Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.     

The distribution of different molecular species of collagen in fibrous, elastic and hyaline cartilages of the pig.

The distribution of type II collagen, considered to be characteristic of cartilaginous tissues, was determined in various specialized cartilages of the mature pig. The tissues examined were: (1) fibrocartilage of the semilunar meniscus of the knee; (2) elastic cartilage of the external ear; (3) hyaline cartilage of (a) the synovial joint (b) the thyroid plate of the larynx, and (c) the nasal septum. The predominant species of collagen in each tissue, whether type I or type II, was appraised semi-quantitatively by analysis of purified collagen solubilized by pepsin and of peptide fragments produced by cyanogen bromide. Cyanogen bromide-derived peptides were characterized by column chromatography on CM-cellulose and by electrophoresis in sodium dodecyl sulphate-polyacrylamide gels. The proportion of each type of collagen was determined precisely by isolating the homologous small peptides alpha1(II)CB6 [nomenclature of Miller (1973) Clin. Orthop. 92, 260-280], by column chromatography on phosphocellulose and determining their relative proportions by amino acid analysis. Thus collagen of the fibrocartilage of the meniscus proved to be all type I; type II was not detected. In contrast, collagen of elastic cartilage of the outer ear, after rigorous exclusion of perichondrium, was type II. Similarly, type II was the only collagen detected in all the mature hyalline cartilages examined.     

Isolation and characterization of a collagen-binding glycoprotein from chondrocyte membranes

A collagen-binding glycoprotein was isolated from purified chick chondrocyte surface membranes by affinity chromatography on type II collagen-Sepharose. The purified glycoprotein has an apparent mol. wt. of 31,000 and binds to native chick collagen types I, II, III, V and M. Although it contains 30% carbohydrates, the majority of which is fucose, it is hydrophobic and soluble only in detergents. The integral membrane protein character of the 31-K protein became apparent from its ability to insert into lecithin vesicles. Liposome-inserted 31-K protein binds 125I-labelled type II collagen in the presence of 0.5 M NaCl, while detergent-solubilized 31-K protein is dissociated from type II collagen by 0.05-0.1 M NaCl. Electron microscopic studies employing the rotary shadowing technique indicate that 31-K protein particles bind to the ends of collagen molecules. We propose that this glycoprotein serves as anchorage site for extracellular collagen to the chondrocyte membrane and thus may be involved in cell-matrix interactions in cartilage.     

Isolation and purification of cyanogen bromide-derived peptides of type II collagen directly from tissue (Swarm chondrosarcoma)

A preparative procedure is described for isolating type II collagen-fragments directly from tissue. Swarm chondrosarcoma from rat, a cartilagenous tissue rich in type II collagen, was digested by cyanogen bromide in 70% formic acid. The resulting crude extract was desalted (G 25 column chromatography) and lyophylized. The yield of peptide mixture was about 1 250 mg obtained from 100 g tissue. The method of purification commonly used for type II collagen prior to cyanogen bromide-cleavage yielded 20 mg peptides from 100 g tissue. Separation of the cyanogen bromide-derived fragments was performed by gel filtration. The column was run at 43 degrees C (denaturing-temperature of collagens) to avoid fibril formation, and a volatile buffer was used (ammonium formate buffer, pH 7.5) so that the effluent fractions could be easily lyophylized. Two-dimensional gel electrophoresis of the main peaks of the column profile demonstrated that this purification step resulted in a good separation of the fragment mixture, although additional steps may be necessary for complete separation of the peptides. The most striking advantages of the method for direct digestion of tissue outlined here are the increase in yield (about 60-fold) and the reduction of purification steps (avoiding type II collagen purification).     

Regulation of insulin secretion by energy metabolism in pancreatic B-cell mitochondria. Studies with a non-metabolizable leucine analogue.

