Hepatic vascular malformations in hereditary hemorrhagic telangiectasia: Doppler sonographic screening in a large family

The purpose of this study was to investigate the ultrastructure of the extracellular matrix of human cornea and sclera by using the atomic force microscope (AFM). Specimens of human cornea (n=16) and sclera (n=10) were obtained from a cornea bank or from enucleated eyes (n=1; clinical and histopathological diagnosis: choroidal melanoma) and fixed in Karnovsky solution. The AFM resolved individual collagen fibrils in corneal and scleral tissue. Scleral collagen fibrils had a diameter ranging from 118.3 to 1268.0 nm and showed clear banding with a mean axial D-periodicity of 77.02 nm. The mean gap depth between the two overlaps was larger in the sclera than in the cornea. The diameter of corneal collagen fibrils ranged from 48.0 to 113.0 nm. In contrast to the sclera, the corneal collagen fibrils did not exhibit clear banding as their surface pattern. Closely attached fibrils with a beaded to globular structure were predominant in the cornea. The mean axial D-periodicity of the corneal collagen fibrils was 68.50 nm. In both tissues, the AFM resolved structures resembling cross-bridges between adjacent fibrils. The corneal collagen fibrils showed fibrillar properties that were different from those of the sclera, and that therefore might be essential for the spatial organization responsible for the optical quality of the cornea.     

Collagen fibrils in the human corneal stroma: structure and aging

PURPOSE: Transparency and biomechanical properties of the cornea depend on the structure and organization of collagen fibrils. The authors determined diameter, axial period, and lateral molecular spacing of collagen fibrils in human corneal stroma as a function of age. METHODS: Seventeen normal human corneas were investigated in their native state by means of small-angle and wide-angle x-ray scattering. RESULTS: The mean radius of collagen fibrils, the axial period of collagen fibrils, and the lateral intermolecular Bragg spacing were found to be age dependent. The authors determined fibril radii of 16.1 +/- 0.5 nm in persons older than 65 years of age (n = 10) and 15.4 +/- 0.5 nm (mean +/- SD) in persons younger than 65 years (n = 7) (P < 0.022). The related age-dependent values were 66.4 +/- 0.7 nm (> 65 years) and 65.2 +/- 0.8 nm (< 65 years) for the axial period (P < 0.006) and 1.515 +/- 0.010 nm (> 65 years) and 1.499 +/- 0.013 nm (< 65 years) for the intermolecular Bragg spacing (P < 0.022). CONCLUSIONS: Aging is related to a three-dimensional growth of collagen fibrils in the human corneal stroma. The age-related growth of the fibril diameter was mostly a result of an increased number of collagen molecules and, in addition, to some expansion of the intermolecular Bragg spacing probably resulting from glycation-induced cross-linking. The observed expansion of the fibrils in an axial direction may result from reduction of the molecular tilting angle within collagen fibrils. The observed alterations of the collagen framework may have implications for refractive surgery and ocular tonometry achieved through related changes in the biomechanical properties of the cornea.     

Scheie's syndrome: the architecture of corneal collagen and distribution of corneal proteoglycans

Processes that modulate the regular architecture and, hence, transparency of the cornea are poorly understood, although proteoglycans are thought to be involved. Scheie's syndrome displays corneal opacification and systemic accumulation of glycosaminoglycans. The manifestations of these two occurrences were examined in relation to the corneal stroma. Collagen architecture was investigated by transmission electron microscopy and synchroton x-ray diffraction. Cuprolinic blue staining located sulfated glycosaminoglycan deposits that disrupted the extracellular matrix. Unlike normal cornea, which contained collagen fibrils of remarkably uniform diameter (26.0 +/- 2.4 nm), there was a large range of fibril sizes in the Scheie's syndrome stroma (19.9 to 52.0 nm). Moreover, the distribution of fibril diameters appeared bimodal. X-ray diffraction confirmed the discovery of abnormally large stromal collagen. The results suggest a link in Scheie's syndrome between proteoglycan content/distribution and stromal disruption, and between stromal disruption and corneal opacification.     

