding assay confirmed the greater association between collagen fibrils and collagenase in the presence of La3+. Ca2+ was not required for binding of the uninhibited enzyme to collagen, but acted to stabilize collagenase against thermoinactivation.     

Scleraldehyde as a stabilizing agent for collagen scaffold preparation

Schizophyllan is a natural polysaccharide, produced by fungi of the genus Schizophyllum. Periodate oxidation specifically cleaves the vicinal glycols in schizophyllan to form their dialdehyde derivatives. The present study investigates the interaction of scleraldehyde with Type I collagen membrane. The formation of the inter and intra interaction between scleraldehyde and the collagen fibres results in significant increase in viscosity of collagen. Crosslinking efficiency of scleraldehyde was found to increase with concentration of scleraldehyde. Scleraldehyde interacted collagen membrane exhibited an increase in thermal stability by 29 °C at pH 8. The gelling time of collagen fibrils was found to decrease with increase in concentration of scleraldehyde due to shift in nucleation centre. Swelling degree of collagen membrane was also found to decrease with increase in concentration of scleraldehyde. Scleraldehyde treated collagen membrane exhibited 93% resistance to collagenase. The modified collagen membrane exhibited non-toxicity towards the fibroblasts cells. The modified collagen membrane by scleraldehyde finds application as a stabilizing agent in scaffold preparation.     

An electrostatic model for collagen fibrils. The interaction of reconstituted collagen with Ca++, Na+, and Cl−

A model for the electrostatic properties of hydrated collagen fibrils, based on the concept of a “penetrable” protein, has been evaluated through studies of collagen fibrils that had been chemically modified to change their electrostatic properties,. A value of 0.28 ± 0.07 ml/g was found for the intrafibrillar space sterically inaccessible to a molecule that had an equivalent spherical radius of 4.5 Å. The net intrinsic charge on reconstituted collagen is +14 mol/mol under physiological conditions, but decreases, at constant pH, with ionic strength. A value of 7.1 for the pK of the histidine and α-amino groups in reconstituted collagen was obtained through the application of the electrostatic model to this effect. The values obtained for calcium binding parameters for collagen fibrils, under solution conditions in which the nonspecific electrostatic properties of collagen fibrils were eliminated (3–5 M tetramethyl ammonium chloride), were in agreement with values obtained in 0.16 M NaCl solutions calculated through the use of the electrostatic theory. These are 0.73 ± 0.23 and 56.2 ± 12.3 sites per molecule with intrinsic association constants of 1101 ± 386 and 21.4 ± 5.2 M^?1, respectively. The model also predicts that an average 4-mV potential difference exists between the reconstituted collagen fibrils and physiological solutions, and that collagen fibrils under such conditions have piezoelectriclike properties. The pattern of interaction of ions with collagen fibrils is such that an allosteric mechanism for the catalytic step in the mineralization of collagen is a possibility.     

Collagen II containing a Cys substitution for Arg-alpha1-519. Analysis by atomic force microscopy demonstrates that mutated monomers alter the topography of the surface of collagen II fibrils.

A recombinant human procollagen II was prepared that contained a substitution of Cys for Arg at alpha1-519 and that was found in five families with early onset generalized osteoarthritis with or without features of a mild chondrodysplasia. Previously, the presence of mutated monomers in mixtures with wildtype collagen II was shown to increase the lag period for fibril assembly. Also, the fibrils were more loosely packed and some thick fibrils lacked a D-periodic banding pattern. Here we re-examined the fibrils using a combination of transmission electron microscopy and atomic force microscopy. The presence of the mutated monomers increased the diameter of the thin filaments that were consistently formed in association with the thick fibrils of collagen II. In addition, the presence of the mutated monomers increased the depth of the gap regions in all fibrils with a distinct D-periodic banding pattern. The results, therefore, may indicate that the mutated monomers formed two or three additional outer layers of monomers in 0D-period staggers on the surface of the fibrils. Apparently, the mutated monomers were bound on the surface through intermolecular disulfide bonds.     

Spatial organization of collagen in annelid cuticle: order and defects

The epidermis of Paralvinella grasslei (Polychaete, Annelida) is covered by an extracellular matrix, the cuticle, mainly composed as in other annelids of superimposed layers of non-striated collagen fibrils. The collagen fibrils of annelid cuticle are shown to be composed of parallel and sinuous microfibrils (thin sections and freeze-fracture replicas). The 3-dimensional organization of collagen is characterized by 2 different types of geometrical order: (a) Fibrils form a quasiorthogonal network, whose structure is comparable to that of a "plywood"; (b) Fibrils are helical, and goniometric studies show that microfibrils present a definite order within each fibril, which is termed "cylindrical twist". These 2 characteristics are those which have recently been evidenced in "blue phases", i.e., liquid crystals which are closely related to cholesteric liquid crystalline phases. Non-fluid analogues of cholesteric liquids are widespread among invertebrate cuticles and the presence of blue phase analogues suggests that a self-assembly mechanisms is involved in cuticle morpho-genesis, which is derived from that governing blue phase growth. The cuticular network presents local rearrangements of fibrils called "defects", despite the fact that they are elaborate structures which trigonal and pentagonal singularities. Branched fibrils are regularly observed. We discuss the involvement of these pattern disruptions in the cuticle growth process.     

