Occurrence of collagen and proteoglycan forms of type IX collagen in chick embryo cartilage. Production and characterization of a collagen form-specific antibody.

Type IX collagen from chick embryonic cartilage is a proteoglycan bearing a single chondroitin sulfate chain covalently linked to the alpha 2(IX) polypeptide chain. We have isolated type IX collagen metabolically labeled with [3H]proline using an antibody to type IX collagen and have found that the molecule is synthesized in two forms, a collagen form (COLIX) and a proteoglycan form (PGIX). In cultured chondrocytes, the two forms of type IX collagen showed a different ability to be deposited in the matrix. We have suggested the possibility that both forms may arise from an alternative substitution of a chondroitin sulfate chain to the NC3 domain of the alpha 2(IX) chain. Based on the reported amino acid sequence at the NC3 domain of alpha 2(IX), we have synthesized undecapeptides containing the sequence around the glycosaminoglycan attachment site of the alpha 2(IX) chain. Antibody against the peptide, which was raised in rabbit, only recognized COLIX and made it possible to distinguish COLIX from PGIX. Evidence shows that this could be due to a difference in antigenicity of the NC3 domain of the alpha 2(IX) chain between COLIX and PGIX caused by the substitution of a chondroitin sulfate chain to the serine residue in this domain. Therefore, this antibody may be useful as a probe for studies on the functions of glycosaminoglycan substitution in type IX collagen.     

Isolation and characterization of type IX collagen-proteoglycan from the Swarm rat chondrosarcoma.

Type IX collagen was partially purified from the Swarm rat chondrosarcoma by a series of a conventional salting-out procedures. The preparation was further separated by anion exchange chromatography into an unbound and a bound fraction in an A230 ratio of about 5:1. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the bound fraction appeared as a broad band, whose molecular mass ranged from 250 to 270 kDa. Digestion with chondroitinase ABC reduced the apparent molecular mass of the bound fraction to about 250 kDa, a value comparable to the molecular mass of the unbound fraction. Tryptic peptide maps of the protein moieties of unbound and bound forms showed that their molecular structures were basically identical. A monoclonal antibody specific for LMW, one of the pepsin-resistant fragments of the rat sarcoma type IX, reacted with both the unbound and bound fractions. Together the results indicate that the unbound and bound fractions represent a type IX collagen devoid of the chondroitin sulfate chain and its proteoglycan form with covalently bound chondroitin sulfate, respectively. The extent of glycosaminoglycan attachment to type IX collagen molecules in rat chondrosarcoma (about 16%) is quite different from the extents described in chick embryo cartilage (about 80%), chick vitreous humour (100%) and bovine cartilage (less than 5%). Further studies on the neoplastic tissue will offer additional information regarding the biological basis and biological consequences of the glycosaminoglycan attachment to type IX collagen molecules.     

Synthesis of type IX collagen: effect of beta-xylosides

Type IX collagen contains a chondroitin sulfate side chain and therefore may be considered as a proteoglycan. We investigated the effect of beta-xylosides on type IX collagen synthesis. Treatment of chondrocytes with beta-xylosides results in the loss of synthesis of large and small molecular weight proteoglycans, but the synthesis of type IX collagen was unaffected. It is likely that the mechanism of addition of sugar residues to type IX collagen is distinct from that of other cartilage proteoglycans.     

Isolation and sequence analysis of the glycosaminoglycan attachment site of type IX collagen

Type IX collagen from chick embryonic cartilage is unique among the collagens in that it contains chondroitin sulfate covalently linked to the alpha 2(IX) polypeptide chain. We have isolated and sequenced the glycosaminoglycan-containing peptide released by collagenase digestion from type IX collagen, labeled biosynthetically with [35SO4] and 3H-aminoacids. This peptide was purified by gel filtration and, following chondroitinase ABC digestion, by reverse-phase high performance liquid chromatography. The amino acid sequence obtained for this peptide has 23 residues, beginning and ending with a collagenous sequence, indicating that it spans an internal noncollagenous domain. Comparison of this sequence with the one predicted from cDNA clone pYN 1738 for the alpha 1(IX)chain and pYN 1731 and pDM 222 for the alpha 2(IX)chain revealed the peptide to be the noncollagenous NC3 domain of alpha 2(IX). The glycosylated sequence Val-Glu-Gly-Ser*-Ala-Asp- of type IX collagen does not have the Ser-Gly normally functioning as the attachment sequence but does have an acidic residue preceding the serine which should improve the acceptability of this sequence for the xylosyltransferase. That it is an adequate acceptor can be inferred from the observation that type IX collagen carries a glycosaminoglycan chain on over 70% of the molecules isolated.     