Increased diameters of collagen fibrils precipitated in vitro in the presence of decorin from various connective tissues

Proteoglycans were isolated from bovine skin, sclera, deep flexor tendon and the periphery of the temporomandibular joint disc with urea. Decorin was purified from each of these extracts by ion-exchange, hydrophobic-interaction and gel-filtration chromatography. Purities were assessed by amino acid analysis and by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the protein cores released by digestion with chondroitin-ABC-lyase. In these respects the decorins were indistinguishable. However the glycosaminoglycan chains released by digesting the proteoglycans with papain varied widely in mobility on SDS-PAGE: that from skin decorin migrating fastest and that from tendon decorin slowest. The effects of each of the decorins on collagen fibrillogenesis in vitro were similar, all reducing the rate of fibril growth (by 55 to 71%, depending on the source of the proteoglycan) and increasing the diameters of the fibrils formed (by 27 to 66%). Core protein alone, isolated from skin decorin, reduced the rate of fibril growth as effectively as intact decorin, but had no effect on the diameter of fibrils formed. The dermatan sulphate chain and the protein thus appear to play different roles in the interaction of intact decorin with collagen. These data suggest that decorin found in fibrous connective tissues may increase Type I collagen fibril diameters, resulting in tissues that are better able to withstand tensile forces.     

Perlecan binds to the beta-amyloid proteins (A beta) of Alzheimer's disease, accelerates A beta fibril formation, and maintains A beta fibril stability

Perlecan is a specific heparan sulfate proteoglycan that accumulates in the fibrillar beta-amyloid (A beta) deposits of Alzheimer's disease. Perlecan purified from the Engelbreth-Holm-Swarm tumor was used to define perlecan's interactions with A beta and its effects on A beta fibril formation. Using a solid-phase binding immunoassay, freshly solubilized full-length A beta peptides bound immobilized perlecan at two sites, representing both high-affinity [K(D) = approximately 5.8 x 10(-11) M for A beta (1-40); K(D) = approximately 6.5 x 10(-12) M for A beta (1-42)] and lower-affinity [K(D) = 3.5 x 10(-8) M for A beta (1-40); K(D) = 4.3 x 10(-8) M for A beta (1-42)] interactions. An increase in the binding capacity of A beta (1-40) to perlecan correlated with an increase in A beta amyloid fibril formation during a 1-week incubation period. The high-capacity binding of A beta (1-40) to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans and was completely abolished by heparin, but not by chondroitin-4-sulfate. Using a thioflavin T fluorometry assay, perlecan accelerated the rate of A beta (1-40) amyloid fibril formation, causing a significant increase in A beta fibril assembly over a 2-week incubation period at 1 h (2.8-fold increase), 1 day (3.6-fold increase), and 3 days (2.8-fold increase) in comparison with A beta (1-40) alone. Perlecan also initially accelerated the formation of A beta (1-42) fibrils within 1 h and maintained significantly higher levels of A beta (1-42) thioflavin T fluorescence throughout a 2-week experimental period in comparison with A beta (1-42) alone, suggesting perlecan's ability to maintain amyloid fibril stability. Perlecan's effects on A beta (1-40) fibril formation and maintenance of A beta (1-42) fibril stability occurred in a dose-dependent manner and was also mediated primarily by perlecan's glycosaminoglycan chains. Perlecan was the most effective enhancer and accelerator of A beta fibril formation when compared directly with other amyloid plaque components, including apolipoprotein E, alpha1-antichymotrypsin, P component, C1q, and C3. This study, therefore, demonstrates that perlecan not only binds to the predominant isoforms of A beta, but also accelerates A beta fibril formation and stabilizes amyloid fibrils once formed, confirming pivotal roles for perlecan in the pathogenesis of A beta amyloidosis in Alzheimer's disease.     

