FBJ virus-induced osteosarcoma contains type I, type I trimer, type III as well as type V collagens

The FBJ osteosarcoma (a virus-induced osteosarcoma named after its discoverers, Finkel, Biskis, and Jinkins) contains an extensive extracellular matrix. Collagens were extracted by digestion with pepsin in dilute acetic acid from tumors grown in lathyritic mice and fractionated by differential salt precipitation, yielding five fractions. Fraction 1 (precipitated at acidic 0.7 M and neutral 2.0 M NaCl) gave rise mainly to alpha 1(III) chain on phosphocellulose column chromatography. The alpha 1(III) chain was identified by its typical behavior on interrupted electrophoresis and analysis of the CNBr-cleaved peptides. The alpha 1(III) chain of the FBJ tumor had a high content of hydroxylysine and neutral saccharide. Fraction 2 (precipitated at acidic 0.7 M and neutral 4.5 M NaCl) yielded alpha 1(I) and alpha 2(I) chains on the phosphocellulose column from which alpha 1(I) was eluted as a broad peak, conceivably reflecting a high content of hydroxylysine and neutral saccharide. Fraction 4 (precipitated at acidic 1.2 M and neutral 4.5 M NaCl) yielded type V collagen, which also featured an exceptionally high content of neutral saccharide (Yamagata, S., et al. (1982) Biochem. Biophys. Res. Commun. 105, 1208-1214). The proportions of type I, type I trimer, type III, and type V collagens extracted by pepsin digestion from FBJ tumor were calculated to be 33, 29, 26, and 12%, respectively. The FBJ tumor is free from invasion by blood vessels, shows no deposition of calcium, and thus has the appearance of cartilage. But type II collagen, a specific gene product of cartilage, could not be identified in any of the fractions analyzed. Contrary to its appearance, collagen type analyses indicate that FBJ osteosarcoma is literally induced from osteogenic cells.     

Identification of collagen alpha1(I) trimer and normal type I collagen in a polyoma virus-induced mouse tumor.

A bone- and cartilage-forming mouse tumor, induced by transforming salivary epithelial cells with polyoma virus, contained large quantities of collagen. Two types of collagen molecules were isolated which had different solubilities in salt. One was type I collagen with a chain composition [α1(I)]₂ α2 and the other was an unusual form of type I collagen with a chain composition [α1(I)]₃. This would appear to be the first in vivo demonstration of α1 type I trimer.     

Intracellular trafficking and degradation of unassociated proalpha2 chains of collagen type I

Procollagen I is a trimer consisting of two proalpha1(I) chains and one proalpha 2(I) chain. In certain cases of mild osteogenesis imperfecta, abnormal proalpha1(I) chains are degraded very soon after synthesis. As a consequence, the cells produce excess proalpha2(I) chains, which cannot form trimers and are not secreted. The objective of this work was to determine the intracellular fate of unassociated proalpha2(I) chains. Mov13 mouse fibroblasts, which do not synthesize proalpha1(I) mRNA, but do produce proalpha2(I) mRNA, were incubated with radioactive amino acids using pulse-chase protocols, and proteins were analyzed by gel electrophoresis, autoradiography, and Western blotting. Mov13 cells produced proalpha2(I) chains that were degraded intracellularly within 30 min. Degradation was inhibited when cells were treated with brefeldin-A, which blocks transit from endoplasmic reticulum to Golgi. Fixed cells exposed to various immunofluorescence markers and imaged by confocal laser scanning microscopy showed that proalpha2(I) chains colocalized with Golgi and lysosome markers. Degradation was inhibited and chains were secreted when cells were treated with wortmannin, which blocks trafficking to lysosomes. These results demonstrate that unassociated proalpha2(I) chains leave the endoplasmic reticulum, transit the Golgi, and enter lysosomes where they are degraded.     

