Molecular organization of type IV collagen: polymer liquid crystal-like aspects

A new X-ray diffraction pattern from type IV collagen is described, which can be interpreted on the basis of crystalline and liquid crystalline origins of the reflections. Bovine anterior lens capsules extracted with 1 NaCl and oriented by extension of 60% under constant load gave medium angle X-ray diffraction patterns showing many of the characteristics typical of liquid crystals. Prominent features, apart from those wide angle features attributable to the collagen triple helix, are (1) a four-point pattern of broad reflections at d-spacing 3.9 nm, and layer line spacing near 5 nm. (2) A broad intense equatorial peak centred at 1.24 nm, indicative of LIQUID=like lateral molecular associations (3) A set of five sharp, streaked meridional reflections (previously obscured by the broad peak near 5 nm in unextracted capsules). (4) A further six higher angle reflections of a diffuse, arced and broad appearance on the meridian. The sharp streaked meridional reflections emanate from a long-range periodicity of units 8–9 nm in diameter. These features form a self-consistent system if interpreted on the basis of staggered liquid crystal-like array of collagen molecules, in which case the first five meridionals and remaining broad reflections, sampled on the meridian, can all be indexed as orders of 21 nm.     

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.     

Short and long range order in basement membrane type IV collagen revealed by enzymic and chemical extraction

Oriented bovine lens capsules give X-ray diffraction patterns suggesting a considerable degree of order in the collagenous components, predominantly type IV collagen. Here we report the effects of preliminary treatment of lens capsules before orientation. Extraction with 4 guanidinium hydrochloride or with heparinase/hyaluronidase reveals the same collagenous diffraction patterns previously seen after extraction with 1 NaCl. There is a four-point pattern of d-spacing 3.9 nm, indicating liquid crystal cybotactic nematic organization, along with sharp streaked meridional reflections which index as orders of 21 nm. This suggests that the removal of basement membrane proteoglycans results in a reduction in diffuse scatter and clarification of the pattern. Extraction of the lens capsules with trypsin or dithiothreitol greatly reduces the intensity of the four-point pattern while leaving the meridional pattern unaffected. This strengthens the evidence that the 21 nm period has its origins in the collagen IV helix. Reduction in the four-point pattern could arise if disruption of non-helical NC1 domains or 7S overlap regions allows slippage of the collagen molecules on orientation, weakening the proposed 1 nm intermolecular stagger. Ultra-low angle diffraction patterns of extracted lens capsules show meridional reflections which index as a long-range axial repeat of approximately 95 nm. This is consistent with a model of microfibrils of type IV collagen in which the NC1 domains bind to the collagen helix at approximately 100 nm intervals, as has been previously suggested.     

Structural organization of collagen in Metridium senile

The structure and distribution of collagen fibres in Metridium senile mesoglea has been investigated using high and small angle X-ray diffraction techniques on conventional and synchrotron sources. The mesoglea collagen axial spacing appears very close to that of rat tail tendon, which is at variance with the values previously obtained from electron microscopic observations. The different intensity distribution of the small angle X-ray diffraction maxima recorded for mesoglea and rat tail tendon indicates a different distribution of electron density inside the repeating period. Furthermore the absence of the first order, the weak second order and the strong third and sixth orders in the patterns of wet and dry mesogleal collagen could explain that only a periodicity of 20–22 nm corresponding to one-third of the true axial period observed in the electron micrographs. The analysis of the reflections at 0.29 and 1.1–1.4 nm characteristics of the collagen molecular structure have been used to determine the distribution and orientation of the collagen fibres in unstretched and stretched samples     

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.     

Three-dimensional structure of type IV collagen in the mammalian lens capsule.

The anterior lens capsule provides a thick, easily handled model system for the study of the organization of type IV collagen, the main component of basement membranes. We have used the technique of rapid freezing, deep-etch, and rotary replication to study the three-dimensional organization of the collagen skeleton in mammalian lens capsule after a variety of extraction procedures. In all cases the collagen appeared as a densely packed three-dimensional branching network of fine microfibrils. The organization of the microfibrils appears to show some regularity, with branch points approximately 40 nm apart. Most junctions are three-way and the network forms predominantly five-sided figures. This closely resembles the collagenous network described by Yurchenco and Ruben (1987, 1988) in human amniotic basement membrane and EHS tumor matrix, but extends their findings to another system for which X-ray diffraction data are available. The three-dimensional network is discussed in terms of molecular packing of type IV collagen in light of the information available from the diffraction data.     

