Structural and immunological characterization of type IV collagen isolated from chicken tissues.

Previously, a type IV collagen fraction was isolated from chicken gizzard and further fractionated into three components called F1, F2 and F3 [Mayne, R. and Zettergren, J.G. (1980) Biochemistry, 19, 4065-4072]. F1 and F2 were consistently isolated in a 2:1 proportion, and the existence of a small native fragment of structure (F1)2F2 was proposed. In the present series of experiments, a type IV collagen fraction was isolated from the chicken kidney and shown to consist almost entirely of F1 and F2 which were again present in a 2:1 proportion. Identical one-dimensional peptide maps for F1 and F2 from both sources were obtained by polyacrylamide gel electrophoresis of peptides obtained after cleavage with cyanogen bromide or Staphylococcus aureus V8 protease. The denaturation temperature of a preparation containing F1 and F2 in native form was determined by optical rotatory dispersion and a single melting curve was observed with a melting temperature of 33 degrees C. This result provides further supportive evidence that F1 and F2 exist as a native fragment (F1)2F2. Antibodies were prepared in rabbits against a type IV collagen fraction isolated from chicken gizzard, and immunofluorescent staining of a wide variety of basement membranes was demonstrated. Experiments were performed in which various type IV collagen fractions were observed in the electron microscope after rotary shadowing. The lengths of (F1)2F2 and F3 were 147 nm and 174 nm respectively, the sum of these lengths (321 nm) corresponding closely to the length of the major triple-helical domain of type IV collagen (326-328 nm). A specific cleavage site was located at a distance of 215 nm from the 7-S domain which, together with the length of (F1)2F2, gives a total length of 362 nm. This value corresponds closely to the maximum length of the arms which originate from the 7-S domain (355 nm) when type IV collagen was solubilized with a low concentration of pepsin. The results suggest that (a) type IV collagen isolated from the chicken gizzard is closely related, if not identical, to type IV collagen isolated from other tissues; (b) there is a single type IV collagen molecule of chain organization[alpha 1(IV)]2 alpha2(IV); (c) the order of the pepsin-resistant fragments within a type IV molecule is 7S-F3-(F1)2F2.

The isolation and partial characterization of chondronectin, an attachment factor for chondrocytes.

We have previously demonstrated that the adhesion of embryonic chick sternal chondrocytes to a type II-collagen substrate is not mediated by fibronectin but rather by a distinct attachment factor which we have named chondronectin. Here we describe the isolation, properties, and biological activity of chondronectin prepared from chicken serum. Chondronectin is shown to be a glycoprotein with an estimated Mr = 180,000. After reduction, it migrates as subunits of Mr = 70,000. Antibodies directed against chondronectin inhibited the attachment of chondrocytes to type II collagen. Chondronectin is immunologically distinct from either fibronectin or laminin. Immunofluorescence studies on frozen sections of embryonic chick sternal cartilage and of cultured sternal chondrocytes showed that chondronectin is cell-associated rather than a major matrix component.

Rates of synthesis of basement membrane proteins by differentiating teratocarcinoma stem cells and their modulation by hormones.

The embryonal carcinoma mouse cell line F-9 was used as a convenient model for a quantitative study of the production of the basement membrane proteins laminin and type IV collagen. Both proteins could be identified in the culture medium and cell layer by radioimmuno assays, metabolic labeling and immunofluorescence. More than 95% of the material is secreted into the medium. Lack of ascorbic acid inhibits secretion of type IV collagen but not of laminin. Induction of differentiation into endoderm-like cells by retinoic acid consistently caused after a lag period of 2-3 days a 5-10 fold increase in the production of basement membrane proteins but not of total protein. Dibutyryl cyclic AMP further potentiated this specific effect particularly with respect to type IV collagen synthesis. Insulin, epidermal growth factor and nerve growth factor produced only moderate increases (10-60%) in the amount of laminin and type IV collagen. Effects of these hormones were only observed with certain doses and were quite variable between different experiments.

Extracellular matrix formation by chondrocytes in monolayer culture.

In previous studies were have reported on the secretion and extracellular deposition of type II collagen and fibronectin (Dessau et al., 1978, J. Cell Biol., 79:342-355) and chondroitin sulfate proteoglycan (CSPG) (Vertel and Dorfman, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:1261-1264) in chondrocyte cultures. This study describes a combined effort to compare sequence and pattern of secretion and deposition of all three macromolecules in the same chondrocyte culture experiment. By immunofluorescence labeling experiments, we demonstrate that type II collagen, fibronectin, and CSPG reappear on the cell surface after enzymatic release of chondrocytes from embryonic chick cartilage but develop different patterns in the pericellular matrix. When chondrocytes spread on the culture dish, CSPG is deposited in the extracellular space as an amorphous mass and fibronectin forms fine, intercellular strands, whereas type II collagen disappears from the chondrocyte surface and remains absent from the extracellular space in early cultures. Only after cells in the center of chondrocyte colonies shape reassume spherical shape does the immunofluorescence reveal type II collagen in the refractile matrix characteristic of differentiated cartilage. By immunofluorescence double staining of the newly formed cartilage matrix, we demonstrate that CSPG spreads farther out into the extracellular space that type II collagen. Fibronectin finally disappears from the cartilage matrix.