Growth of sea cucumber collagen fibrils occurs at the tips and centers in a coordinated manner

Collagen fibrils are the principle source of mechanical strength in the mutable dermis of the sea cucumber Cucumaria frondosa. To obtain information about the mechanism by which collagen molecules self-assemble into fibrils, we have isolated single intact fibrils with lengths in the range 14-444 microm. These fibrils have been studied by scanning transmission electron microscopy, yielding data that show how cross-sectional mass, and hence the number of molecules in the cross-section, depend on axial location. In an individual fibril, the two ends always display similar mass distributions. The two tips of each fibril must therefore maintain identity in shape and size throughout growth. The linear relationship between cross-sectional mass and distance from the adjacent end shows that a growing tip is (like the tip of a vertebrate collagen fibril) paraboloidal in shape. Comparison of data from many different fibrils, over a wide range of lengths, however, revealed that the paraboloidal tip becomes blunter as the fibril grows in length. In contrast to vertebrate fibrils, those from C. frondosa do not have a central shaft region of constant cross-sectional mass. Rather, the cross-sectional mass increases to a maximum in the center of each fibril. The maximum cross-sectional mass of the fibrils increases exponentially with increasing fibril length. The centrosymmetry, the paraboloidal shape of the tips, and the hyperbolic increase in maximum cross-sectional mass with fibril length, is evidence for a co-ordinated regulation of length and diameter, which differs from the kind of regulation that gives rise to collagen fibrils in vertebrates (chickens and mice).     

Serum amyloid P component prevents proteolysis of the amyloid fibrils of Alzheimer disease and systemic amyloidosis

Extracellular deposition of amyloid fibrils is responsible for the pathology in the systemic amyloidoses and probably also in Alzheimer disease [Haass, C. & Selkoe, D. J. (1993) Cell 75, 1039-1042] and type II diabetes mellitus [Lorenzo, A., Razzaboni, B., Weir, G. C. & Yankner, B. A. (1994) Nature (London) 368, 756-760]. The fibrils themselves are relatively resistant to proteolysis in vitro but amyloid deposits do regress in vivo, usually with clinical benefit, if new amyloid fibril formation can be halted. Serum amyloid P component (SAP) binds to all types of amyloid fibrils and is a universal constituent of amyloid deposits, including the plaques, amorphous amyloid beta protein deposits and neurofibrillary tangles of Alzheimer disease [Coria, F., Castano, E., Prelli, F., Larrondo-Lillo, M., van Duinen, S., Shelanski, M. L. & Frangione, B. (1988) Lab. Invest. 58, 454-458; Duong, T., Pommier, E. C. & Scheibel, A. B. (1989) Acta Neuropathol. 78, 429-437]. Here we show that SAP prevents proteolysis of the amyloid fibrils of Alzheimer disease, of systemic amyloid A amyloidosis and of systemic monoclonal light chain amyloidosis and may thereby contribute to their persistence in vivo. SAP is not an enzyme inhibitor and is protective only when bound to the fibrils. Interference with binding of SAP to amyloid fibrils in vivo is thus an attractive therapeutic objective, achievement of which should promote regression of the deposits.     

Denaturation of type I collagen fibrils is an endothermic process accompanied by a noticeable change in the partial heat capacity

Thermal transitions of type I collagen fibrils were investigated by differential scanning calorimetry and spectrophotometry of turbidity within a wide range of external conditions. The advanced microcalorimeter allowed us to carry out the measurements at low concentrations of collagen (0.15-0.3 mg/mL). At these concentrations of collagen and under fibril-forming conditions, the melting curves display two pronounced heat adsorption peaks (at 40 and 55 degreesC). The low-temperature peak was assigned to the melting of monomeric collagen, while the high-temperature peak was assigned to the denaturation of collagen fibrils. It was shown that the denaturation of fibrils, in contrast to the monomeric collagen, is accompanied by a noticeable change in the partial specific heat capacity. Surprisingly, comparison of the collagen calorimetric curves in the fibril-forming and nonforming conditions revealed that DeltaCp of fibril denaturation is caused by a decrease in the Cp of collagen at premelting temperatures. This suggests the existence of an intermediate structural state of collagen in a transparent solution preceding fibril formation. Our study also shows that collagen fibrils formed prior to heating have thermodynamic parameters different from those of fibrils formed and denatured during heating in the calorimeter. Analysis of the data allowed us to determine the denaturation enthalpy of the mature fibrils and to conclude that the enthalpy plays a more important role in fibril stabilization than was previously assumed. The observed large DeltaCp value of fibril denaturation as well as the difference between thermodynamic parameters of the mature and newly formed fibrils is readily explained by the presence of water molecules in the fibril structure.     

