Specific inactivation of prolyl 4-hydroxylase and inhibition of collagen synthesis by oxaproline-containing peptides in cultured human skin fibroblasts

The crucial role of collagen in fibrotic disorders has prompted attempts to develop drugs that inhibit collagen accumulation. Peptides containing the unphysiological amino acid 5-oxaproline (Opr) have recently been found to act as specific syncatalytic inactivators of pure prolyl 4-hydroxylase, the enzyme that catalyzes the formation of 4-hydroxyproline in collagens. The present study indicates that oxaproline-containing peptides benzyloxycarbonyl-Phe-Opr-Gly-benzyl ester (I) and benzyloxycarbonyl-Phe-Opr-Gly-ethyl ester (II) inactivate prolyl 4-hydroxylase in cultured human skin fibroblasts, peptide I being about twice as potent as peptide II. Inactivation by 50% was observed after culturing with about 20-40 microM concentrations of peptide I for 48 h. The inactivation appears to be specific, as no changes were found in the activities of two other intracellular enzymes of collagen synthesis, lysyl hydroxylase and galactosylhydroxylysyl glucosyltransferase. Synthesis of 4-hydroxyproline by the cells was markedly decreased, and 4-hydroxyproline-deficient procollagen accumulated intracellularly, whereas no changes were found in the incorporation of [14C]leucine into protein after culturing of the cells with a 30 microM concentration of peptide I for 48 h. No changes were seen in the viability of the cells or the release of lactate dehydrogenase from them into the culture medium. No significant changes were found in the steady-state levels of the mRNAs for the pro-alpha 1 chains of type I and type III procollagens or for the alpha and beta subunits of prolyl 4-hyroxylase or fibronectin after culturing with 75 microM peptide I for 48 h. The data indicate that inactivation of cellular prolyl 4-hydroxylase has marked effects on cellular 4-hydroxyproline formation and collagen secretion but no effects on the steady-state levels of mRNAs for type I and III procollagens or the two types of subunit of prolyl 4-hydroxylase.     

The disulphide-bonded nature of procollagen and the role of the extension peptides in the assembly of the molecule.

1. The molecular weights of chick tendon and cartilage procollagens, and their constituent polypeptides, were determined by gel filtration and gel electrophoresis. The values obtained are in good agreement and indicate that the mol.wts. of the secreted procollagens (types I and II) and their individual pro-alpha-chains are of the order of 405 000-445 000 and 137 000-145 000 respectively.2. Digestion of tendon procollagen with human rheumatoid synovial collagenase gave products consistent with the presence of large non-helical peptide extensions at both N-and C-termini. Electrophoretic analysis gave apparent mol.wts. of 17 500 and 36 000 for the respective N- and C-terminal extensions of pro-alpha1(I)-and pro-alpha2-chains, and inter-chain disulphide bonds were restricted to the C-terminal location. 3. During the biosynthesis of procollagen by tendon and cartilage cells a close correlation was observed between the extent of inter-chain disulphide bonding and the proportion of procollagen polypeptides having a triple-helical conformation. These processes appeared to commence in the rough endoplasmic reticulum and be completed in the smooth endoplasmic reticulum, but the rate at which they occur in cartilage cells is markedly slower than that found in tendon cells. 4. When the intracellular [14C]procollagen polypeptides present in the rough-endoplasmic-reticulum fractions of tendon and cartilage cells were analysed under non-reducing conditions on agarose/polyacrylamide composite gels, no significant pools of dimeric intermediates were detected. 5. In both cell types, inter-chain disulphide-bond formation occurred even when hydroxylation, and hence triple-helix formation, was inhibited. The presence of pro-alpha1- and pro-alpha2-components in a ratio of 2:1 in the disulphide-linked unhydroxylated procollagen isolated from tendon cells demonstrated that correct chain association occurs in the absence of hydroxylation. This observation is consistent with a model for the assembly of pro-gamma112-chains in which the recognition and selection of pro-alpha1-and pro-alpha2-chains in a 2:1 ratio are directed by the non-helical C-terminal extension peptides of tendon procollagen.     

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.     

