Recombinant expression of hydroxylated human collagen in Escherichia coli

Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.     

Concomitant hydroxylation of proline and lysine residues in collagen using purified enzymes in vitro

Concomitant hydroxylation of proline and lysine residues in protocollagen was studied using purified enzymes. The data suggest that prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) and lysyl hydroxylase (peptidyllysine, 2-oxoglutarate; oxygen 5-oxidoreductase, EC 1.14.11.4) are competing for the protocollagen substrate, this competition resulting in an inhibition of the lysyl hydroxylase but not of the prolyl 4-hydroxylase reaction. When the same protocollagen was used for these hydroxylases, the affinity of prolyl 4-hydroxylase to the protocollagen substrate was about 2-fold higher than that of lysyl hydroxylase. Hydroxylation of lysine residues in protocollagen had no effect on the affinity of prolyl 4-hydroxylase, whereas hydroxylation of proline residues decreased the affinity of lysyl hydroxylase to one-half of the value determined before the hydroxylation. When enzyme preparations containing different ratios of lysyl hydroxylase activity to prolyl 4-hydroxylase activity were used to hydroxylase protocollagen substrate, it was found that in the case of a low ratio the hydroxylation of lysine residues seemed to proceed only after a short lag period. Accordingly, it seems probable that most proline residues are hydroxylated to 4-hydroxyproline residues before hydroxylation of lysine residues if the prolyl 4-hydroxylase and lysyl hydroxylase are present as free enzymes competing for the same protocollagen substrate.     

Characterization of recombinant human prolyl 3-hydroxylase isoenzyme 2, an enzyme modifying the basement membrane collagen IV

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.     

A transient post-translationally modified form of cartilage type II collagen is ignored by self-reactive T cells

Lysine residues in type II collagen (CII) are normally hydroxylated and subsequently glycosylated in the chondrocyte. The immunodominant T cell epitope of CII involves such post-translationally modified lysine at position 264 that has been shown to be critical in the pathogenesis of murine collagen-induced arthritis and also in human rheumatoid arthritis. In this study we identified a line of transgenic mice expressing a TCR specific for hydroxylated rat CII epitope. They were crossed with transgenic mice expressing the rat CII epitope, either specifically in cartilage (MMC mice) or systemically (TSC mice), to analyze T cell tolerance to a post-translationally modified form of self-CII. The mechanism of T cell tolerance to the hydroxylated CII epitope in TSC mice was found to involve intrathymic deletion and induction of peripheral tolerance. In contrast, we did not observe T cell tolerance in the MMC mice. Analysis of CII prepared from rat or human joint cartilage revealed that most of the lysine 264 is glycosylated rather than remaining hydroxylated. Therefore, we conclude that the transient post-translationally modified form of cartilage CII does not induce T cell tolerance. This lack of T cell tolerance could increase the risk of developing autoimmune arthritis.     

Collagen lysyl hydroxylation occurs within the cisternae of the rough endoplasmic reticulum.

Resolution of the heavy microsomal fraction of lung tissue by Ficoll density gradient centrifugation yielded a rough endoplasmic reticulum microsomal fraction containing the highest specific activity of detergent-released lysyl hydroxylase. This same microsomal fraction was previously shown to contain the highest specific activity of detergent-released prolyl hydroxylase activity. When hydroxylation was inhibited during the biosynthesis of collagen, this microsomal fraction contained lysine-rich, hydroxylysine-deficient, collagenase-digestible substrate that could be hydroxylated in the absence of detergent. The results indicate coordinate localization of both prolyl and lysyl hydroxylation reactions within the cisternae of the rough endoplasmic reticulum.     

Prolyl hydroxylase of earthworms. Substrate specificity of an enzyme from the subcuticular epithelium.

