Crystalline fibril structure of type II collagen in lamprey notochord sheath

We report here the existence of a crystalline molecular packing of type II collagen in the fibrils of the lamprey notochord sheath. This is the first finding of a crystalline structure in any collagen other than type I.The lamprey notochord sheath has a composition similar to that of cartilage, with type II collagen, a minor collagen component with 1α, 2α and 3α chains, and cartilage-like proteoglycan. The high degree of orientation of fibrils in the notochord makes it possible to use X-ray diffraction to determine collagen fibril organization in this type II-containing tissue. The low angle equatorial scattering shows the fibrils are all about 17 nm in diameter and have an average center-to-center separation of 31 nm. These results are supported by electron microscope observations. A set of broad equatorial diffraction maxima at higher angles represents the sampling of the collagen molecular transform by a limited crystalline lattice, extending over a lateral dimension close to the diameter of one fibril. This indicates that each 17 nm fibril contains a crystalline array of molecules and, although a unit cell is difficult to determine because of the broad overlapping reflections, it is clear that the quasi-hexagonal triclinic unit cell of type I collagen in rat tail tendon is not consistent with the data. The meridional diffraction pattern showed 26 orders with the characteristic 67 nm periodicity found for tendon. However, the intensities of these reflections differ markedly from those found for tendon and cannot be explained by an unmodified gap/ overlap model within each 67 nm period. Both X-ray diffraction and electron microscope data indicate a low degree of contrast along the fibril axis and are consistent with a periodic binding of a non-collagenous component in such a way as to obscure the gap region.     

Calculated X-ray diffraction pattern from a quasi-hexagonal model for the molecular arrangement in collagen

The near-equatorial region of the medium-angle X-ray diffraction pattern from native rat tail tendon contains sharp reflections which indicate that the collagen molecules are arranged in a crystalline manner within the fibrils. A successful indexing of these reflections would indicate that crystallographic unit cell in the fibrils while the intesities of the reflections are determined by the arrangement of the collagen molecules within a unit cell. It is shown that the quasi-hexagonal model proposed by Hulmes and Miller¹ with slight modifications accounts for the positions of the reflections [i.e. their (R, Z) values]. Previous models used mainly the R-values of the reflections published by Miller and Parry². This model gives a better account of the R-values of the reflections than previous models and, in addition, accounts for the Z-values and the intensities of the reflections. This represents the determination of the three-dimensional structure of the collagen in a native animal tissue, rat tail tendon, to 1 nm resolution.     

Variations in collagen fibril structure in tendons

Variation of collagen fibril structure in tendon was investigated by x-ray diffraction. Anatomically distinct tendons from single species, as well as tendons from different species, were examined to determine the variations that exist in both the axial and lateral structure of the collagen fibrils. The meridional diffraction is derived from the axial collagen fibril structure. Anatomically distinct tendons of a particular species give meridional patterns that are indistinguishable within experimental error. The meridional diffraction patterns from tendons of different mammals are similar but show small species-specific variations, most noticeably in the 14th–18th orders. Tendons of birds also give meridional patterns that are similar to each other, but the avian patterns differ considerably from the mammalian ones. Avian tendons give stronger odd and weaker even low orders, a feature consistent with a reduced gap:overlap ratio, and have a distinctive intensity pattern for the higher meridional orders. Interpretation of these differences has been approached using biochemical data, diffraction by reconsituted fibers of purified collagen, and Fourier transform analysis. From these methods, it appears that the variations observed in the lower orders (2nd–8th) and in the higher orders (29th–52nd) are probably related to differences in the primary structure of the Type I collagen found in the different species. The variations observed in the 14th–18th orders appear not to be related to features within the triple-helical domain of the molecule. Equatorial diffraction yields information on the lateral packing of collagen molecules in the fibrils, and considerable variation was seen in different tendons. Rat tail tendon gives sharp Bragg reflections, demonstrating the presence of a crystalline lateral arrangement of molecules in the fibril. For the first time, sharp lattice reflections similar to those in rat tail tendon have been observed in nontail tendons, including rat achilles tendon, rabbit leg tendon, and wing and leg tendons of quail. In the rabbit and quail tendons, one of the strong equatorial reflections characteristic of the rat tendon pattern, at 1.26 nm, was absent. The positions of the equatorial maxima, which are a measure of intermolecular spacing, varied considerably, being smallest in the specimens displaying crystalline packing. The intermolecular distance in chiken and turkey leg tendons is longer than that found in mammalian tendons, or in avian wing tendons, which supports the hypothesis that a larger intermolecular spacing is characteristic of tendons that calcify. Thus, x-ray diffraction indicates there are reproducible differences in both the axial and lateral structure of collagen fibrils among different tendons. This work on tendon, a tissue containing almost exclusively Type I collagen as its major component, should serve as a basis for analyzing the structure of other connective tissues, which contain different genetic types of collagen and larger amounts of noncollagenous components.     