This study compares the collagen types present in rabbit ear cartilage with those synthesized by dissociated chondrocytes in cell culture. The cartilage was first extracted with 4M-guanidinium chloride to remove proteoglycans. This step also extracted type I collagen. After pepsin solubilization of the residue, three additional, genetically distinct collagen types could be separated by fractional salt precipitation. On SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis they were identified as type II collagen, (1 alpha, 2 alpha, 3 alpha) collagen and M-collagen fragments, a collagen pattern identical with that found in hyaline cartilage. Types I, II, (1 alpha, 2 alpha, 3 alpha) and M-collagen fragments represent 20, 75, 3.5, and 1% respectively of the total collagen. In frozen sections of ear cartilage, type II collagen was located by immunofluorescence staining in the extracellular matrix, whereas type I collagen was closely associated with the chondrocytes. Within 24h after release from elastic cartilage by enzymic digestion, auricular chondrocytes began to synthesize type III collagen, in addition to the above-mentioned collagens. This was shown after labelling of freshly dissociated chondrocytes with [3H]proline 1 day after plating, fractionation of the pepsin-treated collagens from medium and cell layer by NaCl precipitation, and analysis of the fractions by CM(carboxymethyl)-cellulose chromatography and SDS/polyacrylamide-gel electrophoresis. The 0.8 M-NaCl precipitate of cell-layer extracts consisted predominantly of type II collagen. The 0.8 M-NaCl precipitate obtained from the medium contained type I, II, and III collagen. In the supernatant of the 0.8 M-NaCl precipitation remained, both in the cell extract and medium, predominantly 1 alpha-, 2 alpha-, and 3 alpha-chains and M-collagen fragments. These results indicate that auricular chondrocytes are similar to chondrocytes from hyaline cartilage in that they produce, with the exception of type I collagen, the same collagen types in vivo, but change their cellular phenotype more rapidly after transfer to monolayer culture, as indicated by the prompt onset of type III collagen synthesis.     

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.     

Covalent structure of collagen. Amino acid sequence of an arthritogenic cyanogen bromide peptide from type II collagen of bovine cartilage

Bovine articular type II collagen was prepared by limited pepsin digestion, differential salt fractionation and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type II collagen alpha chains yielded twelve distinct peptides designated CB1-12. The peptide alpha 1(II)-CB11 was isolated by carboxymethylcellulose chromatography and Sephadex G-75S gel filtration. Automated Edman degradation together with chymotrypsin, thermolysin and trypsin digestion enabled identification of its complete amino acid sequence. Compared with type I and type III collagen, the data show similarity with alpha 1(I)-CB8 and alpha 1(III)-CB6-1-8-10-2 peptides, respectively. The peptide is located within residues 124-402 of the alpha 1(II) collagen chain and with its identification, now extends the known amino acid sequence of bovine type II cartilage collagen to 660 amino acid residues including alpha 1(II)-CB1-2-6-12-11-8-10 (partial). This corresponds to alpha 1(I)-CB0-1-2-4-5-8-3-7 (partial; 1-660) and alpha 1(III)-CB3A-3B-3C-7-6-1-8-10-2-4-5 (partial; 1-660) of bovine alpha 1(I) and alpha 1(III) collagen chains.     

Immunological and biochemical studies of collagen type transition during in vitro chondrogenesis of chick limb mesodermal cells

This work describes an approach to monitor chondrogenesis of stage-24 chick limb mesodermal cells in vitro by analyzing the onset of type II collagen synthesis with carboxymethyl-cellulose chromatography, immunofluorescence, and radioimmunoassay. This procedure allowed specific and quantitative determination of chondrocytes in the presence of fibroblasts and myoblasts, both of which synthesize type I collagen. Chondrogenesis was studied in high-density cell preparations on tissue culture plastic dishes and on agar base. It was found that stage-24 limb mesenchymal cells initially synthesized only type I collagen. With the onset of chondrogenesis, a gradual transition to type II collagen synthesis was observed. In cell aggregates formed over agar, type II collagen synthesis started after 1 day in culture and reached levels of 80-90 percent of the total collagen synthesis at 6-8 days. At that time, the cells in the center of the aggregates had acquired the typical chondrocyte phenotype and stained only with type II collagen antibodies, whereas the peripheral cells had developed into a "perichondrium" and stained with type I and type II collagen antibodies. On plastic dishes plated with 5 X 10(6) cells per 35mm dish, cartilage nodules developed after 4-6 days, but the type II collagen synthesis only reached levels of 10-20 percent of the total collagen. The majority of the cells differentiated into fibroblasts and myoblasts and synthesized type I collagen. These studies demonstrate that analysis of cell specific types of collagen provides a useful method for detailing the specific events in the differentiation of mesenchymal cells in vitro.     

