A role for prolyl 3-hydroxylase 2 in post-translational modification of fibril-forming collagens

The fibrillar collagen types I, II, and V/XI have recently been shown to have partially 3-hydroxylated proline (3Hyp) residues at sites other than the established primary Pro-986 site in the collagen triple helical domain. These sites showed tissue specificity in degree of hydroxylation and a pattern of D-periodic spacing. This suggested a contributory role in fibril supramolecular assembly. The sites in clade A fibrillar α1(II), α2(V), and α1(I) collagen chains share common features with known prolyl 3-hydroxylase 2 (P3H2) substrate sites in α1(IV) chains implying a role for this enzyme. We pursued this possibility using the Swarm rat chondrosarcoma cell line (RCS-LTC) found to express high levels of P3H2 mRNA. Mass spectrometry determined that all the additional candidate 3Hyp substrate sites in the pN type II collagen made by these cells were highly hydroxylated. In RNA interference experiments, P3H2 protein synthesis was suppressed coordinately with prolyl 3-hydroxylation at Pro-944, Pro-707, and the C-terminal GPP repeat of the pNα1(II) chain, but Pro-986 remained fully hydroxylated. Furthermore, when P3H2 expression was turned off, as seen naturally in cultured SAOS-2 osteosarcoma cells, full 3Hyp occupancy at Pro-986 in α1(I) chains was unaffected, whereas 3-hydroxylation of residue Pro-944 in the α2(V) chain was largely lost, and 3-hydroxylation of Pro-707 in α2(V) and α2(I) chains were sharply reduced. The results imply that P3H2 has preferred substrate sequences among the classes of 3Hyp sites in clade A collagen chains.     

Molecular mechanism of alpha 1(I)-osteogenesis imperfecta/Ehlers-Danlos syndrome: unfolding of an N-anchor domain at the N-terminal end of the type I collagen triple helix

We demonstrate that 85 N-terminal amino acids of the alpha1(I) chain participate in a highly stable folding domain, acting as the stabilizing anchor for the amino end of the type I collagen triple helix. This anchor region is bordered by a microunfolding region, 15 amino acids in each chain, which include no proline or hydroxyproline residues and contain a chymotrypsin cleavage site. Glycine substitutions and amino acid deletions within the N-anchor domain induce its reversible unfolding above 34 degrees C. The overall triple helix denaturation temperature is reduced by 5-6 degrees C, similar to complete N-anchor removal. N-propeptide partially restores the stability of mutant procollagen but not sufficiently to prevent N-anchor unfolding and a conformational change at the N-propeptide cleavage site. The ensuing failure of N-proteinase to cleave at the misfolded site leads to incorporation of pN-collagen into fibrils. Similar, but weaker, effects are caused by G88E substitution in the adjacent triplet, which appears to alter N-anchor structure as well. As in Ehlers-Danlos syndrome (EDS) VIIA/B, fibrils containing pN-collagen are thinner and weaker causing EDS-like laxity of large and small joints and paraspinal ligaments. However, distinct structural consequences of N-anchor destabilization result in a distinct alpha1(I)-osteogenesis imperfecta (OI)/EDS phenotype.     

Collagen polymorphism: isolation and partial characterization of alpha 1(I)-trimer molecules in normal human skin.

Human skin has previously been shown to contain at least two genetically distinct types of collagen, type I and III. Here the presence of an additional form of collagen, α1(I)-trimer, is demonstrated. Skin collagen was solubilized by limited pepsin digestion and then fractionated by sequential precipitation with 1.5, 2.5, and 4.0 m NaCl at pH 7.4. The α-chain subunits of collagen were isolated by gel filtration and carboxymethylcellulose chromatography under denaturing conditions. The 1.5 and 2.5 m NaCl precipitates contained predominantly type I collagen with a chain composition of [α1(I)]₂α2. In the 1.5 m precipitate a small amount of type III collagen was also recovered. In contrast, the 4.0 m NaCl fraction consisted almost exclusively of α-chains which on the basis of cyanogen bromide peptide mapping were shown to be identical with α1(I). The amino acid composition of these chains was also similar to that of α1(I), except that hydroxylysine was increased and lysine was correspondingly decreased. The content of 3-hydroxyproline was also increased. These results suggest that the α-chains in α1(I)-trimer are the same gene products as α1 in type I collagen, but that the co-translational or post-translational hydroxylation of lysyl residues is more extensive in α1(I)-trimer. Estimation of the quantitative amounts of α1(I)-trimer indicated that this collagen accounts for less than 5% of the total collagen in adult human skin. It is speculated, however, that α1(I)-trimer collagen may play a role in the stability and tensile strength of normal human skin and other tissues, and defects in its biochemistry might be associated with diseases of connective tissue.     