Cellular proteins bind to multiple sites of the leader region of cauliflower mosaic virus 35S RNA

Recently, several studies have proposed models describing the mechanisms of Alzheimer's beta-amyloid fibril formation in vitro. However, these models are somewhat controversial and no exact kinetic analyses measuring the polymerization velocity as an indicator of the reaction, have thus far been available. We first formed beta-amyloid fibrils from a synthetic peptide, beta-amyloid(1-40), and determined the optimum conditions for quantitative fluorometry of these beta-amyloid fibrils with thioflavine T. Optimum fluorescence measurements of beta-amyloid fibrils were obtained at the excitation and emission wavelengths of 446 and 490 nm, respectively, with the reaction mixture containing 5 microM thioflavine T and 50 mM of glycine-NaOH buffer, pH 8.5. We then focused our study on the extension phase of beta-amyloid fibril formation in vitro. When beta-amyloid fibrils were incubated with monomeric beta-amyloid(1-40) in conditions where de novo seed formation does not occur, the extension of beta-amyloid fibrils was observed with electron microscopy. Quantitative fluorometry revealed that: (a) extension of amyloid fibrils proceeded by a pseudo-first-order exponential increase as measured by the fluorescence of thioflavine T; (b) the rate of extension was maximum around pH 7.5, and was dependent on the incubation temperature. Between 20 and 37 degrees C, good linearity was observed between the common logarithm of the initial rate and the reciprocal of the absolute temperature; (c) the rate of polymerization was found to be proportional to the product of beta-amyloid fibrils number concentration and the beta-amyloid(1-40) concentration; (d) the net rate of extension was the sum of the rates of polymerization and depolymerization. These results show that beta-amyloid fibril formation can be explained by a first-order kinetic model: i.e., the extension of beta-amyloid fibrils proceeds via the consecutive association of beta-amyloid(1-40) onto the ends of existing fibrils.     

Refractive indices of the collagen fibrils and extrafibrillar material of the corneal stroma

Ultrastructural data from x-ray diffraction studies of the cornea were used to estimate the refractive indices of the collagen fibrils and extrafibrillar material of human, ox, trout, and rabbit corneas. X-ray diffraction measurements of the size and spacing of the collagen fibrils and the separation between the constituent molecules of the fibrils were taken from a previous species study. The tissue volume fractions occupied by the stromal components were estimated and their refractive indices were calculated using the Gladstone-Dale law of mixtures. For the fibrils and extrafibrillar material, the refractive indices in the human cornea were 1.411 and 1.365; for the ox 1.413 and 1.357; for the rabbit 1.416 and 1.357; and for the trout 1.418 and 1.364, respectively. An alternative estimate based on the physical properties and chemical composition of bovine cornea, accounting for interfibrillar type VI collagen and cellular water, produced similar estimates of 1.416 and 1.356 for the fibrils and extrafibrillar material, respectively.     

Biochemical and structural analyses of the extracellular matrix fibrils of Myxococcus xanthus

It is characteristic of myxobacteria to produce large amounts of extracellular material. This report demonstrates that this material in Myxococcus xanthus is fibrillar and describes the structure and chemical composition of the fibrils. The extracellular matrix fibrils are the mediators of cell-cell cohesion in M. xanthus. As such, the fibrils play an important role in the cell-cell interactions that form the basis for the social and developmental lifestyle of this organism. The fibrils are composed of protein and carbohydrate in a 1.0:1.2 ratio. Combined, the two fractions accounted for greater than 85% of the mass of isolated fibrils, and the fibrils were found to compose up to 10% of the dry weight of cells grown at high density on a solid surface. The polysaccharide portion of the fibrils was shown to be composed of five different monosaccharides: galactose, glucosamine, glucose, rhamnose, and xylose. Glucosamine, one of the component monosaccharides of the fibrils and a known morphogen for M. xanthus, inhibited cohesion to a level near that of Congo red (the positive control for cohesion inhibition). Glucose and xylose also inhibited cohesion but less than did glucosamine. Analysis of the morphology of the fibrils, the periodicities within the distribution of fibril diameters observed by field emission scanning electron microscopy, and the observation of fibrils on hydrated cells strongly suggested that the extracellular matrix of M. xanthus was indeed arranged as fibrils. Furthermore, results suggested that the fibrils were constructed as carbohydrate structures with associated proteins.     