Interaction of proteoglycans with the pericellular (1 alpha, 2 alpha, 3 alpha) collagens of cartilage

Interaction between cartilage proteoglycan and the collagen(s) composed of 1 alpha, 2 alpha, and 3 alpha chains was studied in vitro. Most of the collagen was insoluble under the conditions of assay (0.15 M NaCl, 0.008 M phosphate buffer, pH 7.4; 4 degrees C) and was in the form of fibrils 20 nm in diameter or thinner. The larger fibrils had 60-70 nm periodicity, characteristic of native collagens. Proteoglycan monomers which had been labeled by incubating cartilage slices in vitro with Na2 35SO4 were used to assay the interaction. The insoluble collagen fraction bound proteoglycan from solution. At proteoglycan:collagen ratios lower than 1:2, binding was rapid and linear, and the dissociation constant was 1.7 X 10(-9) M. At higher proteoglycan:collagen ratios, more proteoglycan was bound, but at a slower rate. Binding of proteoglycan to collagen did not require fibrils, since soluble 1 alpha, 2 alpha, and 3 alpha containing collagen also bound to proteoglycan and formed an insoluble complex. Denatured collagens did not bind proteoglycan or compete for binding with normal collagen. Optimum binding occurred with intact proteoglycan, but proteoglycan which had been treated with protease was also bound at low levels. Both protease-treated proteoglycan and free chondroitin sulfate competed with intact proteoglycan in the binding assays, but neither chondroitinase ABC-treated proteoglycan nor the oligosaccharides produced by digestion of chondroitin sulfate with testicular hyaluronidase altered the binding of proteoglycan to collagen. Hyaluronic acid did not compete with radioactive proteoglycan, but heparin and dextran sulfate were extremely effective inhibitors of binding. These data suggest a relatively nonspecific interaction between sulfated polyanions and 1 alpha, 2 alpha, and 3 alpha containing collagens. However, given the location of these collagens near the chondrocyte surface, the interaction of fibrillar 1 alpha, 2 alpha, 3 alpha collagen with proteoglycan is likely to occur and to be of biological importance.     

An analysis by rotary shadowing of the structure of the mammalian vitreous humor and zonular apparatus

An electron microscopic analysis of human and bovine vitreous humor after rotary shadowing showed the presence of both collagen fibrils and an extensive loose network of hyaluronan molecules. No interaction between the collagen fibrils and the hyaluronan molecules was observed under the conditions used for rotary shadowing. Periodic "struts" were present on the surface of the collagen fibrils. These struts showed an organization the same as that previously observed for type IX collagen on the surface of collagen fibrils from chicken cartilage and vitreous. However, the knob of the noncollagenous NC4 domain of cartilage type IX collagen was not observed at the ends of the struts in a manner identical to that of chicken vitreous humor. Zonular fibrils were dissected out from bovine eyes and shown by rotary shadowing to contain a beaded fibril which is similar in morphology to the "elastin-associated" microfibrils of many connective tissues. Experiments in which the zonular fibrils were stretched and fixed prior to rotary shadowing showed that the distance between each bead is variable and can be accounted for by the bowing out of overlapping filaments which connect each bead.     

D-periodic distribution of collagen type IX along cartilage fibrils

It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.     

A large chondroitin sulfate proteoglycan (PG-M) synthesized before chondrogenesis in the limb bud of chick embryo

Extraction of stage 22-23 chick embryo limb buds that had been metabolically labeled with [35S]sulfate yielded heparan sulfate proteoglycan, small chondroitin sulfate proteoglycan, and large chondroitin sulfate proteoglycan (designated PG-M). PG-M constituted over 60% of the total macromolecular [35S]sulfates. It was larger in hydrodynamic size, richer in protein, and contained fewer chondroitin sulfate chains as compared to the predominant proteoglycan (PG-H, Mr congruent to 1.5 X 10(6)) of chick embryo cartilage. The chondroitin sulfate chains were notable for their large size (Mr greater than or equal to 60,000) and high content of nonsulfated chondroitin units (about 20% of the total hexosamine). Hexosamine-containing chains corresponding in size to N-linked and O-linked oligosaccharides were also present. The core protein was rich in serine, glutamic acid (glutamine), and glycine which together comprised about 38% of the total amino acids. Following chondroitinase AC II (or ABC) digestion, core molecules were obtained which migrated on sodium dodecyl sulfate gel electrophoresis as a doublet of bands with approximately Mr = 550,000 (major) and 500,000, respectively. The Mr = 550,000 core glycoprotein was structurally different from the core glycoprotein (Mr congruent to 400,000) of PG-H, as ascertained by tryptic peptide mapping and immunochemical criteria. Immunofluorescent localization of PG-M showed that the intensity of PG-M staining progressively became higher in the core mesenchyme region than in the peripheral loose mesenchyme, closely following the condensation of mesenchymal cells. Since the cell condensation process has been shown to begin with the increase of fibronectin and type I collagen concentration, the similar change in PG-M distribution suggests that PG-M plays an important role in the cell condensation process by means of its interaction with fibronectin and type I collagen.     