Collagen metabolism and spicule formation in sea urchin micromeres

The role of collagen or collagen-like protein(s) in the in vitro formation of the sea urchin embryonic skeleton was investigated using isolated micromeres of Strongylocentrotus purpuratus. Micromeres were cultured in sea water containing 4% horse serum on tissue culture plastic or an extracellular matrix of type I collagen. The effect of proline analogs and an inhibitor of collagen hydroxylation on in vitro spicule formation in both culture systems was monitored. When micromeres are cultured in the presence of proline analogs l-azetidine-2-carboxylic acid and l-3,4-dehydroproline which disrupt collagen metabolism, spicule formation is significantly less inhibited on a collagen substratum than on plastic. Culturing micromeres on plastic in the presence of α,α′-dipyridyl, an inhibitor of collagen hydroxylation, resulted in almost complete inhibition of spicule formation. The inhibition by α,α′-dipyridyl can be overcome by culturing micromeres on collagen substratum. These results do not support the idea of collagen being the calcified organic matrix of the spicule. Rather, they suggest that micromeres synthesize a collagen-like extracellular matrix which is necessary for spicule formation. Inhibition of this activity by proline analogs or a collagen processing inhibitor can be overcome by providing the cells with a previously deposited extracellular matrix.     

A critical crosslink region in human-bone-derived collagen type I. Specific cleavage site at residue Leu95

Collagen was extracted from human adult bone by limited pepsin digestion and collagen types were purified by consecutive salt precipitation first under neutral and then under acid conditions. In SDS/PAGE, all collagen type I preparations showed a protein band [alpha 1s(I)] migrating between alpha 1(I) and alpha 2(I) as well as a band [alpha 2s(I)] migrating in front of alpha 2(I). The collagenous nature of the pepsin-stable alpha 1s(I) protein was clearly demonstrated by digestion with human-leucocyte-derived collagenase, immunoblotting with antibodies against collagen type I and amino acid analysis. Partial amino acid sequencing of alpha 1(I) and alpha 1s(I) identified alpha 1s(I) as a shortened alpha 1(I) chain due to a specific cleavage site between residues Leu95 and Asp96 which is in close vicinity to the hydroxylysine-derived crosslink at position 87. In circular dichroism, the proportion of thermally labile collagen molecules was proportional to the amount of shortened alpha 1(I) and alpha 2(I) chains, respectively. The melting temperature was found to be 36 +/- 0.5 degrees C as judged from circular dichroism and susceptibility to proteolysis. Our data provide clear evidence that a shortened alpha 1-derived collagen chain can be extracted from human adult bone whereas it is hardly found in human skin. The unique cleavage site might provide important information about the collagen I molecule embedded in the calcified matrix of human bone.     

A targeted mutation at the known collagenase cleavage site in mouse type I collagen impairs tissue remodeling