X-ray diffraction of reconstituted collagen fibers

The X-ray diffraction of fibers reconstituted from purified rat tail tendon collagen has been compared with that of native rat tail tendon. The axial structure is very similar in the two specimens, while the ordered lateral array found in the native state is only poorly reproduced in the reconstituted fiber. Thus, the axial order is determined by the collagen molecules alone, while the native lateral packing may depend, in part at least, on other tissue components.     

Interpretation of the meridional X-ray diffraction pattern from collagen fibrils in corneal stroma

The low angle X-ray diffraction pattern from corneal stroma can be interpreted as arising from the equivalent of sharp meridional reflections due to the packing of molecules along the collagen fibrils and an equatorial pattern due to the packing of these fibrils within lamellae.Axial electron density profiles for corneal collagen fibrils have been produced by combining intensity data from the meridional pattern with two independent sets of phases. The first set was obtained using an electron microscopical technique, whereas the second set consisted of calculated tendon collagen phases given in the literature. Substantial agreement between the two electron density profiles was found.A quantitative analysis of the difference between the electron density profiles of rat tail tendon and corneal collagen showed that the step between the gap and overlap regions is smaller in cornea than in tendon. This is probably due to the binding of non-collagenous material in the gap region as occurs in bone and other tissue. Two peaks corresponding to regions where electron density is greater in the cornea are situated at the gap/overlap junctions. A third region where the corneal collagen is more electron dense is located near the centre of the gap region. The proximity of these peaks to the positions of hydroxylysine residues along the fibril axis suggests that they may be the major sites at which sugars are bound to corneal collagen.     

Structural dynamic of native tendon collagen

The dynamic behaviour of collagen fibrils is revealed by time-resolved X-ray investigations of native rat tail tendon fibres in tensile tests.     

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.     

Neutron diffraction studies of collagen in fully mineralized bone

Neutron diffraction measurements have been made of the equatorial and meridional spacings of collagen in fully mineralized mature bovine bone and demineralized bone collagen, in both wet and dry conditions. The collagen equatorial spacing in wet mineralized bovine bone is 1.24 nm, substantially lower than the 1.53 nm value observed in wet demineralized bovine bone collagen. Corresponding spacings for dry bone and demineralized bone collagen are 1.16 nm and 1.12 nm, respectively. The collagen meridional long spacing in mineralized bovine bone is 63.6 nm wet and 63.4 nm dry. These data indicate that collagen in fully mineralized bovine bone is considerably more closely packed than had been assumed previously, with a packing density similar to that of the relatively crystalline collagens such as wet rat tail tendon. The data also suggest that less space is available for mineral within the collagen fibrils in bovine bone than had previously been assumed, and that the major portion of the mineral in this bone must be located outside the fibrils.     

Negative staining of rat tail tendon collagen fibrils with uranyl formate

Negative staining of rat tail tendon collagen fibrils with uranyl formate appears to reveal more detail in the axial banding pattern than any other positive or negative staining method hitherto employed. In addition, uranyl formate and other uranyl solutions appear to reveal fine, closely spaced, longitudinal filaments which may represent the individual tropocollagen molecules.     