Identification of the type IX collagen polypeptide chains. The alpha 2(IX) polypeptide carries the chondroitin sulfate chain(s)

Type IX collagen has recently been shown to contain glycosaminoglycan chain(s) and furthermore to be immunologically identical with proteoglycan Lt (Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1985) J. Biol. Chem. 260, 4758-4763). Here we demonstrate that the chondroitin sulfate carrying 115-kDa polypeptide of type IX collagen corresponds to the alpha 2(IX) chain. In addition the 84- and 68-kDa polypeptides were identified as the alpha 1(IX) and the alpha 3(IX) chains, respectively. This conclusion is based on a comparison of the tryptic fingerprints of the 84-, 115-, and 68-kDa chains of type IX collagen on high performance liquid chromatography with the similarly treated C2, C3, and C5 chains of the peptic fragment HMW. In addition, we provide evidence that both the C3 and C4 components of HMW are derived from the alpha 2(IX) chain.     

The cell surface proteoglycan from mouse mammary epithelial cells bears chondroitin sulfate and heparan sulfate glycosaminoglycans

The cell surface proteoglycan fraction isolated by mild trypsin treatment of NMuMG mouse mammary epithelial cells contains largely heparan sulfate, but also 15-24% chondroitin sulfate glycosaminoglycans. We conclude that this fraction contains a unique hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate glycosaminoglycans because (i) the proteoglycan behaves as a single species by sizing, ion exchange and collagen affinity chromatography, and by isopycnic centrifugation, even in the presence of 8 M urea or 4 M guanidine hydrochloride, (ii) the behavior of the chondroitin sulfate in these separation techniques is affected by heparan sulfate-specific probes and vice versa, and (iii) proteoglycan core protein bearing both heparan sulfate and chondroitin sulfate is recognized by a single monoclonal antibody. Removal of both types of glycosaminoglycan reduces the proteoglycan to a core protein of approximately 53 kDa. The proteoglycan fraction is heterogeneous in size, largely due to a variable number and/or length of the glycosaminoglycan chains. We estimate that one or two chondroitin sulfate chains (modal Mr of 17,000) exist on the proteoglycan for every four heparan sulfate chains (modal Mr of 36,000). Synthesis of these chains is reportedly initiated on an identical trisaccharide that links the chains to the same amino acid residues on the core protein. Therefore, some regulatory information, perhaps residing in the amino acid sequence of the core protein, must determine the type of chain synthesized at any given linkage site. Post-translational addition of these glycosaminoglycans to the protein may provide information affecting its ultimate localization. It is likely that the protein is directed to specific sites on the cell surface because of the ability of the glycosaminoglycans to recognize and bind extracellular components.     

Peptide-specific antibodies identify the alpha 2 chain as the proteoglycan subunit of type IX collagen

Type IX collagen is a recently characterized product of chondrocytes. The molecules of this collagen are heterotrimers of three genetically distinct polypeptide chains. One of the three chains contains chondroitin and/or dermatan sulfate glycosaminoglycan chains, giving the molecule a proteoglycan character. In fact, Type IX collagen has been identified with the proteoglycan Lt (PG-Lt), first isolated by Noro, A., Kimata, K., Oike, Y., Shinomura, T., Maeda, N., Yano, S., Takahashi, N., and Suzuki, S. (1983) J. Biol. Chem. 258, 9323-9331 from chick embryonic tibia and femur. Based on amino acid sequences predicted from the nucleotide sequences of cDNA and genomic clones specific for two of the chains of Type IX collagen, we have synthesized oligopeptides representing portions of the two chains. In addition, an oligopeptide has been made based on a partial amino acid sequence of the third chain. Antibodies against the synthetic peptides have been generated in rabbits, and the polyclonal sera have allowed identification of the three genetically distinct polypeptide subunits of Type IX collagen. In addition, labeling with [35S]sulfate and treatment with chondroitinase ABC demonstrates that glycosaminoglycan chains are present on the subunit that has been given the designation alpha 2(IX).     