A relatively crude enzyme preparation derived from the subcuticular epithelium of earthworms catalyzed the formation of 4-hydroxyproline from prolyl residues in unhydroxylated natural collagens and in several synthetic collagen-like polypeptides. The specificity of hydroxylation differed from that of all vertebrate polyl hydroxylases in that (Gly-Pro-Ala)n was a much better substrate than (Gly-Ala-Pro)n. In contrast, however, only the so-called Y position proline (Gly-X-Y) was hydroxylated in Gly-Pro-Pro sequences derived either from natural collagen or from synthetic polypeptides; specificity of hydroxylation for the latter sequence is identical with that of the vertebrate enzymes. Little or no formation of 3-hydroxyproline could be demonstrated in preparations of the enzyme active as a 4-hydroxylase. In contrast with an earlier report from another laboratory, using a crude extract of earthworm body wall, we were unable to demonstrate either significant 3-hydroxyproline formation or efficient 4-hydroxylation of X position prolyl residues in synthetic polypeptides with the internal sequence Gly-Pro-Pro.     

Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in X-Lys-Gly sequences. We report here on the isolation of cDNA clones coding for the enzyme from a chick embryo lambda gt11 library. Several overlapping clones covering all the coding sequences of the 4-kilobase mRNA and virtually all the noncoding sequences were characterized. These clones encode a polypeptide of 710 amino acid residues and a signal peptide of 20 amino acids. The polypeptide has four potential attachment sites for asparagine-linked oligosaccharides and 9 cysteine residues, at least one of which is likely to be involved in the binding of the Fe2+ atom to a catalytic site. A surprising finding was that no significant homology was found between the primary structures of lysyl hydroxylase and prolyl 4-hydroxylase in spite of the marked similarities in kinetic properties between these two enzymes. A computer-assisted comparison indicated only an 18% identity between lysyl hydroxylase and the alpha-subunit of prolyl 4-hydroxylase and a 19% identity between lysyl hydroxylase and the beta-subunit of prolyl 4-hydroxylase. Visual inspection of the most homologous areas nevertheless indicated the presence of several regions of 20-40 amino acids in which the identity between lysyl hydroxylase and one of the prolyl 4-hydroxylase subunits exceeded 30% or similarity exceeded 40%. Southern blot analyses of chick genomic DNA indicated the presence of only one gene coding for lysyl hydroxylase.     

Hydroxylation of prolyl residues in type II procollagen in vitro and in cellulo. Lack of preferential hydroxylation of specific regions of the protein

[14C]Proline-labeled protocollagen, the unhydroxylated form of procollagen, was isolated from cartilage cells incubated with alpha, alpha'-dipyridyl. For examination of the initial steps in the hydroxylation of the protein, it was incubated in vitro with prolyl hydroxylase so that an average of 1.3-2.7 prolyl residues per chain was hydroxylated. The partially hydroxylated alpha chain were cleaved with cyanogen bromide, and the fragments were separated by polyacrylamide gel electrophoresis or column chromatography. The cyanogen bromide fragments were hydroxylated to the same degree. The results indicated, therefore, that in the initial hydroxylation of alpha chains in vitro, there was no preferential hydroxylation of any specific regions of the protein. In a second series of experiments, cartilage cells were incubated with [14C]proline and alpha, alpha'-dipyridyl so that prolyl hydroxylase in the cells was extensively, but not completely, inhibited. Partially hydroxylated alpha chains were isolated, and cyanogen bromide fragments of the alpha chains from the cells were assayed for hydroxy[14C]proline. The alpha chains contained an average of two residues of hydroxyproline per chain, and the cyanogen bromide fragments were hydroxylated to about the same degree. The results indicated, therefore, that when prolyl hydroxylase activity in cells is low relative to the rate at which pro alpha chains are synthesized, hydroxylation of prolyl residues occurs as it does in vitro, and there is no preferential hydroxylation of a specific region of the protein.     

Protein Hydroxylation Catalyzed by 2-Oxoglutarate-dependent Oxygenases

The post-translational hydroxylation of prolyl and lysyl residues, as catalyzed by 2-oxoglutarate (2OG)-dependent oxygenases, was first identified in collagen biosynthesis. 2OG oxygenases also catalyze prolyl and asparaginyl hydroxylation of the hypoxia-inducible factors that play important roles in the adaptive response to hypoxia. Subsequently, they have been shown to catalyze N-demethylation (via hydroxylation) of N^ϵ-methylated histone lysyl residues, as well as hydroxylation of multiple other residues. Recent work has identified roles for 2OG oxygenases in the modification of translation-associated proteins, which in some cases appears to be conserved from microorganisms through to humans. Here we give an overview of protein hydroxylation catalyzed by 2OG oxygenases, focusing on recent discoveries.     