Differential unfolding of alpha1 and alpha2 chains in type I collagen and collagenolysis

Collagenolysis plays a central role in many disease processes and a detailed understanding of the mechanism of collagen degradation is of immense interest. While a considerable body of information about collagenolysis exists, the details of the underlying molecular mechanism are unclear. Therefore, to further our understanding of the precise mechanism of collagen degradation, we used molecular dynamics simulations to explore the structure of human type I collagen in the vicinity of the collagenase cleavage site. Since post-translational proline hydroxylation is an important step in the synthesis of collagen chains, we used the DNA sequence for the α1 and α2 chains of human type I collagen, and the known amino acid sequences for bovine and chicken type I collagen, to infer which prolines are hydroxylated in the vicinity of the collagenase cleavage site. Simulations of type I collagen in this region suggest that partial unfolding of the α2 chain is energetically preferred relative to unfolding of α1 chains. Localized unfolding of the α2 chain leads to the formation of a structure that has disrupted hydrogen bonds N-terminal to the collagenase cleavage site. Our data suggest that this disruption in hydrogen bonding pattern leads to increased chain flexibility, thereby enabling the α2 chain to sample different partially unfolded states. Surprisingly, our data also imply that α2 chain unfolding is mediated by the non-hydroxylation of a proline residue that is N-terminal to the cleavage site in α1 chains. These results suggest that hydroxylation on one chain (α1) can affect the structure of another chain (α2), and point to a critical role for the non-hydroxylation of proline residues near the collagenase cleavage site.     

Interaction of fibronectin and its gelatin-binding domains with fluorescent-labeled chains of type I collagen

Fluorescent probes have been used to obtain dissociation constants for the fluid-phase interaction of human plasma fibronectin and several of its gelatin-binding fragments with purified alpha chains of type I rat tail collagen, as well as with a cyanogen bromide fragment (CB7) of the alpha 1 chain in 0.02 M Tris buffer containing 0.15 M NaCl at pH 7.4. Addition of fibronectin to fluorescein-labeled collagen chains caused a dose-dependent increase in the fluorescence anisotropy which continued over several logs of titrant concentration. Scatchard-type plots of the anisotropy response were biphasic indicating the presence of one or more weak sites (Kd greater than microM) along the collagen chain in addition to a strong site characterized by Kd = 1.3 X 10(-8) M at 25 degrees C. Gelatin-binding fragments with Mr = 42,000, 60,000, and 72,000 also produced a biphasic response with Kd values for the high affinity site being 10- to 20-fold greater than for intact fibronectin. Binding of fibronectin and its fragments to fluorescent-labeled CB7 was essentially the same as to the whole alpha 1 chain. In all cases, the anisotropy response could be reversed or prevented by addition of excess unlabeled gelatin or CB7, but not by synthetic peptides spanning the collagenase cleavage site of alpha 1 (I). Studies of the temperature dependence of Kd for binding of fibronectin to the high affinity site on alpha 1 produced a value of +16 kcal/mol for the enthalpy of dissociation below 30 degrees C. Above this temperature, fibronectin appeared to undergo a subtle conformational transition characterization by a reduced affinity for collagen. This transition occurred in whole fibronectin but not in the gelatin-binding fragments and may involve disruption of intramolecular interactions between different domains.     

Proteasomal degradation of unassembled mutant type I collagen pro-alpha1(I) chains.