Use of monoclonal antibodies to locate the chondroitin sulfate chain(s) in type IX collagen

Recent results show that type IX collagen isolated from chicken cartilage is associated with one or perhaps two chondroitin sulfate chains. To locate the chondroitin sulfate chain(s) along the type IX collagen molecule, rotary shadowing was performed in the presence of monoclonal antibodies which recognize stubs of chondroitin sulfate generated after chondroitinase ABC digestion. Monoclonal antibodies 9-A-2 and 2-B-6 which recognize stubs of chondroitin 4-sulfate were found to bind specifically to the NC3 domain of type IX collagen, and this binding was dependent on prior digestion of the preparation with chondroitinase ABC. Monoclonal antibody 1-B-5, which recognizes unsulfated stubs of chondroitin sulfate, did not show any specific binding to type IX collagen either with or without chondroitinase ABC digestion. As a control, monoclonal antibody 2C2 was used, which in previous work was shown to bind specifically to an epitope located close to or at the NC2 domain. Binding of this antibody to NC2 was unaffected by chondroitinase ABC digestion, and no specific binding of the antibody to the NC3 domain was detected either before or after chondroitinase ABC digestion.     

The structure of human collagen type IX and its organization in fetal and infant cartilage fibrils

Human collagen type IX was isolated from the media of organ cultures of fetal or infant hyaline cartilage. It consisted of three distinct, disulfide-bonded polypeptides of 115, 84, and 72 kDa, respectively. Digestion with chondroitinase ABC reduced the apparent molecular mass of the 115-kDa chain to about 65 kDa demonstrating that also human collagen type IX is a proteoglycan. In the electron microscope, the molecule had a rigid rod-like structure with characteristic kinks and with a globular domain at one end. Digestion of human collagen type IX with pepsin leads to somewhat heterogeneous fragments. Affinity-purified antibodies to the mixture of fragments specifically reacted with the fragment HMW without cross-reaction with chicken HMW. LMW of both species were recognized to the same low extent. Mechanically generated fibril fragments from human fetal cartilage were heterogeneous in diameter. Significantly, they could be immunostained for collagen type IX in a D-periodic pattern and regardless of the fibril diameter. Some fibrils were poorly labeled, again independently of the diameter. Therefore, the role of collagen type IX in cartilage probably is not to control directly the lateral growth during fibrillogenesis but rather to stabilize the fibril network.     

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.     

Immunoidentification of type XII collagen in embryonic tissues

We have generated a monoclonal antibody against a synthetic peptide whose sequence was derived from the nucleotide sequence of a cDNA encoding alpha 1(XII) collagen. The antibody, 75d7, has been used to identify the alpha 1(XII) chain on immunoblots of SDS-PAGE tendon extracts as a 220-kD polypeptide, under reducing conditions. Amino-terminal amino acid sequence analysis of an immunopurified cyanogen bromide fragment of type XII collagen from embryonic chick tendons gave a single sequence identical to that predicted from the cDNA, thus confirming that the antibody recognizes the type XII protein. Immunofluorescence studies with the antibody demonstrate that type XII collagen is localized in type I-containing dense connective tissue structures such as tendons, ligaments, perichondrium, and periosteum. With these data, taken together with previous results showing that a portion of the sequence domains of type XII collagen is similar to domains of type IX, a nonfibrillar collagen associated with cross-striated fibrils in cartilage, we suggest that types IX and XII collagens are members of a distinct class of extracellular matrix proteins found in association with quarter-staggered collagen fibrils.     

Axial structure of the heterotypic collagen fibrils of vitreous humour and cartilage

