Conceived and designed the experiments: OA JO. Performed the experiments: OA JO. Analyzed the data: OA JO. Contributed reagents/materials/analysis tools: OA JO. Wrote the paper: OA JO.
Author Joseph Orgel is a section editor and member of the editorial board of PLoS ONE. The Journal had no part or influence in the research reported or in the writing of the originally submitted manuscript. A patent application has been made that pertains to data reported in this manuscript. #12/419,689: “THIN FIBRIL COLLAGEN MATERIAL AND METHOD FOR MAKING THIN FIBRIL COLLAGEN FROM NATIVE COLLAGEN FIBERS.” This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory and destructive joint disorder that affects tens of millions of people worldwide. Normal healthy joints maintain a balance between the synthesis of extracellular matrix (ECM) molecules and the proteolytic degradation of damaged ones. In the case of RA, this balance is shifted toward matrix destruction due to increased production of cleavage enzymes and the presence of (autoimmune) immunoglobulins resulting from an inflammation induced immune response. Herein we demonstrate that a polyclonal antibody against the proteoglycan biglycan (BG) causes tissue destruction that may be analogous to that of RA affected tissues. The effect of the antibody is more potent than harsh chemical and/or enzymatic treatments designed to mimic arthritis-like fibril de-polymerization. In RA cases, the immune response to inflammation causes synovial fibroblasts, monocytes and macrophages to produce cytokines and secrete matrix remodeling enzymes, whereas B cells are stimulated to produce immunoglobulins. The specific antigen that causes the RA immune response has not yet been identified, although possible candidates have been proposed, including collagen types I and II, and proteoglycans (PG's) such as biglycan. We speculate that the initiation of RA associated tissue destruction
The major parts of the joint which are affected most prominently in RA, are articular cartilage and the synovium. Collagen type II fibrils are major structural elements of the cartilage ECM and the cartilage-like notochord of cartilaginous fishes. They form 67 nm periodic fibrils and fibers, with the participation of proteoglycans (PG's), which bind collagen by their core protein and regulate collagen fiber diameter through their anionic glycosaminoglycan (AGAG) chains. These collagen-PG interactions are also essential for fiber/fibril stability and determine a number of their mechanical properties
The synovium, or synovial membrane, is a thin sheet of vascularized mesenchymal tissue that surrounds the joint cavity and produces synovial fluid, which is responsible for joint lubrication and chondrocyte nutrition, since avascular cartilage is impermeable to oxygen and nutrients in mature joints
PG's, such as the small leucine rich repeat proteins (sLRRP's) decorin and biglycan, are essential for stabilization of fibril-bundle structures
Part of the RA inflammatory response is release of proteases, such as matrix metalloproteinases (MMPs) and “a disintegrin and metalloproteinases with thrombospondin motifs” (ADAMTSs), which digest the ECM of the synovium and cartilage. The major targets in cartilage are collagen type II, PG's (decorin, biglycan) and aggrecan; their remnants were detected in body fluids of RA patients
Initial ECM degradation, however, may occur in the absence of proteases. Severe mechanical loads as well as changes in pH may cause cartilage fibrillation
A) Native (prior to fixing) type II collagen fibrils, incubated in TBS as control for fibril samples shown in B–D. Average fibril size is around 35 nm. B) Collagen type II fibrils following short incubation with anti-biglycan antibody. Fibril diameter is 10–15 nm. C) Collagen type II fibrils following incubation in GHCl. Although severely disrupted, the fibril decomposition appears less complete than that of the antibody incubation (B). D) Collagen type II sample following mechanical disruption. Disruption of native fibril structure is highly localized, with large sections still intact. E) Native bovine articular cartilage (prior to fixing and staining for TEM). F) Bovine articular cartilage post 1 hour treatment with anti-biglycan. Black arrows point to thin-fibrils, white arrows point to normal sized thick-fibrils.
Some parts of the antibody treated samples maintain a loose alignment of the thin-fibrils allowing them to be analyzed with small angle X-ray diffraction (A), and insert B. An 11 and 4.5 nm packing function are apparent, which appear to correspond to the approximate diameter of the thin-fibrils (insert of C) and microfibrils (D). Native thick fibrils are shown in C as a comparison to the decomposition product (thin-fibrils).