Degradation of type I collagen, the most abundant collagen, is initiated by collagenase cleavage at a highly conserved site between Gly775 and Ile776 of the alpha 1 (I) chain. Mutations at or around this site render type I collagen resistant to collagenase digestion in vitro. We show here that mice carrying a collagenase-resistant mutant Col1a-1 transgene die late in embryo-genesis, ascribable to overexpression of the transgene, since the same mutation introduced into the endogenous Col1a-1 gene by gene targeting permitted normal development of mutant mice to young adulthood. With increasing age, animals carrying the targeted mutation developed marked fibrosis of the dermis similar to that in human scleroderma. Postpartum involution of the uterus in the mutant mice was also impaired, with persistence of collagenous nodules in the uterine wall. Although type I collagen from the homozygous mutant mice was resistant to cleavage by human or rat fibroblast collagenases at the helical site, only the rat collagenase cleaved collagen trimers at an additional, novel site in the nonhelical N-telopeptide domain. Our results suggest that cleavage by murine collagenase at the N-telopeptide site could account for resorption of type I collagen during embryonic and early adult life. During intense collagen resorption, however, such as in the immediate postpartum uterus and in the dermis later in life, cleavage at the helical site is essential for normal collagen turnover. Thus, type I collagen is degraded by at least two differentially controlled mechanisms involving collagenases with distinct, but overlapping, substrate specificities.     

Procollagen and collagen produced by a teratocarcinoma-derived cell line,TSD4: Evidence for a new molecular form of collagen

Procollagen and collagen were isolated from the culture medium and cell layer of line TSD4 (obtained from mouse teratocarcinoma OTT6050). SDS-polyacrylamide gel electrophoresis of the highly purified procollagen fraction demonstrated that the fraction is composed of θ chains (150,000 daltons), pro α chains (130,000 daltons), and α chains (100,000 daltons). Limited pepsin digestion of this fraction yielded a single species of collagen molecules having a chain composition (α1)₃, as did collagen isolated from the cell layer. Each α1 chain appears to be slightly larger than α1 chains from calf or human type I and type III collagen. Amino acid analysis and cyanogen bromide peptide profiles of pepsin-treated TSD4 collagen demonstrated significant differences from those of other collagens (II, III, IV) of the type α1(X)₃, although similar to that of the α1 chain of type I collagen, [α1(I)]₂α2. Taken together, acrylamide gel electrophoresis, amino acid composition, electron microscopy, and cyanogen bromide peptide analysis indicate that this material represents a new molecular species of collagen not previously characterized, probably related to [α1(I)]₃.     

Isolation and characterization of a peptide containing the site of cleavage of the chick skin collagen alpha 1[I] chain by animal collagenases.

A 36-residue peptide containing the bond cleaved by animal collagenases was isolated from a digest of chick skin collagen α1-CB7 by Staphylococcus V8 protease. This cleavage site peptide, in contrast to the 36-residue α1-CB2, showed no tendency to renature to the triple helical form, as monitored by molecular sieve chromatography and the determination of circular dichroism spectra. These results provide a direct demonstration that the conformation of the α1[I] chain immediately around the collagenase cleavage site in the native molecule must be of a lower degree of helicity than other portions of the chain. This is considered to be an important factor in the collagenase specificity, in providing access to the sensitive bonds, but enzyme binding sites, probably located in the adjacent region(s) of maximum helicity, are also considered necessary to produce the maximum reaction rate.     

The cleavage site preference of the porcine pepsin on the N-terminal α1 chain of bovine type I collagen: a focal analysis with mass spectrometry

Bovine type I collagen consists of two α1 and one α2 chains, containing the internal triple helical regions and the N- and C-terminal telopeptides. In industries, it is frequently digested with porcine pepsin to produce a triple helical collagen without the telopeptides. However, the digestion mechanism is not precisely understood. Here, we performed a mass spectrometric analysis of the pepsin digest of the N-terminal telopeptide pQLSYGYDEKSTGISVP (1–16) in the α1 chain. When purified collagen was digested, pQLSYGY (1–6) and pQLSYGYDEKSTG (1–12) were identified, while DEKSTG (7–12) was not. When the N-terminal telopeptide mimetic synthetic peptide pQLSK(MOCAc)GYDEKSTGISK(Dnp)P-NH₂ was digested, pQLSK(MOCAc)GYDEKSTG (1–12) and ISK(Dnp)P-NH₂ (13?16) were readily identified, pQLSK(MOCAc)GY (1?6) and DEKSTGISK(Dnp)P-NH₂ (7?16) were weakly detected, and DEKSTG (7–12) was hardly identified. These results suggest that pepsin preferentially cleaves Tyr6–Asp7 and less preferentially Gly12–Ile13. They also suggest that the former cleavage requires native collagen structure, while the latter cleavage does not.     