Immunofluorescence analysis of collagen, fibronectin, and basement membrane protein in scleroderma skin.

Scleroderma skin and the subcutaneous tissue was studied by indirect immunofluorescence with specific antibodies against interstitial collagens and procollagens, against fibronectin and against the basement membrane proteins Type IV collagen and laminin. Staining for Type I procollagen and fibronectin was distinctly increased in the lower dermis and subcutaneous tissue. When compared with normal skin the data suggests that fibrosis may start around capillaries and in close proximity to adipose cells. Additional changes in the distribution to Type IV collagen and laminin were found in some patients and probably reflect the alterations in small blood vessels.

Changes in the patterns of collagens and fibronectin during limb-bud chondrogenesis.

The distribution and sequence of appearance of fibronectin and of type-I and type-II collagen in the developing cartilage models of embryonic chick hind-limb buds was studied by immunofluorescence, using specific antibodies directed against these proteins. Fibronectin and type-I collagen are evenly distributed throughout the intercellular space of the mesenchyme prior to condensation of core mesenchyme of the limb anlage and formation of the cartilage blastema. With the onset of the condensation process fibronectin and type-I collagen appear to increase in the cartilage blastema compared to the surrounding loose mesenchyme, reaching a maximal density at the time of cartilage differentiation. The latter process is marked by the appearance of type-II collagen in the cartilage blastema. As cartilage differentiation progresses, type-I collagen is gradually replaced by type-II collagen; fibronectin disappears and is completely absent from mature cartilage. The transient appearance of type-I collagen and fibronectin suggests a temporal role in cell-matrix or cell-cell interactions in chondrogenesis, since it had been shown that (a) type-I collagen substrates stimulate cell proliferation and cartilage differentiation in limb-bud mesenchyme cell cultures; (b) fibronectin mediates attachment of cells to collagen substrates; and (c) fibronectin is directly involved in cellular interactions in chondrocyte cultures.

Synthesis of types I, III and AB2 collagen by chick tendon fibroblasts in vitro.

Tendons from 14--17-day-old chick embryos contain predominantly type I collagen and about 5% AB2 collagen; type III collagen is not detectable by biochemical methods, such as sodium dodecyl sulfate/polyacrylamide gel electrophoresis or cyanogen bromide pattern, but can be visualized by immunofluorescence staining with collagen-type-specific antibodies. Similarly, freshly dissociated tendon cells secrete only type I collagen into the culture medium but no significant amounts of type III collagen [Uitto, J., Lichtenstein, J. R., and Bauer, E. A. (1976) Biochemistry, 15, 4935--4942]. Transfer of tendon cells from chick embryos to monolayer conditions, however, initiated synthesis of type III collagen in about 10% of the cells within three days, as visualized by immunofluorescence staining. Secretion of type III collagen into the culture medium can also be detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. With increasing number of passages the number of cells producing type III collagen reached levels of about 80% after the third passage, while 90% of all cells stained positively for type I collagen. This is reflected by an increase of production of type III collagen as determined by CM-cellulose chromatography. Using velocity sedimentation, the secretion of type III procollagen and of pN-collagen (carrying the amino-terminal extension only), into the culture medium of a second-passage tendon cell culture was detected. This study provides new evidence that the phenotype of cells may alter during transfer from the environment in vivo to conditions in vitro and that additional changes may occur with time in culture.

Synthesis of type III collagen by fibroblasts from the embryonic chick cornea.

Synthesis of collagen types I, II, III, and IV in cells from the embryonic chick cornea was studied using specific antibodies and immunofluorescence. Synthesis of radioactively labeled collagen types I and III was followed by fluorographic detection of cyanogen bromide peptides on polyacrylamide slab gels and by carboxymethylcellulose chromatography followed by disc gel electrophoresis. Type III collagen had been detected previously by indirect immunofluorescence in the corneal epithelial cells at Hamburger-Hamilton stages 20--30 but not in the stroma at any age. Intact corneas from embryos older than stage 30 contain and synthesize type I collagen but no detectable type III collagen. However, whole stromata subjected to collagenase treatment and scraping (to remove epithelium and endothelium) and stromal fibroblasts from such corneas inoculated in vitro begin synthesis of type III collagen within a few hours while continuing to synthesize type I collagen. As demonstrated by double-antibody staining, most corneal fibroblasts contain collagen types I and III simultaneously. Collagen type III was identified biochemically in cell layers and media after chromatography on carboxymethylcellulose be detection of disulfide-linked alpha l (III)3 by SDS gel electrophoresis. The conditions under which the corneal fibroblasts gain the ability to synthesize type III collagen are the same as those under which they lose the ability to synthesize the specific proteoglycan of the cornea: the presence of corneal-type keratan sulfate.