Coordinate models of the biglycan-type II collagen fibril complex based on the decoron-type I collagen fibril structures published recently (1) are shown with a model Fab (green) unit attaching to the biglycan (blue) epitope (colored red, A and ‘top’ view B). Because the epitope is located within a solvent filled channel of the collagen fibril
A) Section of native human articular cartilage, incubated in TBS, that has collagen type II fibrils of regular 30–50 nm diameter (control for samples B–D). B) Section of human articular cartilage treated with ABC lyase for 24 h with some thin fibrils of collagen type II. C) Section of human articular cartilage treated with Guanidine hydrochloride for 24 h with presence of thin 10–15 nm fibrils and normal thick fibrils (fibril bundles). D) Section of human articular cartilage, treated with anti-biglycan antibody for 24 h, shows some collagen type II thin fibrils as well as fibrils of regular 30–50 nm diameter. Arrows point to decomposing fibrils.
A) Control AFM data of type I collagen fibrils: native type I fibrils. B) Control AFM data of type I collagen fibrils: Anti- PG core protein antibody conjugated with 30 nm particles attached to type I fibrils. Note that the fibrils are intact and that the gold particles are clearly discernible as densely packed globules. C) AFM image of native lamprey type II fibrils. D) AFM image of lamprey type II fibrils after treatment with gold particle conjugated anti- biglycan antibodies as in B. Compare with B. No gold particles are visible, whilst in the type I collagen control, they are clearly imaged. E) Unstained TEM of lamprey derived collagen fibrils following anti-biglycan treatment. The antibodies in this preparation were conjugated to 10 nm gold particles and were still able to decompose the thick fibrils into thin-fibrils, but no gold particles are visible after the preparation is washed in TBS. F) Unstained TEM of biglycan aggregates attached to gold particle conjugated antibodies. Gold particles are clearly visible as dense black spots that do not appear in E.
Native fibrils of lamprey notochord do not show any detectable difference between its collagen type II fibers and those seen from the tissues of mammals
TEM images of lamprey tissues treated with the biglycan antibodies showed (
We suggest, that anti-biglycan antibodies attach to biglycan core-proteins on the surface of thick-fibrils and that this interaction disrupts the bonding between the core-protein and the collagen molecules comprising the fibrils. Even though GAG bridges remain intact, the loosening of the biglycan core-protein – collagen interaction ‘unties the string’ that holds the thick-fibrils together (
In order to test the hypothesis that disruption of the biglycan core-protein – collagen interaction accounts for the fibril-debundling, lamprey notochord samples were treated with Guanidine hydrochloride and ABC lyase respectively as a positive control. Guanidine hydrochloride causes protein denaturation, therefore its action would mimic the hypothesized action of the biglycan antibody, albeit in a more caustic and less specific manner. TEM images of Guanidine treated notochord and cartilage illustrated the same type of degradation of collagen fibrils, although the incubation time had to be much longer (24 h instead of 1 h for the antibody) to achieve a similar degree of decomposition. In contrast, ABC lyase removes the AGAG chain from the protein core and should produce the similar results albeit via a different structural mechanism. Thin fibrils in both notochord and articular cartilage samples were seen in TEM images after this treatment, although damage was relatively mild in comparison to the antibody-mediated decomposition. In addition to these chemical methods of tissue degradation, mechanical degradation was also examined. Friction was applied to native lamprey notochord tissues and the results of this damage were analyzed by TEM and compared with the other experiments. Mechanical impact, used for this study, may correspond to damage of the articular cartilage due to trauma or normal wear. The presence of thin fibrils were observed (absence of regular 30–50 nm fibrils) in certain areas, which had higher load, although some parts of tissue still had normal architecture in comparison to undamaged, non-treated control samples. Finally, significant tissue degradation (and biglycan release) was observed in the presence of protease inhibitors but not in the presence of alternative antibodies such as anti-collagen (see SI
Lamprey notochord is a cartilage-like tissue that spans the length of the chordate back, located beneath and parallel to the central nervous system between the brain and tail. Although it is the main axial skeleton at the embryonic stage, the notochord is replaced by the vertebral column in most vertebrates. However, in some chordates it remains into adulthood (e.g., lamprey, lungfish, sturgeon, and some sharks). The mature notochord contains a soft cellular inner part, surrounded by protective fibrous sheath, composed of three layers: inner basal lamina, thick collagenous (cartilage-like) layer, and elastic filamentous membrane
Variations in the different level of damage (thin/normal collagen type II fibrils), obvious in TEM images of mammalian and lamprey tissues, can be explained by differences in the molecular composition of these tissues that in turn influences the tissue architecture. Lamprey notochord contains primarily collagen type II and biglycan. Human and bovine articular cartilage contain biglycan, fibromodulin, decorin, and other ECM molecules, which regulate fibrillogenesis, fiber diameter, support fibers, and give the tissue specific mechanical properties. Cartilage and meniscus contain more biglycan than decorin and the ratio changes from zone to zone. The superficial zone contains about 32% of decorin and 38% of biglycan of all PG content, the inner deep zone contains about 23% of decorin and 53% of biglycan of all PGs, and middle zone has 28% of decorin and 52% of biglycan of all PG's
Although our observations are
Given that the concentration of the antibody used may be physiologically relevant, the nature of its disruptive affect may be the same
Collagen fibrils are assembled in such way that the MMP collagenase cleavage-site is protected by the C-telopeptide in folded conformation
The observed tissue destruction in lamprey notochord and analogous fibrillar decomposition in articular cartilage as a response to anti-biglycan treatment, may have significance to human health and aging. Elevated levels of anti-biglycan antibodies are detected in the body fluids of arthritis patients
Antibodies against biglycan (and perhaps other sLRRP protein cores, although biglycan appears to be the most vulnerable as decorin appears unaffected despite its close homology) binds to the PG-core protein and disrupts the hydrogen-bonding network on the interior of the concave surface that binds to the collagen fibril.