Identification ofClostridium histolyticum collagenase hyperreactive sites in type I,II, and III collagens: Lack of correlation with local triple helical stability

The class I and IIClostridium histolyticum collagenases (CHC) have been used to identify hyperreactive sites in rat type I, bovine type II, and human type III collagens. The class I CHC attack both collagens at loci concentrated in the N-terminal half of these collagens starting with the site closest to the N-terminus. The class II CHC initiate collagenolysis by attacking both collagens in the interior to produce a mixture of C-terminal 62,000 and a N-terminal 36,000 fragments. Both fragments are next shortened by removal of a 3000 fragment. These results are very similar to those reported earlier for the hydrolysis of rat type I collagen by these CHC, indicating that the three collagens share many hyperreactive sites. Similar reactions carried out with the respective gelatins show that they are cleaved at many sites at approximately the same rate. Thus, the hyperreactivity of the sites identified must be attributed to their environment in the native collagens. N-terminal sequencing of the fragments produced in these reactions has allowed the identification of 16 cleavage sites in the α1(I), α2(I), α1(II), and α1(III) collagen chains. An analysis of the triple helical stabilities of these cleavage site regions as reflected by their imino acid contents fails to yield a correlation between reactivity and triple helical stability. The existence of these hyperreactive CHC cleavage sites suggests that type I, II, and III collagens contain regions that have specific nontriple helical conformations. The sequence of these sites presented here now makes it possible to investigate these conformations by computational and peptide mimetic techniques.     

The gelatinolytic activity of rat uterus collagenase

The collagenase produced by rat uterine cells in culture has been examined for its ability to degrade denatured collagen. Acting as a gelatinase, rat uterus collagenase was able to successfully degrade the denatured chains of collagen types I through V. In addition, the enzyme produced multiple cleavages in these chains and displayed values for Km of 4-5 microM, compared to values of 1-2 microM when native collagen was used as substrate. Furthermore, rat uterus collagenase degraded the alpha 2 chain of denatured type I collagen at a significantly faster rate than the alpha 1 chain, as previously observed for human skin fibroblast collagenase. In contrast to the action of human skin collagenase, however, the rat uterus enzyme was found to be a markedly better gelatinase than a collagenase, degrading the alpha chains of denatured type I guinea pig skin collagen at rates some 7-15-fold greater than native collagen. Human skin collagenase degrades the same denatured chains at rates ranging from 13-44% of its rate on native collagen. Rat uterus collagenase, then, is approximately 50 times better a gelatinase than is human skin collagenase. In addition to its ability to cleave denatured collagen chains at greater rates than native collagen, the rat uterus collagenase also attacked a wider spectrum of peptide bonds in gelatin than does human skin collagenase. In addition to cleaving the Gly-Leu and Gly-Ile bonds characteristic of its action on native collagen, rat uterus collagenase readily catalyzed the cleavage of Gly-Phe bonds in gelatin. The rat enzyme was also capable of cleaving Gly-Ala and Gly-Val bonds, although these bonds were somewhat less preferred by the enzyme. The cleavage of peptide bonds other than Gly-Leu and Gly-Ile appears to be a property of the collagenase itself and not a contaminating protease. Thus, it appears that the collagenase responsible for the degradation of collagen during the massive involution of the uterus might also act as a gelatinase to further degrade the initial products of collagenolysis to small peptides suitable for further metabolism.     

Cleavage of Type II and III collagens with mammalian collagenase: site of cleavage and primary structure at the NH2-terminal portion of the smaller fragment released from both collagens.