The third activity for lysyl hydroxylase 3: galactosylation of hydroxylysyl residues in collagens in vitro.

Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in posttranslational modifications of collagens. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity. Lysines and hydroxylysines form collagen cross-links. Hydroxylysine derived cross-links, usually as glycosylated forms, occur especially in weight-bearing and mineralized tissues. The detailed functions of the hydroxylysyl and hydroxylysyl linked carbohydrate structures are not known, however. Hydroxylysine linked carbohydrates are found mainly in collagens, but recent reports indicate that these structures are also present and probably have an important function in other proteins. Earlier we have shown that human LH3, but not isoforms LH1, LH2a and LH2b, possesses both LH and glucosyltransferase activity (J. Biol. Chem. 275 (2000) 36158). In this paper we demonstrate that galactosyltransferase activity is also associated with the same gene product, thus indicating that one gene product can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation. In vitro mutagenesis experiments indicate that Cys(144) and aspartates in positions 187-191 of LH3 are important for the galactosyltransferase activity. Our results suggest that manipulation of the gene for LH3 can be used to selectively alter the glycosylation and hydroxylation reactions, and provides a new tool to clarify the functions of the unique hydroxylysine linked carbohydrates in collagens and other proteins.     

Prolyl hydroxylation in elastin is not random

BackgroundThis study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues.MethodsEnzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools.ResultsIt was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%–24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied.ConclusionsThe results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues.General significanceThe study for the first time shows that prolyl hydroxylation is highly regulated in elastin.     

Lysyl hydroxylase 2 is a specific telopeptide hydroxylase, while all three isoenzymes hydroxylate collagenous sequences.

Lysyl hydroxylase (LH), with three isoenzymes in vertebrates, catalyzes the formation of hydroxylysine by acting on -X-Lys-Gly- triplets in the collagenous domains of proteins of the collagen superfamily and also in -X-Lys-Ala- or -X-Lys-Ser- sequences in the telopeptides located at the ends of the polypeptide chains in some fibril-forming collagens. The hydroxylysine residues are essential for the stability of collagen crosslinks and act as carbohydrate attachment sites. The extent of lysine hydroxylation varies between collagen types, between tissues in the same collagen type and in certain diseases, suggesting that the LH isoenzymes may have different substrate specificities. We studied here the hydroxylation of synthetic peptides representing various hydroxylation sites in type I and IV collagens by purified recombinant LHs in vitro and of a recombinant full-length type I procollagen chain coexpressed with each LH in insect cells. All three LHs hydroxylated peptides representing collagenous sequences of type I and IV collagens, although with different K(m) and V(max) values. Furthermore, all three hydroxylated the collagenous domain of the coexpressed type I procollagen chain to a similar extent. None of the isoenzymes hydroxylated peptides representing the N and C telopeptides of type I collagen, but LH2, unlike the other two isoenzymes, hydroxylated the N telopeptide in the coexpressed procollagen chain. Hydroxylation of the telopeptide lysines by LH2 thus occurs only in the context of a long peptide. These data provide the first direct evidence that LH2 is a specific telopeptide hydroxylase, while all three LHs act on collagenous sequences.     

Mimivirus collagen is modified by bifunctional lysyl hydroxylase and glycosyltransferase enzyme