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.     

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.     

Low angle X-ray diffraction studies on stained rat tail tendons

Low angle X-ray diffraction patterns of rat tail tendons with heavy metal stains added were examined to help clarify the effects of fixation and staining on collagen fibrils. Fixing and staining of rat tail tendon fibers gives an X-ray pattern with an intensified 3.8 nm row and the preservation of most equatorial features found in the native pattern. The presence of the native pattern features suggests the value of fixation in preserving native structure before staining. Staining of rat tail tendon fibers without prior fixation led to the disappearance of the native equatorial features and the appearance of a new broad row line corresponding to a spacing of around 10.0--17.5 nm. This observation suggests that some alteration has taken place in the native structure and may be related to electron microscopic observations of units of 10.0--20.0 nm in collagen fibrils under some disruptive or developmental conditions.     

Closest packing of two-stranded coiled-coils as a model for the collagen fibril.

We propose that in the collagen fibril, the triple-helical molecules form two-stranded coiled-coils of period 5 × 670A?. Coiled-coils are packed on a tetragonal lattice and are axially staggered with ten in the unit cell (observed side 55A?) so that it carries the 670A?periodicity of the fibril. When nearest neighbours have opposing supercoil hands, the observed tetragonal lattice represents closest packing of two-stranded coiled-coils. This proposal is consistent with the row line spacings measured from the low angle X-ray diffraction pattern of tendon and explains the systematic absences and the two undisputed equatorial reflections. Unlike explanations for the diffraction pattern which invoke a five-stranded microfibril, our interpretation is consistent with its equatorial intensity distribution.     

Phasing the meridional diffraction pattern of type I collagen using isomorphous derivatives

The meridional X-ray diffraction pattern of wet rat tail tendon contains information about the one-dimensional structure, or axial projection of electron density distribution of the type I collagen fibril. Using synchrotron radiation we have determined the intensities of the first 50 meridional X-ray diffraction reflections. The approach of isomorphous addition with reagents, selected using criteria of chemical reactivity, which label at fewer sites than the stains used in previous studies was applied to phase these 50 reflections to produce a one-dimensional electron density distribution map of a single D-repeat of the collagen fibril. This method is not model-dependent and thus constitutes the first unambiguous determination of the meridional phases of type I collagen.     

Structure and orientation of collagen fibres in human mitral valve

The structure and distribution of collagen fibres in chordae tendineae, anterior leaflet and annulus fibrous of human mitral valve has been investigated using high and small angle X-ray diffraction. The molecular packing of collagen in native mitral valve components is very similar to that in native rat tail tendon. The distribution and orientation of collagen fibres in unstretched and stretched specimens has been deduced by the arcing of the high and small angle meridional reflections. Collagen fibres, which are aligned along the chordae tendineae, are preferentially distributed along the branchings of the chordae into the anterior leaflet and then course towards the annulus fibrous. However, in the anterior leaflet a considerable amount of collagen fibres are organized in a tridimensional isotropic network even after high deformation of the tissue.     

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.     

The structure of calcified tendon

Summary The small angle X-ray scattering from unheated and heated calcified chicken tendon has been accurately measured using an automatically recording Kratky camera. A two-dimensional optical diffraction pattern of electron micrographs has been recorded and is in excellent agreement with the small angle X-ray diffraction pattern.The wide and small angle X-ray diffraction pattern, the Fraunhofer diffraction pattern, electron microscopic appearance, and a Fourier synthesis are mutually consistent, and indicate that the crystalline fraction of apatite in normal calcified tendon exists in the form of well oriented crystallites of about 330 Å in length and 50–60 Å in width with the long axis being parallel to the local fibre axis. The separate crystallites are largely located in parallel rows which repeat every 660 Å in the longitudinal direction of the tendon.Analysis of the small angle equatorial scattering shows the crystallites to be clustered together into three well-defined groups with diameters of about 60 Å, 190 Å, and 400 Å. It is suggested that the largest clusters are located in between the collagen fibrils and correspond to the primary calcification.This work was carried out under the supervision of Professor Arne Engström (Head of the Department of Medical Physics, Karolinska Institutet, Stockholm) while the author held a Longterm Fellowship from the European Molecular Biology Organization.     