Hierarchical assembly and the onset of banding in fibrous long spacing collagen revealed by atomic force microscopy.

The mechanism of formation of fibrillar collagen with a banding periodicity much greater than the 67 nm of native collagen, i.e. the so-called fibrous long spacing (FLS) collagen, has been speculated upon, but has not been previously studied experimentally from a detailed structural perspective. In vitro, such fibrils, with banding periodicity of approximately 270 nm, may be produced by dialysis of an acidic solution of type I collagen and alpha(1)-acid glycoprotein against deionized water. FLS collagen assembly was investigated by visualization of assembly intermediates that were formed during the course of dialysis using atomic force microscopy. Below pH 4, thin, curly nonbanded fibrils were formed. When the dialysis solution reached approximately pH 4, thin, filamentous structures that showed protrusions spaced at approximately 270 nm were seen. As the pH increased, these protofibrils appeared to associate loosely into larger fibrils with clear approximately 270 nm banding which increased in diameter and compactness, such that by approximately pH 4.6, mature FLS collagen fibrils begin to be observed with increasing frequency. These results suggest that there are aspects of a stepwise process in the formation of FLS collagen, and that the banding pattern arises quite early and very specifically in this process. It is proposed that typical 4D-period staggered microfibril subunits assemble laterally with minimal stagger between adjacent fibrils. alpha(1)-Acid glycoprotein presumably promotes this otherwise abnormal lateral assembly over native-type self-assembly. Cocoon-like fibrils, which are hundreds of nanometers in diameter and 10-20 microm in length, were found to coexist with mature FLS fibrils.     

Three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium phosphate crystals of pickerel (Americanus americanus) and herring (Clupea harengus) bone

High-voltage (1.0 MV) electron microscopy and stereomicroscopy, electron probe microanalysis, electron diffraction and three-dimensional computer reconstruction, have been used to examine the spatial relationship between the inorganic crystals of calcium phosphate and the collagen fibrils of pickerel and herring bone. High-voltage stereo electron-micrographs were obtained of cross-sections of the cylinder-shaped intramuscular bones in uncalcified regions, in regions where only one or only several crystals had been deposited in some of the fibrils, and in successive sections containing progressively more mineral crystals until the stage of full mineralization was reached. High-resolution electron probe microanalysis confirmed that the electron-dense particles contained calcium and phosphorus. In the earliest stages of mineralization and progressing throughout the mineralization process, the crystals are located only within the collagen fibrils; crystals are not observed free in the extracellular spaces between collagen fibrils. The progressive increase in the mass of mineral deposited in the bone tissue with time occurs, essentially, completely within the collagen fibrils including the stage of full mineralization. At this stage, cross-sectional profiles of collagen fibrils are completely obliterated by mineral. A small number of crystals that are located on or close to the surface of the fibrils appear to extend a very short distance into the spaces between the fibrils. These ultrastructural observations of the very onset of calcification in which nucleation of the calcium phosphate crystals is clearly shown to begin within specific volumes of collagen fibrils, and of the subsequent temporal and spatial sequences of this phenomenon, which shows that calcification continues wholly within the collagen fibrils until maximum calcification is achieved, add important information on the basic physical chemical mechanism of the calcification and the structural elements that are involved. The spatial and temporal independence of the sites where mineralization is initiated establishes that such ultrastructural locations within individual collagen fibrils represent independent, physical chemical nucleation loci. The findings are totally inconsistent with the proposal that crystals must first be deposited in matrix vesicles, or other components such as mitochondria, and subsequently released and propagated in the interfibrillar space, until they eventually reach and impregnate the hole zone regions of the collagen fibrils. Three-dimensional computer reconstruction of serial transverse and longitudinal sections demonstrates periodic swellings along the collagen fibrils, corresponding to the hole zone region of their axial period as mineralization proceeds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of inorganic phosphate and sulfate anions with collagen

We use direct infrared measurements to determine the number of binding sites, their dissociation constants, and preferential interaction parameters for inorganic phosphate and sulfate anions in collagen fibrils from rat tail tendons. In contrast to previous reports of up to 150 bound phosphates per collagen molecule, we find only 1-2 binding sites for sulfate and divalent phosphate under physiological conditions and approximately 10 binding sites at low ionic strength. The corresponding dissociation constants depend on NaCl concentration and pH and vary from approximately 50 microM to approximately 1-5 mM in the physiological range of pH. In fibrils, bound anions appear to form salt bridges between positively charged amino acid residues within regions of high excess positive charge. In solution, we found no evidence of appreciable sulfate or phosphate binding to isolated collagen molecules. Although sulfate and divalent phosphate bind to fibrillar collagen at physiological concentrations, our X-ray diffraction and in vitro fibrillogenesis experiments suggest that this binding plays little role in the formation, stability and structure of fibrils. In particular, we demonstrate that the previously reported increase in the critical fibrillogenesis concentration of collagen is caused by preferential exclusion of "free" (not bound to specific sites) sulfate and divalent phosphate from interstitial water in fibrils rather than by anion binding. Contrary to divalent phosphate, monovalent phosphate does not bind to collagen. It is preferentially excluded from interstitial water in fibrils, but it has no apparent effect on critical fibrillogenesis concentration at physiological NaCl and pH.