Collagens, the most abundant proteins in animals, are modified by hydroxylation of proline and lysine residues and by glycosylation of hydroxylysine. Dedicated prolyl hydroxylase, lysyl hydroxylase, and collagen glycosyltransferase enzymes localized in the endoplasmic reticulum mediate these modifications prior to the formation of the collagen triple helix. Whereas collagen-like proteins have been described in some fungi, bacteria, and viruses, the post-translational machinery modifying collagens has never been described outside of animals. We demonstrate that the L230 open reading frame of the giant virus Acanthamoeba polyphaga mimivirus encodes an enzyme that has distinct lysyl hydroxylase and collagen glycosyltransferase domains. We show that mimivirus L230 is capable of hydroxylating lysine and glycosylating the resulting hydroxylysine residues in a native mimivirus collagen acceptor substrate. Whereas in animals from sponges to humans the transfer of galactose to hydroxylysine in collagen is conserved, the mimivirus L230 enzyme transfers glucose to hydroxylysine, thereby defining a novel type of collagen glycosylation in nature. The presence of hydroxylysine in mimivirus proteins was confirmed by amino acid analysis of mimivirus recovered from A. polyphaga cultures. This work shows for the first time that collagen post-translational modifications are not confined to the domains of life. The utilization of glucose instead of the galactose found throughout animals as well as a bifunctional enzyme rather than two separate enzymes may represent a parallel evolutionary track in collagen biology. These results suggest that giant viruses may have contributed to the evolution of collagen biology.     

Crystal Structure of the 2-Oxoglutarate- and Fe(II)-Dependent Lysyl Hydroxylase JMJD6

Lysyl and prolyl hydroxylations are well-known post-translational modifications to animal and plant proteins with extracellular roles. More recent work has indicated that the hydroxylation of intracellular animal proteins may be common. JMJD6 catalyses the iron- and 2-oxoglutarate-dependent hydroxylation of lysyl residues in arginine-serine-rich domains of RNA-splicing-related proteins. We report crystallographic studies on the catalytic domain of JMJD6 in complex with Ni(II) substituting for Fe(II). Together with mutational studies, the structural data suggest how JMJD6 binds its lysyl residues such that it can catalyse C-5 hydroxylation rather than N^?-demethylation, as for analogous enzymes.     

A connective tissue disorder caused by mutations of the lysyl hydroxylase 3 gene

Lysyl hydroxylase 3 (LH3, encoded by PLOD3) is a multifunctional enzyme capable of catalyzing hydroxylation of lysyl residues and O-glycosylation of hydroxylysyl residues producing either monosaccharide (Gal) or disaccharide (Glc-Gal) derivatives, reactions that form part of the many posttranslational modifications required during collagen biosynthesis. Animal studies have confirmed the importance of LH3, particularly in biosynthesis of the highly glycosylated type IV and VI collagens, but to date, the functional significance in vivo of this enzyme in man is predominantly unknown. We report here a human disorder of LH3 presenting as a compound heterozygote with recessive inheritance. One mutation dramatically reduced the sugar-transfer activity of LH3, whereas another abrogated lysyl hydroxylase activity; these changes were accompanied by reduced LH3 protein levels in cells. The disorder has a unique phenotype causing severe morbidity as a result of features that overlap with a number of known collagen disorders.     

Spondylocheiro dysplastic form of the Ehlers-Danlos syndrome--an autosomal-recessive entity caused by mutations in the zinc transporter gene SLC39A13

We present clinical, radiological, biochemical, and genetic findings on six patients from two consanguineous families that show EDS-like features and radiological findings of a mild skeletal dysplasia. The EDS-like findings comprise hyperelastic, thin, and bruisable skin, hypermobility of the small joints with a tendency to contractures, protuberant eyes with bluish sclerae, hands with finely wrinkled palms, atrophy of the thenar muscles, and tapering fingers. The skeletal dysplasia comprises platyspondyly with moderate short stature, osteopenia, and widened metaphyses. Patients have an increased ratio of total urinary pyridinolines, lysyl pyridinoline/hydroxylysyl pyridinoline (LP/HP), of approximately 1 as opposed to approximately 6 in EDS VI or approximately 0.2 in controls. Lysyl and prolyl residues of collagens were underhydroxylated despite normal lysyl hydroxylase and prolyl 4-hydroxylase activities; underhydroxylation was a generalized process as shown by mass spectrometry of the alpha1(I)- and alpha2(I)-chain-derived peptides of collagen type I and involved at least collagen types I and II. A genome-wide SNP scan and sequence analyses identified in all patients a homozygous c.483_491 del9 SLC39A13 mutation that encodes for a membrane-bound zinc transporter SLC39A13. We hypothesize that an increased Zn(2+) content inside the endoplasmic reticulum competes with Fe(2+), a cofactor that is necessary for hydroxylation of lysyl and prolyl residues, and thus explains the biochemical findings. These data suggest an entity that we have designated "spondylocheiro dysplastic form of EDS (SCD-EDS)" to indicate a generalized skeletal dysplasia involving mainly the spine (spondylo) and striking clinical abnormalities of the hands (cheiro) in addition to the EDS-like features.     