Collagenase cleavage of human Type II and III collagens has been studied using a highly purified preparation of rabbit tumor collagenase. Progress of the reactions in solution was followed by viscometry and the results indicated that under the conditions employed Type III collagen molecules were cleaved at approximately five times the rate of Type II molecules. Cleavage products of the reactions were isolated in denatured form by agarose molecular sieve chromatography. The molecular weights and amino acid compositions of the products demonstrated that Type II and III molecules had been cleaved at the characteristic three-quarter, one-quarter locus, giving rise to a large fragment derived from the NH2-terminal portion of the molecule and a smaller fragment representing the COOH-terminal region. The amino acid sequence at the NH2-terminal portion of the smaller fragment derived from Type II collagen was determined to be Ile-Ala-Gly-Gln-Arg, and the corresponding region from Type III collagen was found to have the sequence Leu-Ala Gly-Leu-Arg. These sequences for alpha1(II) and alpha1(III) chains adjacent to the site of collagenase cleavage along with previous data for alpha1(I) and alpha2 chains indicate that the minimum specific sequence required for collagenase cleavage is Gly-Ile-Ala or Gly-Leu-Ala. Inspection of the available sequence data for collagen alpha chains indicates that the latter sequences are found in at least three additional locations at which collagenase cleavage does not occur. Each of the sequences which are apparently not substrates for collagenase, however, are followed by a Gly-X-Hyp sequence. We suggest, then, that a minimum of five residues in collagen alpha chains COOH-terminal to the cleavage site comprise the substrate recognition site.     

Structural properties of a collagenous heterotrimer that mimics the collagenase cleavage site of collagen type I

Collagens contain sequence- and conformation-dependent epitopes responsible for their digestion by collagenases at specific loci. A synthetic heterotrimer construct containing the collagenase cleavage site of collagen type I was found to mimic perfectly native collagen in terms of selectivity and mode of enzymatic degradation. The NMR conformational analysis of this molecule clearly revealed the presence of two structural domains, i.e. a triple helix spanning the Gly-Pro-Hyp repeats and a less ordered portion corresponding to the collagenase cleavage site where the three chains are aligned in extended conformation with loose interchain contacts. These structural properties allow for additional insights into the very particular mechanism of collagen digestion by collagenases.     

Characterization of the mechanisms by which gelatinase A, neutrophil collagenase, and membrane-type metalloproteinase MMP-14 recognize collagen I and enzymatically process the two alpha-chains

The turnover of native collagen has been ascribed to different members of the matrix metalloproteinase (MMP) family. Here, the mechanisms by which neutrophil collagenase (MMP-8), gelatinase A (MMP-2), and the ectodomain of MT1-MMP (ectMMP-14) degrade fibrillar collagen were examined. In particular, the hydrolysis of type I collagen at 37 degrees C was investigated to identify functional differences in the processing of the two alpha-chain types of fibrillar collagen. Thermodynamic and kinetic parameters were used for a quantitative comparison of the binding, unwinding, and hydrolysis of triple helical collagen. We demonstrate that the MMP family has developed at least two distinct mechanisms for collagen unwinding and cleavage. MMP-8 and ectMMP-14 display a similar mechanism (although with different catalytic parameters), which is characterized by binding (likely through the hemopexin-like domain) and cleavage of alpha-1 and/or alpha-2 chains without distinguishing between them and keeping the gross conformation of the triple helix (at least during the first cleavage step). On the other hand, MMP-2 binds preferentially the alpha-1 chains (likely through the fibronectin-like domain, which is not present in MMP-8 and ectMMP-14), grossly altering the whole triple helical arrangement of the collagen molecule and cleaving preferentially the alpha-2 chain. These distinctive mechanisms underly a drastically different mode of interaction with triple helical fibrillar collagen I, according to which the MMP domain is involved in binding. These findings can be related to the different role exerted by these MMPs on collagen homeostasis in the extracellular matrix.     

Letter to the editor: Influence of proline hydroxylation upon the thermal stability of collagen fragment alpha1CB2

A polarimetric study of a 36-residue peptide, α1CB2, obtained by CNBr cleavage of rat-tail tendon collagen, has been made. The equilibrium and kinetic data for this peptide are compared with those found for rat-skin collagen peptide α1CB2. The marked difference in thermal stability is interpreted in terms of the differing degree of hydroxylation of proline residues in otherwise identical peptides.     