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.     

Structural analysis of turkey tendon collagen upon removal of the inorganic phase

Calcified leg flexor tendons in which the inorganic phase content had been lowered by progressive demineralization were studied by small angle X-ray diffraction and thermogravimetry. The X-ray diffraction results agree very well with the data previously obtained on calcified turkey tendon indicating that the method used to decalcify tendons provides good correspondence with the process of calcification. Up to five thermal processes can be detected in the thermogravimetric scans: (1) water release; (2) collagen decomposition; (3 and 4) combustion of the residual organic components; (5) carbonate removal from the apatitic phase. The temperature of collagen decomposition decreases at lower inorganic phase content in agreement with the higher thermal stability of calcified collagen fibrils compared with uncalcified ones. The decrease of collagen thermal stability upon decalification is paralleled by a decrease of the structural order of the collagen fibrils as indicated by small angle X-ray diffraction data. Decalcification down to about 40% wt of inorganic phase does not significantly alter the inorganic blocks that are regularly arranged inside the gap zone of the collagen. Further removal of inorganic phase down to about 15% wt provokes a variation of the intensity distribution of the small angle meridional reflections that can be ascribed to a reduction of the mean height of the inorganic blocks. At inorganic phase contents below 15% wt the gap region is more free to contract upon air drying as a result of the reduction of the mean length of the inorganic blocks.     

X-ray diffraction analysis of collagen fibers processed with glutaraldehyde and glycerol

Rat tail tendon collagen fixed with glutaraldehyde and treated with glycerol has been studied by X-ray diffraction technique. The evaluation of the distribution of the areas at higher and less density of molecular packing in the collagen fibrils has been carried out through the analysis of the intensity distribution of the low angle X-ray diffraction maxima. The results show that this treatment usually employed in the freeze-etching technique induces a modification of the degree of order in specific regions inside the axial period D.     

Isolation, ultrastructure, and partial characterization of collagen from the perineurium of the Florida lobster, Panulirus argus.

Highly concentrated extracellular filaments in the perineurium of the Florida spiny lobster, Panulirus argus, were isolated using ultracentrifugation and linear sucrose gradients. The pellet obtained was highly enriched for the filaments as observed by transmission electron microscopy. Fibril diameter and axial periodicity measurements were obtained from filaments positively and negatively stained with uranyl acetate. A period between 14.0 and 25.0 nm and an average fibril diameter of 15.0 nm were observed. The filaments proved resistant to solubilization by most conventional agents and by several collagenases. NaOH (0.1 M at 100 degrees C) safely dissolved the filaments for measurements of protein content by the Lowry method and carbohydrate content with anthrone reagent. These tests revealed a protein content of approximately 84% and a high carbohydrate content of approximately 15%. Polyacrylamide electrophoresis of an acid-pepsin filament extract revealed a highly concentrated band (approximately 100,000) corresponding to the alpha-1 and alpha-2 bands of vertebrate type I collagen. Wide angle X-ray diffraction yielded meridional reflections that confirmed the filaments as collagen when compared with mammalian collagen X-ray diffraction. The amino acid composition was determined with a computer-assisted Beckman amino acid analyzer, which showed a glycine content of 279 residues/1000. Hydroxylysine and hydroxyproline were present in lower concentrations than expected.     

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.     

Immunological identification of types I and III collagen in bovine lens epithelium and its anterior lens capsule

To define the molecular structure of bovine lens epithelium and its anterior lens capsule, we investigated the composition of lens capsule basement membrane proteins. Immunofluorescence and immunogold techniques were used to demonstrate the presence of type I and type III collagen in the lens capsule and in primary explant epithelial cultures grown on protein-binding membranes. Immunofluorescence staining with specific antibodies indicated that type I and type III collagen were constituents of lens basement membrane. We observed that deposition of type III collagen was more than type I collagen. The synthesis of fibrillar collagen by lens epithelium and its deposition in the lens capsule was established by localization of fibrillar collagen by transmission immunoelectron microscopy. These results demonstrate for the first time that normal lens epithelium synthesize fibrillar collagen which is an intrinsic component of the anterior lens capsule basement membrane.