Histological staining as a measure of stress in collagen fibers

It has been reported previously that collagen fibers will stain either red or green by Masson's and other trichrome methods depending on whether they have been respectively stressed or relaxed prior to fixation. This was shown in skin [1, 2, 3] tendon [4, 5] bone [6] and films of collagen [7]. If this stain-stress dependence is of a unique quantitative nature, then staining could be used as a tension probe for collagen fibers. Relaxed and stressed collagen bundles of rat tail tendon and rat Achilles tendon have been stained using various staining periods, and results indicate that the change in staining may be associated with denser packing of the fibers in the bundle under stress rather than directly due to the stress itself. Denser packing may reduce the rate of penetration of the counterstain thus causing the staining differences. Since this rate of penetration is dependent on a number of other variables (unrelated to stress), it is concluded that collagen staining is not a reliable tension probe.     

Cross-linkage sites in type I collagen fibrils studied by neutron diffraction

Cross-links in tendon collagen are essential for the biomechanical strength of healthy tissue. The nature and position of these cross-links has long been a subject for conjecture. We have approached this problem in a non-destructive manner, by studying neutron diffraction from collagen fibrils that have been specifically deuterated by reduction at keto-amine and Schiff base groups with sodium borodeuteride (NaB2H4). The intensities of the first 23 meridional reflections were recorded for both native and reduced tendons. These data were used to calculate the neutron-scattering density profile of the 67 nm (D) repeat of type I collagen fibrils in rat tail tendon. This approach not only succeeds in determining the location of the cross-linkage sites with respect to the fibril structure, as projected onto the fibre axis, but also presents a novel form of the isomorphous derivative solution to the phase problem.     

Glutaraldehyde-induced changes in the axially projected fine structure of collagen fibrils

The fine structure of the collagen fibril, as seen in axial projection, is changed by treatment with glutaraldehyde. The changes are detectable in electron-optical staining patterns and in the intensities of the low-angle meridional X-ray diffraction maxima. Current knowledge of the amino acid sequence of collagen and of the axial arrangement of molecules in fibrils permits interpretation in terms of specific alterations to the axial distribution of electron density along the fibril. Analysis of fibril staining patterns from glutaraldehyde-treated calf skin collagen shows that uptake of staining ions in positive staining patterns is inhibited at residues known to interact with glutaraldehyde (lysyl, hydroxylysyl and probably histidyl side-chains) and on other charged residues in the immediate neighbourhood of the glutaraldehyde-reactive residues. This can be seen as a "stain-exclusion effect" due to the presence of bulky polymeric complexes of glutaraldehyde molecules at cross-linking sites. Such stain exclusion accounts for the drastic changes in the negative staining pattern following treatment with glutaraldehyde. The intensity changes observed in the low-angle meridional X-ray reflections from rat tail tendon, similarly treated, also can be explained by the presence of these bulky complexes. Existing data have been used to predict a model of the altered electron density profile indicating the axial distribution of glutaraldehyde along a D-period of moist tendon collagen.     

Preliminary X-ray crystallographic data on phospholipase C from Bacillus cereus.

Low-angle X-ray diffraction shows that, despite the well-defined regular axially projected structure, there is no long-range lateral order in the packing of molecules in native (undried) or dried elastoidin spicules from the fin rays of the spurhound Squalus acanthias. The equatorial intensity distribution of the X-ray diffraction pattern from native elastoidin indicates a molecular diameter of 1.1 nm and a packing fraction for the structure projected on to a plane perpendicular to the spicule (fibril) axis of 0.31 (the value for tendon is much higher at around 0.6). Density measurements support this interpretation. When the spicule dries the packing fraction increases to 0.43 but there is still no long-range order in the structure. The X-ray diffraction patterns provide no convincing evidence for any microfibrils or subfibrils in elastoidin. Gel electrophoresis shows that the three chains in the elastoidin molecule are identical. The low packing fraction for collagen molecules in elastoidin explains the difference in appearance between electron micrographs of negatively stained elastoidin and tendon collagen. In elastoidin, but not in tendon collagen, an appreciable proportion of the stain is able to penetrate between the collagen molecules.     