Isolation of human type X collagen and immunolocalization in fetal human cartilage

Type X collagen was extracted with 1 M NaCl and 10 mM dithiothreitol at neutral pH from fetal human growth plate cartilage and purified to homogeneity by gel filtration and anion-exchange chromatography. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 66,000 under reducing conditions, and as a high-Mr oligomer under non-reducing conditions. Purified collagenase digests most of the molecule; pepsin digestion at 4 degrees C decreases the Mr of the monomer to 53,000. A rabbit antiserum was raised against purified human type X collagen; the IgG fraction was specific for this collagen by criteria of ELISA and immunoblotting after absorption with collagen types I, II, VI, IX and XI. Immunohistological studies localized type X collagen exclusively in the zone of hypertrophic and calcifying cartilage.     

Collagen synthesis by regenerating rabbit ear tissue

The principal collagen types synthesized during two distinct phases of regeneration in rabbit ears have been investigated, in order to relate altered phenotypic expression in connective tissue cells to regeneration of cartilage. To do this, radioactively labeled collagens synthesized in short-term culture by selected regenerating ear tissues were analyzed by ion-exchange chromatography and SDS-gel electrophoresis of the intact collagens and of the cyanogen bromide peptides derived from them. Prior to the appearance of cartilage, rabbit ear holes are filled by an outgrowth of mesenchyme-like cells derived locally from adjacent tissues. These cells produce a mixture of collagens including type I, [α1(I)]₂α2, and the type I trimer, [α1(I)]₃, but not type II collagen. Trimer production represents about one-fourth of the collagen synthesized in either a 4-, 10-, or a 24-hr incubation. Trimer is not made by tissues from healing skin wounds nor is it present in normal, uninjured ear tissues. Type II collagen synthesis was detected in tissues taken from late regenerates containing histologically recognizable cartilage, and direct analysis of regenerated cartilage confirmed the presence of type II collagen in the matrix. Thus, regenerated cartilage in the rabbit ear system contains the normal cartilage collagen, type II, while the proliferating cell mass from which the cartilage develops synthesizes the unusual collagen, [α1(I)]₃.     

Human collagen ''fingerprints'' produced by clostridopeptidase A digestion and high-pressure liquid chromatography.

Samples (1-2mg) of purified human type I, II and III collagens and alpha1(I) and alpha2 chains were digested with clostridiopeptidase A and the released peptides analysed by ion-exchange high-pressure liquid chromatography. Specific 'fingerprints' were produced for each type of collagen. The reproducible nature of these 'fingerprints' and the reconstitution of the type I 'fingerprint' from the 'fingerprints' of the component alpha1(I) and alpha2 chains showed that the specificity of these 'fingerprints' was related to the primary structure of each type of collagen. In addition, some of the differences observed between the 'fingerprints' of the alpha1(I) and alpha2 chains of type I collagen were shown to be suitable for the quantitative analysis of these chains.     

Primary structure of the heparin-binding site of type V collagen

The abilities of collagens, type I, II, III, IV, and V, to bind heparin were examined by heparin-affinity chromatography and binding studies with [35S]heparin. At a physiological pH and ionic strength, only type V collagen bound to heparin. Collagens type I and II showed higher affinities than types III and IV for heparin, but did not bind to a heparin column at a physiological ionic strength. The heparin binding site of type V collagen was located in a 30 kDa CNBr fragment of the alpha 1(V) chain, and the amino acid sequence of this fragment was determined. The 30 kDa fragment contained a cluster of basic amino acid residues, and enzymatic cleavage within this basic domain greatly reduced the heparin-binding activities of the resulting peptides. Thus this basic region is probably the heparin-binding site of type V collagen.     

