Minor Collagen Research Articles

The effect of ascorbate on the synthesis of minor (non-interstitial) collagens by cultured bovine aortic smooth muscle cells

Ultrastructural and biochemical studies were carried out on bovine aortic smooth muscle cells cultured in the presence or absence of ascorbate. In its absence, electron microscopic examination of cultures revealed that the extracellular components consisted primarily of microfibrils. Morphologically identifiable collagen fibrils were only observed in the matrix upon ascorbate supplementation. Smooth muscle cells grown in ascorbate-free media synthesized large amounts of type VI collagen. The identity of the latter was confirmed by ion exchange chromatography, slab gel electrophoresis, and amino acid analysis. Addition of ascorbate resulted in a stimulation of type I collagen production, levels of the type III remained constant, and types V and VI were decreased. Since, in the absence of ascorbate, smooth muscle cells are known to synthesize predominantly elastin, the present data support the contention that the type VI collagen and the microfibrillar component of elastic tissue are either identical or similar.

Type II collagen of lamprey

1. 1. The major collagen in lamprey notochord is type II, as determined by its amino acid composition and solubility properties. 2. 2. This collagen has a distribution of charged residues indistinguishable from higher vertebrate Type II collagens as judged by its SLS banding pattern. 3. 3. Lamprey type II collagen has a higher thermal stability than lamprey skin collagen, in contrast to the identical melting temperatures for these types in mammals. 4. 4. A minor collagen in lamprey notochord has solubility properties, amino acid composition, and electrophoretic mobility similar to that of 1α, 2α, 3α collagen in human cartilage.

Auftreten und Spezifität von Antikörpern gegen verschiedene Kollagentypen (II, Kurzkettige, IX) bei Arthrose

Auftreten und Spezifität von Antikörpern gegen verschiedene Kollagentypen (II, Kurzkettige, IX) bei Arthrose

Morphology of the pericellular capsule in articular cartilage revealed by hyaluronidase digestion

To allow a more valid comparison between our previous ultrastructural data and the immunolocalization of type IX and other minor collagen species in cryosectioned cartilage, we examined both normal and testicular hyaluronidase-digested canine tibial cartilage by electron microscopy. Removal of matrix proteoglycans caused the pericellular capsule to collapse against the cell surface, suggesting that its normal anatomical position is mediated by pericellular matrix hydration. Detailed examination of the pericellular capsule and pericellular channel revealed fine, faintly banded fibrils and an amorphous component somewhat similar in structure to basement membrane collagens. Matrix vesicles and the electron-dense material of the interterritorial matrix were only partially digested by hyaluronidase. We propose that the pericellular capsule is composed of a "felt-like" network of minor collagen species which act synergistically to maintain both the composition of the pericellular matrix and the integrity of the chondrocyte/pericellular matrix complex during compressive loading.

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.

Changes in the synthesis of minor cartilage collagens after growth of chick chondrocytes in 5-bromo-2′-deoxyuridine or to senescence

Analyses were made of the minor collagens synthesized by cultures of chondrocytes derived from 14-day chick embryo sterna. Comparisons were made between control cultures, cultures grown for 9 days in 5-bromo-2′-deoxyuridine (BrdU) and clones of chondrocytes grown to senescence. Separation of minor collagens from interstitial collagens was achieved by differential salt precipitation in the presence of carrier collagens in acid conditions. The precipitate at 0.9 M NaCl 0.5 M acetic acid from control cultures was shown by CNBr peptide analysis to contain only the α1(II) chain of type II collagen, whereas after BrdU treatment or growth to senescence synthesis of only α 1(I) and α2(I) chains occurred. The synthesis of type III collagen was not detected. Analysis of the precipitate at 2.0 M NaCl, 0.5 M HAc from control cultures demonstrated the synthesis of 1α, 2α and 3α chains together with the synthesis of short chain (SC) collagen of M r 43 000 after pepsin digestion. After BrdU treatment or growth to senescence a chains were isolated which possessed the migration positions on polyacrylamide gel electrophoresis (PAGE), or the elution positions on CM-cellulose chromatography, of the α1(V) and α2(V) chains of type V collagen. In addition, for BrdU-treated but not for control cultures, intracellular immunofluorescent staining was observed with a monoclonal antibody which specifically recognizes an epitope present in the triple helix of type V collagen. Synthesis of short chain (SC) collagen was not detected after BrdU treatment or growth to senescence. These results suggest that chick chondrocytes grown in conditions known to cause switching of collagen synthesis from type II to type I collagen also undergo a switch from the synthesis of 1α, 2α and 3α chains to the synthesis of the α1(V) and α2(V) chains of type V collagen. It appears that there are several cartilage-specific collagens which together undergo a regulatory control to the synthesis of collagens typical of other connective tissues.