A generalized packing model for type I collagen

Only tail tendon (TT) collagen has a sharp X-ray diffraction pattern, so that packing models for the equatorial arrangement of molecules in collagen fibrils have been developed primarily for TT collagen. A more general structure is developed applicable to all type I collagen tissues. Comparison of water content-equatorial diffraction spacing plots of several collagens shows all have essentially the same dry state diffraction spacing but differ as water content increases. TT collagen has the least spacing and the sharpest pattern. The interplanar spacing of the Hulmes-Miller quasi-hexagonal model for TT collagen was used to calculate the intermolecular spacing, which matched the observed diffraction spacing for bone matrix collagen. It is inferred that wet bone matrix collagen packs in a rectangular pattern because of the interaction between the many intermolecular crosslinks and the water absorbed on the collagen molecules. This argument also indicates that TT collagen packs into a quasi-hexagonal scheme because there are fewer intermolecular crosslinks than in bone matrix collagen.     

Conformation and sequence evidence for two-fold symmetry in left-handed beta-helix fold

The glycosaminoglycan (GAG) side-chains of small leucine-rich proteoglycans have been postulated to mechanically cross-link adjacent collagen fibrils and contribute to tendon mechanics. Enzymatic depletion of tendon GAGs (chondroitin and dermatan sulfate) has emerged as a preferred method to experimentally assess this role. However, GAG removal is typically incomplete and the possibility remains that extant GAGs may remain mechanically functional. The current study specifically investigated the potential mechanical effect of the remaining GAGs after partial enzymatic digestion.A three-dimensional finite element model of tendon was created based upon the concept of proteoglycan mediated inter-fibril load sharing. Approximately 250 interacting, discontinuous collagen fibrils were modeled as having a length of 400 μm, being composed of rod elements of length 67 nm and E-modulus 1 GPa connected in series. Spatial distribution and diameters of these idealized fibrils were derived from a representative cross-sectional electron micrograph of tendon. Rod element lengths corresponded to the collagen fibril D-Period, widely accepted to act as a binding site for decorin and biglycan, the most abundant proteoglycans in tendon. Each element node was connected to nodes of any neighboring fibrils within a radius of 100 nm, the slack length of unstretched chondroitin sulfate. These GAG cross-links were the sole mechanism for lateral load sharing among the discontinuous fibrils, and were modeled as bilinear spring elements. Simulation of tensile testing of tendon with complete cross-linking closely reproduced corresponding experiments on rat tail tendons. Random reduction of 80% of GAG cross-links (matched to a conservative estimate of enzymatic depletion efficacy) predicted a drop of 14% in tendon modulus. Corresponding mechanical properties derived from experiments on rat tail tendons treated in buffer with and without chondroitinase ABC were apparently unaffected, regardless of GAG depletion. Further tests for equivalence, conservatively based on effect size limits predicted by the model, confirmed equivalent stiffness between enzymatically depleted tendons and their native controls.Although the model predicts that relatively small quantities of GAGs acting as primary collagen cross-linking elements could provide mechanical integrity to the tendon, partial enzymatic depletion of GAGs should result in mechanical changes that are not reflected in analogous experimental testing. We thus conclude that GAG side chains of small leucine-rich proteoglycans are not a primary determinant of tensile mechanical behavior in mature rat tail tendons.     

Structure and function of bone collagen fibrils

The intermolecular volume of fully hydrated collagen fibrils from a number of mineralized and non-mineralized tissues of adult rats has been determined both by an exclusion technique and by a method which involves the monitoring of specific X-ray diffraction parameters. The intermolecular volume of either bone or dentinal fibrils is approximately twice that of either tail or achilles tendon, and the most frequent intermolecular distance in bone or dentine fibrils is approximately 3 Å larger than of the tendons.A number of fibrillar structures are most compatible with the intermolecular volume of rat tail tendon. These include hexagonal molecular packing and orthogonal arrays of microfibrils comprising seven parallel molecular strands. The intermolecular volume of bone or dentinal collagen fibrils, on the other hand, appears to arise from structures having a disordered or pseudo-hexagonal molecular packing, in which the most frequent intermolecular distance is about 19 Å.The space associated with collagen fibrils in adult bone is such that 70 to 80% of the mineral is located within the intermolecular space of the fibrils—approximately equal amounts of mineral being in spaces having lateral dimensions of 25 to 75 Å and 6 to 12 Å, respectively. Particles located in the latter kind of intermolecular space probably constitute, to a large extent, the non-crystalline mineral phase of adult bone.The stereo-chemical constraints on the transport of mineral ions into and within collagen fibrils of bone and tendon support the postulate that bone collagen is an in vivo catalyst for mineral deposition and further suggests that its catalytic activity may be partially regulated through its molecular packing.     