Reduction of disulfide bonds in an amyloidogenic Bence Jones protein leads to formation of "amyloid-like" fibrils in vitro

Motivated by the finding that the amino acid sequence of the Bence Jones protein BJP-DIA was identical to that of the main protein component of the amyloid fibrils obtained from the same patient with AL-amyloidosis, (Klafki, H.-W., Kratzin, H.-D., Pick, A.-I., Eckart, K., Karas, M. & Hilschmann, N. (1992) Biochemistry 31, 3265-3272.), we attempted to create "amyloid-like" fibrils from the Bence Jones protein in vitro, without addition of proteolytic enzymes. Reduction of BJP-DIA, solubilized in PBS, pH 7.4, overnight at 37 degrees C resulted in the formation of a precipitate which had affinity for the dye Congo red. Electron microscopy of negatively stained samples of the reduced protein revealed aggregates of linear unbranched fibrils. SDS-polyacrylamide gel electrophoresis demonstrated that the precipitate consisted almost exclusively of intact light chain molecules. This result makes it possible to deduce a molecular model of these amyloid fibrils generated in vitro.     

Three-dimensional organization of collagen fibrils during corneal stromal wound healing after excimer laser keratectomy

PURPOSE: To investigate the structural changes in corneal stromal collagen fibrils after excimer laser keratectomy in relation to the degree of corneal haze. SETTING: University of Tokyo Hospital, Tokyo, Japan. METHODS: Corneal haze was quantitatively measured by analyzing the light scattering in Scheimpflug images of the corneas of white rabbits after excimer laser keratectomy. Collagen fibril structure was examined using scanning electron microscopy after chemical digestion with sodium hydroxide solution; the same specimens were examined by transmission electron microscopy after re-embedding. RESULTS: Corneal haze reached a peak 4 weeks after excimer laser keratectomy and then gradually decreased. The collagen fibrils of the normal cornea were regularly arranged parallel to the surface of the cornea, with small interfibrillar distances. After excimer laser keratectomy, the arrangement was highly disordered, with increased interfibrillar distances. These structural changes were most prominent 4 weeks after excimer laser keratectomy. CONCLUSION: The structural changes in the collagen fibrils of the corneal stroma, especially the increase in interfibrillar distances and the disordered arrangement, were associated with corneal haze after excimer laser keratectomy.     

A scanning electron microscopic study of the degenerative cartilage in patellar chondropathy

Patellar chondropathy as cartilage degeneration localized in patellar cartilage in young persons is characterized by cartilaginous changes, such as softening, swelling, and fissuring. With a view to structural characterization of early cartilaginous degeneration before erosion, the morphology of affected cartilage was studied under a scanning electron microscope. The surface network of cartilage constituting fibrils had an edematous change, presenting with fibrillation on the medial facet, whereas many fibrils of the central ridge had a collagen bundle, and fissuring of varying size was observed. It appeared that a mechanical force (shearing) acting on the site of the central ridge was associated with the formation of a collagen bundle and its destruction. On the lateral facet, fibrils were arranged perpendicular to the joint surface; the superficial layer of fibrils was worn by hyper-pressure acting on the lateral facet. On the fractured surface, the coarseness of collagen fibrils showed changes that varied with the site and stage of cartilage degeneration. Frequent changes were signs of fibril loosening (coarsening), such as reduction in fibril density (i.e., edematous change), collagen fibril aggregation, and fissuring, and longitudinal restructuring of fibrils. The patellar cartilage in the patients of this series showed a structure adapted to the mechanical force. The initial structural changes of cartilage consisted of collagen fibril aggregation and reduction in fibril density. These changes give rise to matrix rarefaction, which in turn causes cartilage degeneration to progress. These changes were concurrent in both the superficial and middle layers and were not localized as basal degeneration.     