All three chains of 1α2α3α collagen from hyaline cartilage resist human collagenase

A previous report that the 3α collagen chain of hyaline cartilage was cleaved by human collagenase could not be confirmed when the 1α2α3α collagen fraction was freed of all contaminating type II collagen. All three minor collagen chains, 1α, 2α and 3α, were totally resistant to highly purified collagenases from both rheumatoid synovial and gastric mucosal tissues. This finding and CNBr-peptide patterns suggest that, despite the close homology with α1(II), the 3α chain is a unique collagen component, possibly combined with 1α and 2α in heterotrimeric molecules. In contrast, a 3α-like component from fibrocartilage was cleaved by collagenase and gave a CNBr-peptide pattern more typical of α1(II) than of the collagenase-resistant 3α of hyaline cartilage.

Biosynthesis and processing of collagens in different cartilaginous tissues.

The distribution, structure, and biosynthesis of various collagen types have been studied in growth and structural cartilage from young rabbits. The major collagen of cartilage is alpha 1(II); however, all cartilage matrix also contains 1 alpha, 2 alpha, 3 alpha (Type Cm), as well as a high molecular weight disulfide-linked collagen (Type M). Cartilage fragments in organ culture demonstrate synthesis of precursors of collagen alpha chains and processing to their final forms. Although Type Cm collagen is present in the same proportion in the matrix of growth and structural cartilage, in vitro radiolabeling of rabbit cartilage showed that only growth cartilage is capable of actively synthesizing Type Cm, except in the newborn period when synthesis of Type Cm does occur in structural cartilage. A low molecular weight collagen (designated G collagen) is synthesized in vitro by growth cartilage but not by structural or articular cartilage. Preferential distribution of these minor collagens in growth cartilage suggests a role in development during normal cartilage growth.

1 Alpha 2 alpha 3 alpha collagen is arthritogenic.

Native 1 alpha 2 alpha 3 alpha collagen (500 micrograms per rat) was both immunogenic and arthritogenic in Alderley Park rats (46% developed arthritis) but only immunogenic in Sprague-Dawley rats. Conversely, native type II collagen (500 micrograms per rat) was immunogenic and arthritogenic in both strains (64% arthritic in Alderley Park strain, 57% arthritic in Sprague-Dawley strain). The inflammatory polyarthritis induced by 1 alpha 2 alpha 3 alpha collagen was similar to that produced by native type II collagen in clinical appearance, time of onset, and histology. Antibodies raised to native bovine type II collagen cross-reacted with native 1 alpha 2 alpha 3 alpha collagen and vice versa. Thus the minor collagen component of cartilage, the 1 alpha 2 alpha 3 alpha collagen, as well as the major collagen component, type II collagen, are immunogenic and arthritogenic in the rat, with strain differences.

P-HMW-collagen, a minor collagen obtained from chick embryo cartilage without proteolytic treatment of the tissue.

The fragments of minor collagens of cartilages, called HMW and LMW, were isolated after pepsin treatment of sternal cartilages of young chickens and were shown to be entirely triple-helical molecules as judged by their circular dichroic spectra. Studies on renaturation kinetics of HMW suggested that the interchain disulfide bonds in HMW reside at one of the ends of the so-called long arm. Polyclonal antibodies against HMW were raised and affinity purified. These antibodies did not cross-react with type II collagen nor with other minor collagens such as LMW and 1 alpha, 2 alpha, 3 alpha collagen in native or denatured structure. The antibodies were used to identify HMW-related molecules which were synthesized by embryonic chick cartilages in vitro. Some of these molecules were secreted into the organ culture medium and could be recovered from it by ammonium sulfate precipitation. Polyacrylamide gel electrophoresis of this precipitate gave one band of high molecular weight which could be reduced to two bands migrating slightly faster than the alpha 1(II) chain when identified by immunoblotting. These bands could also be identified among about six radiolabelled polypeptides present in the ammonium sulfate precipitate of medium proteins when analysed by polyacrylamide gel electrophoresis followed by fluorography. The same polypeptides could be recovered from the medium by immunoprecipitation with anti-HMW antibodies. Their presence in cartilage tissue was shown by immunoblotting of material extracted from cartilage tissue and separated on polyacrylamide gels. We suggest that the protein containing these polypeptide chains represents the parent molecule of the peptic fragment HMW as it is synthesized in vivo and have designated it p-HMW-collagen.

Collagen of fibrocartilage: a distinctive molecular phenotype in bovine meniscus

Fibrocartilage of bovine knee meniscus was analyzed for major and minor collagen constituents. The main fraction (∼98%) of pepsin-solubilized collagen consisted of type I with a small proportion (< 10%) of type III molecules. The minor fraction (1–2%) isolated by salt precipitation could be further resolved into type V collagen that consisted of α1(V) and α2(V) chains and a type II-like molecule with chains that had all the characteristics of the 3α variant of α1(II) found in hyaline cartilage. The articular surface zone of the meniscus appeared 2–3-fold enriched in these minor collagens compared with deeper tissue, though qualitatively the same distinctive collagen phenotype was evident throughout.