Biglycan core-protein dissociates from the collagen fibrils and the fibril bundle decomposes into thin-fibrils.
Having dissociated from the fibril-bundle, the collagen type II thin-fibrils have a higher exposure of catalytic sites (increased surface area to volume ratio) and become vulnerable to collagenase and gelatinase binding, unwinding and cleavage (see above and refs.
We further speculate, that with the breakdown of these molecular aggregates, a host of normally hidden or low-concentration epitopes, such as PG breakdown products, collagen peptides and so on, would become present in higher concentrations than before. Theoretically, this could initiate a immunological cascade that leads to further tissue autoimmune activity and a worsening of the disease.
Lamprey notochord has a rather simple composition in comparison to mammalian cartilage, but this helps make it both an appealing and suitable model of mammalian systems for initial investigation of normal and pathological processes in cartilage at the fibrillar level. The examined interaction of anti-biglycan antibodies with the notochord ECM can serve as a model for the mechanism of rheumatoid arthritis and has potential as a simplistic RA model system. The visualization of antibody-treated tissues under different conditions inspires a speculative model of tissue destruction similar to that seen in autoimmune-induced rheumatoid arthritis, characterized by collagen matrix decomposition due to PG disruption by the anti-biglycan antibody. This proposed model highlights the crucial position of the biglycan antibody in the development of rheumatoid arthritis and our associated observations provide the first indication of what its role is beyond being a marker of this common and widespread disease.
Adult Sea lampreys were donated by Ludington Biological Station of the U.S. Fish and Wildlife Service and J. Ellen Marsden of the University of Vermont. Human and bovine articular cartilage, stored under physiological conditions, were provided by Tom Schmit and Vincent Wang Rush University. All samples were obtained under the appropriate institutional scientific review boards from the home institutions donating tissues.
Lamprey notochord was extracted from and prepared from dissected lamprey as described previously. Bovine articular cartilage supports scraped from articular surface. Human articular cartilage plugs were harvested from donor tissues. All tissues have been stored in TBS* at 4°C at pH = 7–7.5.
Polyclonal biglycan antibody (Novus, epitope: DRLAIQFGNYKK) with 0.5 mg/ml concentration has been stored in 0.5 ml vials at 4°C. Thin pieces of cartilage/notochord were incubated in antibody solution for 1, 2, and 24 hours. Samples were washed in TBS 2 times for 5 min and stored in TBS pH = 7.0 at 4°C. The specificity of the antibody was previously verified by Chen et al.,
Number of fibril/fiber species per square micron: Number of fibrils/fibers of each type: thin fibrils, thick fibers/fibrils; for each tissue: lamprey notochord, bovine cartilage and human cartilage; in each condition: native (control) and ab treated. The counts are averaged to 1 square micron of area.
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Size of fibril/fiber species: Average diameter of fibrils/fibers of each class for each tissue type (see methods), measured in nm. Note that the presence of large fibril-bundles inflates the determined average size for ‘thick-fibrils’.
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Results for sircol protocol (see SI
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Results for blyscan protocol (see SI
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We thank the Ludington Biological Station of the U.S. Fish and Wildlife Service and J. Ellen Marsden of the University of Vermont for the donation of lamprey specimens, and Dr. Vincent Wang and Dr. Tom Schmidt of Rush University for the donation of cartilage samples. Thanks to Yimei Chen of the Electron Microscopy Center at University of Chicago for help in sample preparation and collection of TEM data and to Jared Lapkovsky for assistance with TEM images obtained on a LVEM5 low-voltage (5 kV) benchtop TEM by Delong America, Montreal, Canada. Our gratitude to collages at IIT for their assistance with gold-labeling and AFM data acquisition; Sandra Bishnoi and Aruna Sundaram. Special thanks to the staff and scientists of the BioCAT group.