Structural study of the calcifying collagen in turkey leg tendons

The calcified turkey leg tendon represents a simple bone-like tissue that is ideally suited to analysis by diffraction methods. In this paper we report some structural studies of the tendon collagen in the uncalcified, fully calcified and partially calcified states. The low-angle meridional X-ray pattern from the uncalcified tendon is very similar to that of the rat tail tendon, and the resulting one-dimensional structure of the collagen fibril exhibits no feature that could be related to its eventual calcification. The structure of the fully calcified tendon, as determined by a combination of X-ray and neutron diffraction analyses, shows that the mineral is associated with the collagen at the level of the hole or gap region. In the calcifying tendon, increases in the amplitudes of the first and second X-ray meridional reflections are correlated with an increase in the mineral content of the collagen. On the basis of simple models, it is shown that this change in the pattern can be explained by a nucleation mechanism of calcification. It is concluded that when collagen becomes calcified the mineral penetrates throughout the fibril and is crystalline in the hole region but amorphous between the collagen molecules. The mechanism of calcification and the mechanical implications of the fully calcified structure are also discussed.     

Fibrous long spacing collagen ultrastructure elucidated by atomic force microscopy.

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.     

Benzo[a]pyrene-collagen interactions

In vitro interactions of benzo[a]pyrene (BaP) with acid-soluble type I collagen from rat tail tendon have been investigated. The fluorescence of BaP increases in the presence of collagen. Bound BaP inhibits the formation of collagen fibrils in solution. When BaP-collagen complexes are irradiated in air with UV (365 nm) light, BaP rapidly undergoes photooxidation with the further inhibition of fibril formation. Viscosity and circular dichroism (CD) studies show that neither BaP nor further UV-irradiation alters the size or helical conformation of the protein. During thermal denaturation of collagen, BaP fluorescence changes. Collagen from young rat tail tendon shows a pronounced drop at about 38 degrees C, whereas that from old rat tail tendon exhibits an increase with a plateau in the same temperature range. These anomalous changes are observed when tyrosine residues, present only in the non-helical terminal telopeptides of collagen, are excited at 275 nm, but not by direct BaP excitation at 387 nm. These findings suggest that the specific hydrophobic telopeptide region, which plays an important role in fibril formation, are affected by bound BaP.     

THE FINE STRUCTURE OF ELASTIC FIBERS

The fine structure of developing elastic fibers in bovine ligamentum nuchae and rat flexor digital tendon was examined. Elastic fibers were found to contain two distinct morphologic components in sections stained with uranyl acetate and lead. These components are 100 A fibrils and a central, almost amorphous nonstaining area. During development, the first identifiable elastic fibers are composed of aggregates of fine fibrils approximately 100 A in diameter. With advancing age, somewhat amorphous regions appear surrounded by these fibrils. These regions increase in prominence until in mature elastic fibers they are the predominant structure surrounded by a mantle of 100 A fibrils. Specific staining characteristics for each of the two components of the elastic fiber as well as for the collagen fibrils in these tissues can be demonstrated after staining with lead, uranyl acetate, or phosphotungstic acid. The 100 A fibrils stain with both uranyl acetate and lead, whereas the central regions of the elastic fibers stain only with phosphotungstic acid. Collagen fibrils stain with uranyl acetate or phosphotungstic acid, but not with lead. These staining reactions imply either a chemical or an organizational difference in these structures. The significance and possible nature of the two morphologic components of the elastic fiber remain to be elucidated.