The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts.

Collagen secretion by chick embryo fibroblasts was measured by incorporating [¹⁴C]proline into proteins and then analyzing the amount of collagen in the cell and medium separately by using purified bacterial collagenase. In order to produce varying levels of hydroxylation, cells were incubated with varying concentrations of ascorbate or with varying concentrations of α,α′-dipyridyl in the presence of saturating ascorbate. Ascorbate stimulated both the hydroxylation of proline in collagen and the secretion of collagen; the concentration of ascorbate required for half-maximal stimulation of both proesses was approximately 4.5 × 10^?7, m. Since the cells could concentrate ascorbate 10-fold, this K_M for proline hydroxylation is 100-fold lower than values reported for purified prolyl hydroxylase (Abbot, M. T., and Udenfriend, S. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.), p. 173, Academic Press New York; Kivirikko K. I., et al. (1968) Biochim. Biophys. Acta, 151, 558–567). Conversely, α,ga′-dipyridyl inhibited both proline hydroxylation and collagen secretion; half-maximal inhibition of both processes was observed at 7 × 10^?5, m. The results of the two types of experiments show that the secretion of collagen becomes directly proportional to proline hydroxylation when approximately 30% of the proline residues in collagen have been hydroxylated compared to maximal hydroxylation of 50%. Since the stability of triple-helical collagen at 37 °C has been shown to be dependent on the hydroxyproline content of the molecule (Rosenbloom, J., et al. (1973) Arch. Biochem. Biophys., 158, 478–484), we suggest that the observed proportionality between secretion and hydroxylation is a reflection of the increased amount of stable triple helical collagen at 37 °C. When the cells were incubated with a concentration of ascorbate that was saturating for secretion and hydroxylation, there was no significant activation of prolyl hydroxylase as measured in a cell-free extract. These experiments suggest that ascorbate effects collagen secretion by acting at the site of proline hydroxylation but not by increasing the activity of prolyl hydroxylase.     

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.     

Differential effects of ascorbate depletion and α,α′-dipyridyl treatment on the stability,but not on the secretion,of type IV collagen in differentiated F9 cells

Ascorbic acid stimulates secretion of type I collagen because of its role in 4-hydroxyproline synthesis, but there is some controversy as to whether secretion of type IV collagen is similarly affected. This question was examined in differentiated F9 cells, which produce only type IV collagen, by labeling proteins with [¹⁴C]proline and measuring collagen synthesis and secretion. Hydroxylation of proline residues in collagen was inhibited to a greater extent in cells treated with the iron chelator α,α′-dipyridyl (97.7%) than in cells incubated without ascorbate (63.1%), but both conditions completely inhibited the rate of collagen secretion after 2–4 h, respectively. Neither treatment affected laminin secretion. Collagen synthesis was not stimulated by ascorbate even after treatment for 2 days. On SDS polyacrylamide gels, collagen produced by α,α′-dipyridyl-treated cells consisted mainly of a single band that migrated faster than either fully (+ ascorbate) or partially (− ascorbate) hydroxylated α1(IV) or α2(IV) chains. It did not contain interchain disulfide bonds or asn-linked glycosyl groups, and was completely digested by pepsin at 15°C. These results suggested that it was a degraded product lacking the 7 S domain and that it could not form a triple helical structure. In contrast, the partially hydroxylated molecule contained interchain disulfide bonds and it was cleaved by pepsin to collagenous fragments similar in size to those obtained from the fully hydroxylated molecule, but at a faster rate. Kinetic experiments and monensin treatment suggested that completely unhydroxylated type IV collagen was degraded intracellularly in the endoplasmic reticulum or cis Golgi. These studies indicate that partial hydroxylation of type IV collagen confers sufficient helical structure to allow interchain disulfide bond formation and resistance to pepsin and intracellular degradation, but not sufficient for optimal secretion. J Cell. Biochem. 67:338–352, 1997. Published 1997 Wiley-Liss, Inc.     

All three chains of 1 alpha 2 alpha 3 alpha collagen from hyaline cartilage resist human collagenase

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