Biochemical and immunological studies of fibroblasts derived from a patient with Ehlers-Danlos syndrome type IV. Demonstrate reduced type III collagen synthesis.

Fibroblasts derived from a skin biopsy of a patient with the Ehlers-Danlos syndrome (EDS) type IV were cultured in monolayer. The amount of collagen synthesized during a 24-h pulse was not different from that found with normal fibroblasts. Chromatographic procedures and immunofluorescence staining showed a normal synthesis of type I procollagen and collagen but a deficiency in synthesis of type III procollagen and collagen. This could be corroborated by radioimmuno assays showing a reduction in type III procollagen by about 90%. The secretion and degradation of collagens was not altered. The results demonstrate that the molecular defect in this particular patient is due to an impairment of the mechanism controlling the gene expression for type III procollagen.

Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix.

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.

[Characterization of a serum protein with selective affinity for denatured collagen (author's transl)]. / Charakterisierung eines Serumproteins mit selektiver Affinität zu denaturiertem Kollagen.

A protein known as antigelatin factor (AGF) was isolated from human serum by affinity chromatography with immobilized denatured collagen. In biochemical and immunological assays AGF showed specificity to denatured, but not to native collagen of the types I, II and III. A close relationship to Cell Attachment Protein and Cold Insoluble Globulin was found in comparative studies.

Similarity of antigelatin factor and cold insoluble globulin.

Antigelatin factor, a protein capable of complexing denatured collagen, was separated from human serum by adsorption onto immobilized collagen. Antiserum raised against the material binding to denatured collagen permitted the development of a radioassay for the determination of antigelatin factor in which the complex of antigelatin factor and denatured 125I-labeled collagen is precipitated with this antiserum. Further purification of antigelatin factor was achieved by chromatography on DEAE-cellulose yielding an electrophoretically homogeneous protein. Its migration rate in dodecyl sulfate-polyacrylamide gel electrophoresis was identical with that of cold insoluble globulin (molecular weight approx. 440 000) prepared from human plasma by a published procedure amended by DEAE-cellulose chromatography. Reduction of disulfide bonds yielded subunits of molecular weight approx. 220 000, indistinguishable from those of cold insoluble globulin. The amino acid composition of both proteins was very similar. Immunological identity of both proteins was demonstrated by gel diffusion against monospecific anti-cold insoluble globulin antiserum. Closely related binding curves were obtained if denatured 125I-labeled collagen was reacted with increasing amounts of either cold insoluble globulin or antigelatin factor and the complexes formed were precipitated with anti-cold insoluble globulin antiserum. In addition, antigelatin factor and cold insoluble globulin mediated the fixation of denatured 125I-labeled collagen to trypsinized macrophages in the same way. Therefore, it is concluded that antigelatin factor and cold insoluble globulin are identical or very closely related proteins.

Identification of the sites in collagen alpha-chains that bind serum anti-gelatin factor (cold-insoluble globulin).

Anti-gelatin factor was prepared from guinea-pig and human serum by affinity chromatography on denatured type-I collagen. As shown previously, this component is related to cold-insoluble globulin. It reacted with 125I-labelled denatured collagen, and the reaction could be inhibited by preincubation with unlabelled collagenous components. In the inhibition assay comparable activities were observed for native and denatured type-I, -II, -III and -IV collagens. There was also no difference in reactivity between collagens of different species. The reactive sites in the collagen alpha-chains were located by inhibition assays on distinct CNBr- and collagenase-derived peptides. The results obtained with fragments from alpha1(I)-, alpha2- and alpha1(II)-chains indicate that the most active region is located between positions 643 and 819 of the alpha1-chain. Lower activities were found for other regions of collagen and may indicate that the factor has the potential to interact with several sites in the alpha-chains. The present data agree with observations by Kleinman, McGoodwin & Klebe [Biochem. Biophys. Res. Commun. (1976) 72, 426-432] on the specificity of a serum factor promoting the attachment of fibroblasts to collagen.

On the mechanism and stereochemistry of the malate-lactate fermentation of Leuconostoc mesenteroides.

During the transformation of (2S, 3R) [3-3H]malate to (S) lactate no tritium exchange takes place. The stereochemical course of the decarboxylation studied with (2S, 3R) [3-2H]-malate in 3HOH/H2O and (2S, 3R) [3-3H]malate in 2H2O occurs with retention and is therefore the same as that determined by other authors for malic enzyme from vertebrates and from Escherichia coli. The malate-lactate fermentation is a useful procedure to prepare chiral methyl groups on a preparative scale starting from (2S, 3R) [3-H]malate.