We have previously shown that type I procollagen pro-alpha1(I) chains from an osteogenesis imperfecta patient (OI26) with a frameshift mutation resulting in a truncated C-propeptide, have impaired assembly, and are degraded by an endoplasmic reticulum-associated pathway (Lamandé, S. R., Chessler, S. D., Golub, S. B., Byers, P. H., Chan, D., Cole, W. G., Sillence, D. O. and Bateman, J. F. (1995) J. Biol. Chem. 270, 8642-8649). To further explore the degradation of procollagen chains with mutant C-propeptides, mouse Mov13 cells, which produce no endogenous pro-alpha1(I), were stably transfected with a pro-alpha1(I) expression construct containing a frameshift mutation that predicts the synthesis of a protein 85 residues longer than normal. Despite high levels of mutant mRNA in transfected Mov13 cells, only minute amounts of mutant pro-alpha1(I) could be detected indicating that the majority of the mutant pro-alpha1(I) chains synthesized are targeted for rapid intracellular degradation. Degradation was not prevented by brefeldin A, monensin, or NH(4)Cl, agents that interfere with intracellular transport or lysosomal function. However, mutant pro-alpha1(I) chains in both transfected Mov13 cells and OI26 cells were protected from proteolysis by specific proteasome inhibitors. Together these data demonstrate for the first time that procollagen chains containing C-propeptide mutations that impair assembly are degraded by the cytoplasmic proteasome complex, and that the previously identified endoplasmic reticulum-associated degradation of mutant pro-alpha1(I) in OI26 is mediated by proteasomes.     

The mammalian chitinase-like lectin, YKL-40, binds specifically to type I collagen and modulates the rate of type I collagen fibril formation

YKL-40 is expressed in arthritic cartilage and produced in large amounts by cultured chondrocytes, but its exact role is unclear, and the identities of its physiological ligands remain unknown. Purification of YKL-40 from resorbing bovine nasal cartilage and chondrocyte monolayers demonstrated the existence of three isoforms, a major and minor form from resorbing cartilage and a third species from chondrocytes. Affinity chromatography experiments with purified YKL-40 demonstrated specific binding of all three forms to collagen types I, II, and III, thus identifying collagens as potential YKL-40 ligands. Binding to immobilized type I collagen was inhibited by soluble native ligand, but not heat-denatured ligand, confirming a specific interaction. Binding of the chondrocyte-derived species to type I collagen was also demonstrated by surface plasmon resonance analysis, and the dissociation rate constant was calculated (3.42 x 10(-3) to 4.50 x 10(-3) s(-1)). The chondrocyte-derived species was found to prevent collagenolytic cleavage of type I collagen and to stimulate the rate of type I collagen fibril formation in a concentration-dependent manner. By contrast, the cartilage major form had an inhibitory effect on type I collagen fibrillogenesis. Digestion with N-glycosidase F, endoglycosidase H and lectin blotting did not reveal any difference in the carbohydrate component of these two YKL-40 species, indicating that this does not account for the opposing effects on fibril formation rate.     

Distribution of extracellular matrix proteins type I collagen, type IV collagen, fibronectin, and laminin in mouse folliculogenesis

The extracellular matrix (ECM) plays a prominent role in ovarian function by participating in processes such as cell migration, proliferation, growth, and development. Although some of these signaling processes have been characterized in the mouse, the relative quantity and distribution of ECM proteins within developing follicles of the ovary have not been characterized. This study uses immunohistochemistry and real-time PCR to characterize the ECM components type I collagen, type IV collagen, fibronectin, and laminin in the mouse ovary according to follicle stage and cellular compartment. Collagen I was present throughout the ovary, with higher concentrations in the ovarian surface epithelium and follicular compartments. Collagen IV was abundant in the theca cell compartment with low-level expression in the stroma and granulosa cells. The distribution of collagen was consistent throughout follicle maturation. Fibronectin staining in the stroma and theca cell compartment increased throughout follicle development, while staining in the granulosa cell compartment decreased. Heavy staining was also observed in the follicular fluid of antral follicles. Laminin was localized primarily to the theca cell compartment, with a defined ring at the exterior of the follicular granulosa cells marking the basement membrane. Low levels of laminin were also apparent in the stroma and granulosa cell compartment. Taken together, the ECM content of the mouse ovary changes during follicular development and reveals a distinct spatial and temporal pattern. This understanding of ECM composition and distribution can be used in the basic studies of ECM function during follicle development, and could aid in the development of in vitro systems for follicle growth.     