Cartilage fibrils of mammals are biochemically heterogeneous: differential distribution of decorin and collagen IX

Cartilage fibrils contain collagen II as the major constituent, but the presence of additional components, minor collagens, and noncollagenous glycoproteins is thought to be crucial for modulating several fibril properties. We have examined the distribution of two fibril constituents-decorin and collagen IX-in samples of fibril fragments obtained after bovine cartilage homogenization. Decorin was preferentially associated with a population of thicker fibril fragments from adult articular cartilage, but was not present on the thinnest fibrils. The binding was specific for the gap regions of the fibrils, and depended on the decorin core protein. Collagen IX, by contrast, predominated in the population with the thinnest fibrils, and was scarce on wider fibrils. Double-labeling experiments demonstrated the coexistence of decorin and collagen IX in some fibrils of intermediate diameter, although most fibril fragments from adult cartilage were strongly positive for one component and lacked the other. Fibril fragments from fetal epiphyseal cartilage showed a different pattern, with decorin and collagen IX frequently colocalized on fragments of intermediate and large diameters. Hence, the presence of collagen IX was not exclusive for fibrils of small diameter. These results establish that articular cartilage fibrils are biochemically heterogeneous. Different populations of fibrils share collagen II, but have distinct compositions with respect to macromolecules defining their surface properties.     

Mucoepidermoid carcinoma of the epibulbar connective tissue with diffuse intraocular epithelial invasion

PATIENT: A 55-year-old man was treated twice with local excision and crycoagulation for a recurrent limbal mass of the left eye. The original histologic diagnosis was squamous cell carcinoma. Three months after the last recurrence the globe was enucleated because of a spontaneous perforation at the corneoscleral limbus with iris prolapse. Histologic examination, including PAS and mucicarmine stains, revealed a mucoepidermoid carcinoma of the epibulbar conjunctiva with infiltration of the cornea, sclera, iris and ciliary body. CONCLUSIONS: Examination of specially stained sections (e.g. with mucicarmine) should be routinely performed for those conjuctival neoplasms that contain a squamous component. Aggressive surgical management, such as early enucleation including normal appearing tissue next to the globe, should be considered for treatment of primary mucoepidermoid carcinoma to avoid later exenteration or metastasis.     

Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis

Tissue deposition of soluble proteins as amyloid fibrils underlies a range of fatal diseases. The two naturally occurring human lysozyme variants are both amyloidogenic, and are shown here to be unstable. They aggregate to form amyloid fibrils with transformation of the mainly helical native fold, observed in crystal structures, to the amyloid fibril cross-beta fold. Biophysical studies suggest that partly folded intermediates are involved in fibrillogenesis, and this may be relevant to amyloidosis generally.     

X-ray diffraction study of in vitro calcification of tendon collagen

Decalcified samples of turkey leg tendon were submitted to in vitro calcification in the presence of metastable solutions of calcium phosphate at different concentrations. The structural relationship between apatitic deposits and collagen fibrils was examined by high- and small-angle X-ray diffraction using conventional and synchrotron radiation sources. At high supersaturation the apatitic crystallites were deposited on the collagen fibrils with their crystallographic c-axis preferentially oriented parallel to the fibril axis. At lower supersaturation, a fraction of the apatitic crystallites also grew with the c-axis preferentially oriented parallel to the collagen fibril axis, whereas other exhibited a preferential orientation perpendicular to the fibril axis. The analysis of the small-angle X-ray diffraction data indicates that the deposition of the apatitic phase in the sample stored in solution at lower supersaturation induced modifications of the collagen electron density distribution in the axial direction, which can be attributed to the deposition of the inorganic crystallites inside the gap region of the collagen structure.     