Minor collagens in arthrotic human cartilage. Change in content of 1 alpha, 2 alpha, 3 alpha and M-collagen with age and in osteoarthrosis.

The content and distribution of 1 alpha, 2 alpha, 3 alpha and M-collagens in human articular cartilage were studied. As controls, normal femoral heads and costal cartilage of autopsy material from newborn to 91-year-old persons were used. The osteoarthrotic cartilage was obtained from patients undergoing total hip replacement aged 45-80. The pepsin-digested cartilage collagen was fractionated by differential salt fractionation. The collagen content of the fractions was determined, and the fractions were separated by polyacrylamide slab gel electrophoresis. In the extracted collagen, the type II collagen varied from 82 to 97 per cent with increasing age. The 1 alpha, 2 alpha and 3 alpha chains decreased. M-collagen, especially of the high molecular weight components, disappeared with age. In osteoarthrosis three types of change - degeneration, new fibrocartilage formation on the surface of osteophyte and reparative cartilage - were separately studied. In all types of osteoarthrosis, an increase of minor collagens was found. In newly formed fibrocartilage, the reappearance of M-collagen was conspicuous. It is proposed that the three types of osteoarthrotic cartilage may be characterized on the basis of content and distribution of minor collagens.

Biochemical properties and immunolocalization of minor collagens in foetal calf cartilage

Type II collagen is the major collagen of cartilage. However, new collagenous chains have been described in different cartilaginous tissues, recently. The la, 2a and 3a chains were extracted from human and bovine hyaline cartilage [1]. Other minor collagenous components were extracted from neonatal pig and human cartilage noted M, CFI, CF2 [2-4], from bovine nasal cartilage and human intervertebral disc noted CPS1, CPS2 [5,6] and from chicken sternal cartilage noted HMW, LMW [7,8] and M 1, M 2 [9]. Here, we report the partial characterization of 3 collagenous fractions obtained after limited pepsin treatment of foetal calf cartilage and isolated according to their solubility properties. The 1.2 M NaC1 fraction contains the la, 2t~ and 3a chains. The 2.0 M NaC1 and 3.0 M NaCl fractions contain at least 7 collagenous chains, which are related to the disulfide-bonded new chains enumerated above, but show some major differences, Antibodies against the 1.2 M and 2.0 M NaC1 fractions were raised in rabbits and their specificity tested by radioimmunoprecipitation. The localization of the corresponding collagenous chains was achieved by indirect immunofluorescence in epiphyseal proper and growth cartilage from foetal calf cartilage. 2. MATERIALS AND METHODS

Two genetically distinct molecular species of octopus muscle collagen

The arm muscular tissue of an octopus, Octopus vulgaris, was subjected to limited proteolysis with pepsin and the solubilized tissue collagen was separated into two fractions by selective salt precipitation. Biochemical characterization of the major collagen fraction precipitating at 0.45 M NaCl (pH 2.6) has demonstrated that it comprises almost a single type of molecular species with structure (α1) 2α2, which was identified by its CNBr-peptide pattern as being similar to Type I-like collagen of octopus skin. On the other hand, the minor collagen fraction precipitating at 0.90 M NaCl (pH 2.6) was found to contain a unique γ-chain-sized component cross-linked by disulfide bonds. Sepharose CL-4B gel filtration of the reduced γ component revealed the presence of an α component, which is virtually identical in amino acid composition to the original γ protein and bears a close resemblance to known basement membrane collagens. This unique α component was genetically distinct from α1 and α2 chains of the major muscle collagen as judged by their CNBr-peptide patterns.

Affinity Chromatography of Collagen on Collagen-Binding Fragments of Fibronectin

The affinity of fibronectin for collagen was exploited in biospecific affinitychromatography of collagen. Matrices with excellent capacity and stability were obtained by coupling collagen-binding fragments of fibronectin to Sepharose. Collagen-binding tryptic (30,000 daltons) and chymotryptic (45,000 daltons) fragments, lacking the binding sites of intact fibronectin for various other substances, were coupled to Sepharose and used to chromatograph gelatin and type I collagen. Gelatin was rapidly and quantitatively bound to these matrices at 4°C and 37°C, while binding of type I collagen took place more slowly and was temperature dependent. The collagen did not bind at 4°C but bound quantitatively at 37°C if preincubated at this temperature. These results suggest that a temperature-dependent perturbation of the triple helical structure of the collagen uncovers the binding site for fibronectin, allowing the collagen to bind to the insolubilized collagen-binding fibronectin fragments. Enzyme affinity assays showed that the conformational change in the fibronectin-binding region of collagen was irreversible. Affinity chromatography on collagen-binding fragments of fibronectin could provide a method for the study of structure and function of collagens and may prove useful for the isolation of minor collagens.

Minor collagens of chicken hyaline cartilage.

Minor collagens of chicken hyaline cartilage.