We have compared the axial structures of negatively stained heterotypic, type II collagen-containing fibrils with computer-generated staining patterns. Theoretical negative-staining patterns were created based upon the "bulkiness" of the individual amino acid side-chains in the primary sequence and the D-staggered arrangement of the triple-helices. The theoretical staining pattern of type II collagen was compared and cross-correlated with the experimental staining pattern of both reconstituted type II collagen fibrils, and fibrils isolated from adult and foetal cartilage and vitreous humour. The isolated fibrils differ markedly in both diameter and composition. Correlations were significantly improved when a degree of theoretical hydroxylysine glycosylation was applied, showing for the first time that this type of glycosylation influences the negative-staining pattern of collagen fibrils. Increased correlations were obtained when contributions from types V/XI and IX collagen were included in the simulation model. The N-propeptide of collagen type V/XI and the NC2 domain of type IX collagen both contribute to prominent stain-excluding peaks in the gap region. With decreasing fibril diameter, an increase of these two peaks was observed. Simulations of the fibril-derived staining patterns with theoretical patterns composed of proportions of types II, V/XI and IX collagen confirmed that the thinnest fibrils (i.e. vitreous humour collagen fibrils) have the highest minor collagen content. Comparison of the staining patterns showed that the organisation of collagen molecules within vitreous humour and cartilage fibrils is identical. The simulation model for vitreous humour, however, did not account for all stain-excluding mass observed in the staining pattern; this additional mass may be accounted for by collagen-associated macromolecules.     

Partial characterization of type X collagen from bovine growth-plate cartilage. Evidence that type X collagen is processed in vivo

Sequential extraction of bovine growth-plate cartilage with 4 M guanidinium chloride and pepsin was used to identify the intact and pepsinized forms respectively of type X collagen. This collagen occurs predominantly as the processed [alpha 1(X)]3 form in vivo, although the procollagen [pro alpha 1(X)]3 form can also be detected. The bovine pro alpha 1(X) and alpha 1(X) chains have Mr values identical to the corresponding chick species (Mr 59,000 and 49,000). However, the pepsinized alpha 1(X)p chains (Mr 47,000) are larger than those of the chick (Mr 45,000), and the bovine collagen type X is further distinguished by being disulphide-bonded within the triple-helical domain.     

Chondroitin sulfate perlecan enhances collagen fibril formation. Implications for perlecan chondrodysplasias

Inactivation of the perlecan gene leads to perinatal lethal chondrodysplasia. The similarity to the phenotypes of the Col2A1 knock-out and the disproportionate micromelia mutation suggests perlecan involvement in cartilage collagen matrix assembly. We now present a mechanism for the defect in collagen type II fibril assembly by perlecan-null chondrocytes. Cartilage perlecan is a heparin sulfate or a mixed heparan sulfate/chondroitin sulfate proteoglycan. The latter form binds collagen and accelerates fibril formation in vitro, with more defined fibril morphology and increased fibril diameters produced in the presence of perlecan. Interestingly, the enhancement of collagen fibril formation is independent on the core protein and is mimicked by chondroitin sulfate E but neither by chondroitin sulfate D nor dextran sulfate. Furthermore, perlecan chondroitin sulfate contains the 4,6-disulfated disaccharides typical for chondroitin sulfate E. Indeed, purified glycosaminoglycans from perlecan-enriched fractions of cartilage extracts contain elevated levels of 4,6-disulfated chondroitin sulfate disaccharides and enhance collagen fibril formation. The effect on collagen assembly is proportional to the content of the 4,6-disulfated disaccharide in the different cartilage extracts, with growth plate cartilage glycosaminoglycan being the most efficient enhancer. These findings demonstrate a role for perlecan chondroitin sulfate side chains in cartilage extracellular matrix assembly and provide an explanation for the perlecan-null chondrodysplasia.     

Isolation and primary structure of PARP, a 24-kDa proline- and arginine-rich protein from bovine cartilage closely related to the NH2-terminal domain in collagen alpha 1 (XI)

A protein rich in proline and arginine (proline/arginine-rich protein (PARP] has been isolated from dissociative extracts of bovine nasal and articular cartilage, and its primary structure has been determined. The protein has 218 amino acids, giving a calculated protein Mr of 24,075. In nasal cartilage, this protein is in molar concentrations equivalent to 1/20-1/10 that of the link protein of cartilage proteoglycan aggregates. PARP has also been isolated from bovine articular cartilage, bovine fetal epiphysis, and nonossified human tarsal bones. PARP is similar to various collagen NH2-terminal domains. It is 49% identical to the NH2-terminal end of collagen alpha 1 (XI), 17% identical to the NC4 domain of collagen alpha 1 (IX), and 14% identical to the NC3 domain of collagen alpha 1 (XII). Four cysteines are conserved between type XI collagen and PARP, and these form two disulfide bonds. Two of the cysteines are also conserved between PARP and collagens IX and XII. The homology between the collagens and PARP makes it possible to speculate on the likely disulfide bond pattern in the collagen NH2-terminal domains. It is probable that PARP is a collagen fragment removed during processing in a manner analogous to chondrocalcin (the C-terminal propeptide of type II collagen).