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 alpha 1(II) chain of type II collagen, whereas after BrdU treatment or growth to senescence synthesis of only alpha 1(I) and alpha 2(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 1 alpha, 2 alpha and 3 alpha chains together with the synthesis of short chain (SC) collagen of Mr 43000 after pepsin digestion. After BrdU treatment or growth to senescence alpha chains were isolated which possessed the migration positions on polyacrylamide gel electrophoresis (PAGE), or the elution positions on CM-cellulose chromatography, of the alpha 1(V) and alpha 2(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 1 alpha, 2 alpha and 3 alpha chains to the synthesis of the alpha 1(V) and alpha 2(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.     

Covalent cross-linking of the NC1 domain of collagen type IX to collagen type II in cartilage

From a study to understand the mechanism of covalent interaction between collagen types II and IX, we present experimental evidence for a previously unrecognized molecular site of cross-linking. The location relative to previously defined cross-linking sites predicts a specific manner of interaction and folding of collagen IX on the surface of nascent collagen II fibrils. The initial evidence came from Western blot analysis of type IX collagen extracted by pepsin from fetal human cartilage, which showed a molecular species that had properties indicating an adduct between the alpha1(II) chain and the C-terminal domain (COL1) of type IX collagen. A similar component was isolated from bovine cartilage in sufficient quantity to confirm this identity by N-terminal sequence analysis. Using an antibody that recognized the putative cross-linking sequence at the C terminus of the alpha1(IX) chain, cross-linked peptides were isolated by immunoaffinity chromatography from proteolytic digests of human cartilage collagen. They were characterized by immunochemistry, N-terminal sequence analysis, and mass spectrometry. The results establish a link between a lysine near the C terminus (in the NC1 domain) of alpha1(IX) and the known cross-linking lysine at residue 930 of the alpha1(II) triple helix. This cross-link is speculated to form early in the process of interaction between collagen IX molecules and collagen II polymers. A model of molecular folding and further cross-linking is predicted that can spatially accommodate the formation of all six known cross-linking interactions to the collagen IX molecule on a fibril surface. Of particular biological significance, this model can accommodate potential interfibrillar as well as intrafibrillar links between the collagen IX molecules themselves, so providing a mechanism whereby collagen IX could stabilize a collagen fibril network.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo.

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.     

Characterization of collagen precursors found in rat skin and rat bone.

Two genetic types of collagenous proteins, type I and type III, were isolated by extraction and differential salt precipitation from rat skin. The yield of collagen precursors was increased by injecting animals with colchicine 30 min before sacrifice to inhibit secretion of collagen. DEAE-cellulose chromatography was used to separate collagen from collagen precursors. Although these preparations contained more type I collagen than type III collagen, there were always more type III than type I precursors. The precursor chains of type I fractions were separated on CM-cellulose chromatography after denaturation. Three precursor forms were found for each collagen alpha chain, a complete chain (proalpha chain), and a precursor chain with only an amino-terminal (pNalpha chain) and carboxy-terminal extension (pCalpha chain). Species differences were demonstrated between rat collagen precursors and other species using rat calvaria (frontal and parietal) bones extracted with either 0.5 N acetic acid or neutral salt buffers containing protease inhibitors. Native rat procollagen elutes earlier than chicken or human procollagen on DEAE-cellulose chromatography and does not separate significantly from the pC collagen form. The collagenase resistant amino terminal peptides of rat pNalpha1 and pNalpha2 were the same size (16 000) but could be separated by DEAE-cellulose chromatography.     