Dermal collagen fibrils are hybrids of type I and type III collagen molecules

It has been suggested that dermal collagen fibrils with 67-nm periodicity consist of hybrids of type I and type III collagens. This is based on the assumption that all these banded fibrils are coated with type III collagen regardless of their diameter. However, conclusive evidence for this form of hybridization is lacking. In order to clarify this problem dermal collagen fibrils were disrupted into microfibrils using 8 M urea. Single and double indirect immunoelectron microscopy showed type III collagen at the periphery of intact collagen fibrils but no labeling with type I collagen antibodies, suggesting that the epitopes for this collagen were masked. Disrupted collagen fibrils revealed type I collagen throughout the fibril except for the periphery which was coated with type III collagen. Almost no type III collagen was noted in the interior of the collagen fibrils. Since type III collagen is present only at the periphery it suggests that this collagen has a different role than type I collagen and may have a regulatory function in fibrillogenesis.     

Growth on type I collagen promotes expression of the osteoblastic phenotype in human osteosarcoma MG-63 cells.

Using MG-63 cells as a model system capable of partial osteoblastic differentiation, we have examined the effect of growth on extracellular matrix. MG-63 cell matrix and purified type I collagen induced a morphological change characterized by long cytoplasmic processes reminiscent of those seen in osteocytes. Concurrent biochemical changes involving bone marker proteins included increased specific activity of cell-associated alkaline phosphatase and increased secretion of osteonectin (up to 2.5-fold for each protein); all changes occurred without alterations in the growth kinetics of the MG-63 cells. The increase in alkaline phosphatase activity was maximal on days 6-8 following seeding; increased osteonectin secretion was most prominent immediately following seeding; all changes decreased as cells reached confluence. Growing cells on type I collagen resulted in an increased induction of alkaline phosphatase activity by 1,25(OH)2D3 (with little change in the 1,25(OH)2D3 induction of osteonectin and osteocalcin secretion), and increased TGF-beta induction of alkaline phosphatase activity as well (both TGF-beta 1 and TGF-beta 2). Both the 1,25(OH)2D3 and TGF-beta effects appeared to be synergistic with growth on type I collagen. These studies support the hypothesis that bone extracellular matrix may play an important role in osteoblastic differentiation and phenotypic expression.     

Binding of human monomeric type I collagen to platelets

Interaction of platelets with subendothelial collagen is important in primary hemostasis and thrombosis. Although activation of platelets by collagen polymers has been widely investigated, only insufficient data are available concerning the binding of genetically distinct collagen types in their triple helical (monomeric) form to platelets. We report on the binding of 125I-labeled human type I collagen to platelets. The binding assay was performed at 20 degrees C in the presence of arginine in order to prevent polymerization of the collagen monomers. The binding of monomeric 125I-labeled human type I collagen is dose- and time-dependent, saturable and specific, since it is competitively inhibited by unlabeled type I collagen, but not by unlabeled human type V collagen. Scatchard analysis reveals a class of specific high affinity binding sites with a Kd of 2.5 X 10(-8) M. These results suggest that platelets interact with type I collagen through specific binding sites, and that there are various different binding sites on the platelet membrane for the genetically distinct collagen types.     

A simple method for quantitative estimation of collagen type III to type I ratio in soft tissues

In the present paper a relatively rapid and simple method for estimating the ratio of collagen type III to type I in soft tissues is proposed. The ratio Gly/Ala is determined in pure collagen samples obtained from pepsin digests of the tissues. Since this ratio varies linearly depending on the composition of the mixtures of the two collagen types, it is shown that the percentage content of the two collagen types is easily calculated.     