Isoform-specific effect of apolipoprotein E on cell survival and beta-amyloid-induced toxicity in rat hippocampal pyramidal neuronal cultures

Dermatosparaxis is a recessive disorder of animals (including man) which is caused by mutations in the gene for the enzyme procollagen N-proteinase and is characterised by extreme skin fragility. Partial loss of enzyme activity results in accumulation of pNcollagen (collagen with N-propeptides) and abnormal collagen fibrils in the fragile skin. How the N-propeptides persist in the tissue and how abnormal fibril morphology results in fragile skin is poorly understood. Using biochemical and quantitative mass mapping electron microscopy we showed that the collagen fibrils in the skin of a dermatosparactic calf contained 57% type I pNcollagen and 43% type I collagen and the fibrils were irregularly arranged in bundles and hieroglyphic in cross-section. Image analysis of the fibril cross-sections suggested that the deviation from circularity of dermatosparactic fibrils was caused by N-propeptides of pNcollagen being located at the fibril surface. Comparison of experimental and theoretical axial mass distributions of the fibrils showed that the N-propeptides were located to the overlap zone of the fibril D-period (where D=67 nm, the characteristic axial periodicity of collagen fibrils). Treatment of the dermatosparactic fibrils with N-proteinase did not remove the N-propeptides from the fibrils, although the N-propeptides were efficiently removed by trypsin and chymotrypsin. However, the N-propeptides were efficiently cleaved by the N-proteinase when the pNcollagen molecules were extracted from the fibrils. These results are consistent with close packing of N-propeptides at the fibril surface which prevented cleavage by the N-proteinase. Long-range axial mass determination along the fibril length showed gross non-uniformity with multiple mass bulges. Of note is the skin fragility in dermatosparaxis, and also the appearance of mass bulges along the fibril long axis symptomatic of the fragile skin of mice which lack decorin. Western blot analysis showed that the dermatosparactic fibrils bound elevated levels of the proteoglycan, compared with normal skin fibrils. The results showed that N-propeptides can distort the morphology of fibrils, that they do not inhibit binding of gap-associated macromolecules (such as decorin) and that the normal mechanical properties of skin are strongly dependent on the close association of near-cylindrical fibrils, thereby enabling maximal fibril-fibril interactions.     

Ehlers-Danlos syndrome type VIIB. Morphology of type I collagen fibrils formed in vivo and in vitro is determined by the conformation of the retained N-propeptide

Previously we showed that fibrils generated from collagen and pNcollagen-ex6 from fibroblasts of an individual with Ehlers-Danlos syndrome (EDS) type VIIB were hieroglyphic in cross-section and all N-propeptides were located at the fibril surface. Hieroglyphs were resolved to near-cylindrical fibrils (that were similar in appearance to the fibrils seen in the tissues of individuals with EDS type VIIB) by treatment with N-proteinase which cleaved the pN alpha 1(I) chains but not the pN alpha 2(I)-ex6 chains (Watson, R. B., Wallis, G. A., Holmes, D. F., Viljoen, D., Byers, P. H., and Kadler, K. E. (1992) J. Biol. Chem. 267, 9093-9100). Here, quantitative scanning transmission electron microscopy (STEM) showed that N-propeptides in hieroglyphs were in a "bent-back" conformation and thus located exclusively in the overlap zone of the fibril D-period (D = 67 nm). In contrast, STEM of fibrils from the dermis of an individual with EDS type VIIB showed that partially cleaved N-propeptides (in which cleaved pN alpha 1(I) remained in noncovalent association with pN alpha 2(I)-ex6 chains) were distributed equally between the gap and overlap zones of the fibrils. Comparison of experimental data with theoretical mass distributions of the fibril based on amino acid sequence data gave a consistent value of 33 nm for the total axial extent for the N-propeptides in hieroglyphic and tissue fibrils irrespective of the location of N-propeptides to the gap or overlap zone. These data exclude the possibility that N-propeptides adopt a random configuration, but rather, that they locate to specific sites in the gap and overlap zones. The results demonstrated that cleavage of pN alpha 1(I) chains in vivo releases the N-propeptides from the constraints of the bent-back conformation. Co-distribution of partially cleaved N-propeptides between gap and overlap zones allows a higher surface packing density of N-propeptides and explains how circularity of large diameter fibrils can be achieved despite the retention of N-propeptides in tissues of individuals with EDS type VIIB.