Collagen-binding proteins secreted by type II pneumocytes in culture

The alveolar epithelial basement membrane is believed to play important roles in lung development, in maintaining normal alveolar architecture, and in guiding repair following lung injury. However, little is known about the formation of this structure, or of the mechanisms which mediate interactions between the epithelium and specific matrix macromolecules. Since type IV collagen is a major structural component of basement membranes, we investigated the production of type IV collagen-binding proteins by primary cultures of rat lung type II pneumocytes. Cultures were labeled for up to 24 h with 3H-labeled amino acids or [3H]mannose. Soluble collagen-binding proteins which accumulated in the culture medium were isolated by chromatography on collagen-Sepharose and examined by SDS-polyacrylamide gel electrophoresis. The major type IV collagen-binding protein (CBP1) was identified as fibronectin. We also identified a novel disulfide-bonded collagen-binding glycoprotein (CBP2; Mr = 45,000, reduced). This protein was not recognized by polyclonal antibodies to fibronectin, and showed no detectable binding to denatured type I collagen. The protein was resolved from fibronectin and partially purified by sequential chromatography on gelatin and type IV collagen-Sepharose. We suggest that type II pneumocyte-derived collagen-binding proteins contribute to the formation of the epithelial basement membrane and/or mediate the attachment of these cells to collagenous components of the extracellular matrix.     

The C-terminus type I collagen is a major binding site for heparin

The binding of collagens and fragments of type I collagen to heparin was studied by gel electrophoresis and affinity chromatography. Samples bound in 150 mM NaCl/10 mM Hepes (pH6.5) were eluted with 2 M NaCl, 6 M urea, or a linear gradient of 0.15–1.0 M NaCl. The triple-helical conformation was shown to be essential for binding. The vertebrate collagenase-generated C-terminal fragment, TC^B was shown to have greater binding affinity for heparin than the N-terminal TC^A fragment. Both type II collagen and the NC1 domain of type IV collagen bound to heparin, whereas pepsin-solubilized tetrameric type IV failed to bind.     

Construction and characterization of type II collagen complementary deoxyribonucleic acid clones

The mRNA for type II collagen was purified from embryonic chick sternum or from purified sternal chondrocytes with guanidine thiocyanate as the extractant. Double-stranded cDNAs to procollagen mRNAs from sternum were synthesized and dC-tailed. After annealing with PstI-cleaved, dG-tailed pBR322, this DNA was used to transform Escherichia coli X1776. Transformed colonies were screened by colony hybridization to type I and II collagen cDNAs. Clones that preferentially hybridized to type II cDNA were characterized further. Four such cDNA clones, pCgII-2, 3, 10 and 12, with inserts of 400, 320, 260 and 750 bp, have been identified as type II collagen cDNA clones by several criteria, including their preference for hybridizing with type II rather than type I collagen mRNAs in hybrid-selected translation experiments.     

Isolation and characteristics of ligand specificity of VLA-1 integrin from human smooth muscles]

Using affinity chromatography with immobilized monoclonal antibodies to the beta 1-subunit of human integrin, a total integrin fraction (subfamily beta 1) was isolated from the detergent extract of human smooth muscle (uterus). Immunoprecipitation and immunoblotting with specific antibodies revealed integrins VLA-1 and VLA-5. The former was isolated in a homogeneous state by chromatography on immobilized type I collagen in the presence of 1 mM Mn2+. The pure receptor yield was 2-4 mg per 400 g of smooth muscle tissue. Analysis of substrate specificity of VLA-1 in the liposome test revealed that this integrin possesses a broad spectrum of ligand specificity and can interact via a Ca2+, Mg(2+)-dependent mechanism with interstitial collagens of I, II and III types and with basal membrane proteins (type IV collagen and laminin). VLA-1 does not interact with fibronectin, thrombospondin or albumin. Denaturation of type I collagen decreases the liposome binding 5-7-fold. The peptide Gly-Arg-Gly-Asp-Ser-Pro added to the incubation mixture does not inhibit the liposome interaction with incorporated VLA-1 integrin, type I collagen and laminin.