Amino acid analysis by hydrophilic interaction chromatography coupled on-line to electrospray ionization mass spectrometry

Summary. Collagens form a common family of triple-helical proteins classified in 21 types. This unique structure is further stabilized by specific hydroxylation of distinct lysyl and prolyl residues forming 5-hydroxylysine and hydroxyproline (Hyp) isomers, mostly 4-trans and 3-trans-Hyp. The molecular distribution of the Hyp-isomers among the different collagen types is still not well investigated, even though disturbances in the hydroxylation of collagens are likely to be involved in several diseases such as osteoporosis and autoimmune diseases. Here, a new approach to analyze underivatized amino acids by hydrophilic interaction chromatography (HILIC) coupled on-line to electrospray ionization mass spectrometry (ESI-MS) is reported. This method can separate all three studied Hyp-isomers, Ile, and Leu, which are all isobaric, allowing a direct qualitative and quantitative analysis of collagen hydrolysates. The sensitivity and specificity was increased by a neutral loss scan based on the loss of formic acid (46 u).     

Production of murine collagen-induced arthritis using Klebsiella pneumoniae O3 lipopolysaccharide as a potent immunological adjuvant.

Collagen-induced arthritis (CIA) was produced in mice with non H-2q and H-2r haplotypes by repeated immunization of porcine type-II collagen (CII) together with Klebsiella O3 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes that appeared in joints of repeatedly immunized mice were characterized by destruction of normal joint structure, synovial hyperplasia with proliferation of synovial cells, and infiltration of inflammatory cells. No such lesions were produced in mice receiving repeated injections of CII alone or KO3 LPS alone. Development of the humoral antibody and the delayed-type hypersensitivity to CII was exclusively found in mice immunized with the mixture of CII and KO3 LPS. It was therefore suggested that arthritis lesions induced by repeated immunization with the mixture of CII and KO3 LPS might be caused by an autoimmune mechanism, and that the experimental model might be useful for characterization of human rheumatoid arthritis (RA).     

PHD3-mediated prolyl hydroxylation of nonmuscle actin impairs polymerization and cell motility

Actin filaments play an essential role in cell movement, and many posttranslational modifications regulate actin filament assembly. Here we report that prolyl hydroxylase 3 (PHD3) interacts with nonmuscle actin in human cells and catalyzes hydroxylation of actin at proline residues 307 and 322. Blocking PHD3 expression or catalytic activity by short hairpin RNA knockdown or pharmacological inhibition, respectively, decreased actin prolyl hydroxylation. PHD3 knockdown increased filamentous F-actin assembly, which was reversed by PHD3 overexpression. PHD3 knockdown increased cell velocity and migration distance. Inhibition of PHD3 prolyl hydroxylase activity by dimethyloxalylglycine also increased actin polymerization and cell migration. These data reveal a novel role for PHD3 as a negative regulator of cell motility through posttranslational modification of nonmuscle actins.     

Underhydroxylated minor cartilage collagen precursors cannot form stable triple helices.

Matrix-free cells from chick-embryo sterna were incubated with various concentrations of 2,2'-bipyridyl, an iron chelator that inhibits prolyl hydroxylase and lysyl hydroxylase. At concentrations in the region of 0.1 mM, significant effects on cartilage collagen hydroxylation and secretion were observed. When the underhydroxylated collagens were subsequently digested with chymotrypsin or chymotrypsin plus trypsin at 4 degrees C for 15 min, the minor cartilage collagen precursors (namely types IX and XI) were extensively degraded; type II procollagen was only partially susceptible and was converted into underhydroxylated collagen. The results demonstrate that there were significant differences in triple-helix stability among cartilage collagens such that the underhydroxylated minor collagen precursors were unable to attain a native structure under conditions where type II procollagen was successful.