Membrane glycoproteins involved in hepatocyte adhesion to collagen type I

Liver membrane glycoproteins with affinity for immobilized collagen type I were subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electroelution of the separated proteins. Electroeluted glycoproteins with ability to neutralize the inhibitory effect of anti-CollCAM antibodies on hepatocyte adhesion to collagen were collected from several consecutive runs and used to raise a high titer antiserum, denoted anti-CollCAM II. IgG from this antiserum inhibited the attachment of hepatocytes to dishes coated with collagen type I, but not to fibronectin- or collagen type IV-coated dishes. When the antibodies were immobilized to Sepharose CL-4B they bound three sets of glycoproteins with apparent Mr's of 105,000, 115,000, and 130,000 as analyzed by SDS-PAGE under nonreducing (NR) conditions. Upon reduction (R) the glycoproteins migrated with apparent Mr's of 115,000, 130,000, and 160,000, respectively. The Mr 105,000-115,000 (NR) glycoproteins effectively neutralized the inhibitory effect exerted by both anti-CollCAM and anti-CollCAM II antibodies, on hepatocyte spreading and attachment to collagen type I substrates. Peptide mapping suggested the Mr 160,000 (R) species to be different from the Mr 115,000 (R).     

Insulin binds to type V collagen with retention of mitogenic activity

The abilities of eight extracellular matrix proteins, fibronectin, vitronectin, laminin, and collagen types I, II, III, IV, and V to bind insulin were examined by binding studies with insulin conjugated with peroxidase. At a physiological pH and ionic strength, type V collagen bound to insulin most strongly. The other types of collagen, laminin, and vitronectin also bound insulin with affinity lower than that of type V collagen. The insulin-binding site of type V collagen was in a 30-kDa CNBr fragment of the alpha 1 (V) chain. Analysis of the amino acid sequence showed that this 30-kDa fragment was identical to the heparin-binding fragment of type V collagen. The insulin-binding sites of laminin and vitronectin were located in the A chain and in the heparin-binding domain, respectively. Insulin bound to type V collagen stimulated the synthesis of DNA by mouse mammary tumor MTD cells, indicating that bound insulin retained mitogenic activity.     

Characterisation of the immune response to type I collagen in scleroderma

This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSE^low). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4⁺, activated (CD25⁺), memory (CD45RO⁺) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-γ but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-γ, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-α, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls.     

Liquid crystallinity in condensed type I collagen solutions. A clue to the packing of collagen in extracellular matrices.

We recently described a new type of assembly of collagen molecules, forming typical liquid crystalline phases in highly concentrated solutions after sonication. The present work shows that intact 300 nm long collagen molecules also form cholesteric liquid crystalline domains, but the time required is much longer, several weeks instead of several days. Differential calorimetry and X-ray diffraction show that sonication does not alter the triple-helical structure of the collagen fragments. In the viscous solutions, observed between crossed polars in optical microscopy, the textures vary as a function of the concentration. Molecules first align near the air interface at the coverslip edge, then as the concentration increases by slow evaporation of the solvent, the birefringence extends inwards and liquid crystalline domains progressively appear. For concentrations estimated to be above 100 mg/ml, typical textures and defects of cholesteric phases are obtained, at lower concentrations zig-zag extinction patterns and banded patterns are observed; all these textures are described and interpreted. The cholesteric packing of collagen fibrils in various extracellular matrices is known, and the relationship that can be made between the ordered phases obtained with collagen molecules in vitro and the related geometrical structures observed between fibrils in vivo is thoroughly discussed.     

Modulation of Pseudomonas aeruginosa adherence to collagen type I and type II by carbohydrates

Abstract This study was undertaken to examine if receptor recognizing saccharides may be involved in the adherence of Pseudomonas aeruginosa to collagen type I and type II. We performed an adherence inhibition assay: cells of individual P. aeruginosa isolates attached to immobilized collagen type I or type II in the presence of monosaccharides, which could serve as blockers of bacterial receptors. Bacterial binding to collagen type I molecules was inhibited to the highest degree by sugar composition d -galactose/ d -mannose/ N -acetylneuraminic acid (5:5:1), whereas attachment of P. aeruginosa to collagen type II was inhibited by composition d -glucose/ d -galactose (1:1). The same strains which were sensitive to inhibition of binding to collagen type II by both collagen types, were also sensitive to blocking by composition d -glucose/ d -galactose. It suggests that saccharides play a role in adherence of P. aeruginosa to collagen type I and type II, and a common receptor for both types of collagen may be available on the surface of P. aeruginosa cells.