Translation of embryonic-chick tendon procollagen messenger ribonucleic acid in two cell-free protein-synthesizing systems

Embryonic-chick tendon poly(A)-containing RNA was translated in the wheat-germ and mRNA-dependent rabbit reticulocyte-lysate systems. The ability of each system to synthesize polypeptides similar to pro-alpha chains of collagen was tested on the bases of electrophoretic mobility and susceptibility to highly purified bacterial collagenase. Very small amounts of polypeptides in the size range of pro-alpha chains were synthesized in the wheat-germ system, whereas efficient synthesis of two polypeptides similar to pro-alpha1 and pro-alpha2 chains was achieved in the reticulocyte lysate. The collagenous nature of the major high-molecular-weight products synthesized was demonstrated by their susceptibility to collagenase and ability to act as a substrate for purified collagen proline hydroxylase. Determinations of the relative amounts of these translation products suggest that the 2:1 ratio of pro-alpha1 and pro-alpha2 chains found in type I procollagen is reflected in proportional amounts of translatable mRNA for pro-alpha1 and pro-alpha2 chains. Comparisons of the electrophoretic mobilities of hydroxylated and unhydroxylated reticulocyte-lysate translation products were made with appropriate standards of hydroxylated and unhydroxylated procollagen polypeptides. The results suggest that, in common with a number of secreted proteins, procollagen is synthesized as pre-pro molecules consistent with the ;Signal Hypothesis'.     

Collagen biosynthesis. Characterization of subcellular fractions from embryonic chick fibroblasts and the intracellular localization of protocollagen prolyl and protocollagen lysyl hydroxylases

1. Subcellular fractions of freshly isolated matrix-free embryonic chick tendon and sternal cartilage cells have been characterized by chemical analysis, electron microscopy and the location of specific marker enzymes. These data indicate the fractions to be of a high degree of purity comparable with those obtained from other tissues, e.g. liver and kidney. 2. When homogenates were assayed for protocollagen prolyl hydroxylase and protocollagen lysyl hydroxylase activities, addition of Triton X-100 (0.1%, w/v) was found to stimulate enzyme activities by up to 60% suggesting that the enzymes were probably membrane-bound. 3. Assay of subcellular fractions obtained by differential centrifugation for protocollagen prolyl hydroxylase activity indicated the specific activity to be highest in the microsomal fraction. Similar results were obtained for protocollagen lysyl hydroxylase activity. 4. Submicrosomal fractions obtained by discontinuous sucrose-gradient centrifugation were assayed for the two enzymes and protocollagen prolyl hydroxylase and protocollagen lysyl hydroxylase were found to be associated almost exclusively with the rough endoplasmic reticulum fraction in both tendon and cartilage cells.     

The molecular defect in an autosomal dominant form of osteogenesis imperfecta. Synthesis of type I procollagen containing cysteine in the triple-helical domain of pro-alpha 1(I) chains

Synthesis of procollagen was examined in skin fibroblasts from a patient with a moderately severe autosomal dominant form of osteogenesis imperfecta. Proteolytic removal of the propeptide regions of newly synthesized procollagen, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, revealed the presence of type I collagen in which two alpha 1(I) chains were linked through interchain disulfide bonds. Fragmentation of the disulfide-bonded alpha 1(I) dimers with vertebrate collagenase and cyanogen bromide demonstrated the presence of a cysteine residue in alpha 1(I)CB8, a fragment containing amino acid residues 124-402 of the alpha 1(I) collagen chain. Cysteine residues are not normally found in the triple-helical domain of type I collagen chains. The heterozygous nature of the molecular defect resulted in the formation of three kinds of type I trimers: a normal type with normal pro-alpha(I) chains, a type I trimer with one mutant pro-alpha 1(I) chain and two normal chains, and a type I trimer containing two mutant pro-alpha 1(I) chains and one normal pro-alpha 2(I) chain. The presence of one or two mutant pro-alpha 1(I) chains in trimers of type I procollagen was found to reduce the thermal stability of the protein by 2.5 and 1 degree C, respectively. In addition to post-translational overmodification, procollagen containing one mutant pro-alpha 1(I) chain was also cleared more slowly from cultured fibroblasts. The most likely explanation for these disruptive changes in the physical stability and secretion of the mutant procollagen is that a cysteine residue is substituted for a glycine in half of the pro-alpha 1(I) chains synthesized by the patient's fibroblasts.     

Synthesis and processing of a type I procollagen containing shortened pro-alpha 1(I) chains by fibroblasts from a patient with osteogenesis imperfecta

Skin fibroblasts from a patient with a lethal form of osteogenesis imprefecta were found to synthesize equal amounts of normal pro-alpha 1(I) chains and pro-alpha 1(I) chains which are about 10% shorter because of a deletion of about 100 amino acids in the middle of the alpha chain domain. The pro-alpha 1(I) chains were incorporated into three different kinds of trimers: a normal type I trimer with normal length pro-alpha 1(I) chains; a type Is trimer with one shortened pro-alpha 1(I) chain and two normal length chains; and a type Iss trimer containing two shortened pro-alpha 1(I) chains and one normal length pro-alpha 2(I) chain. As judged by resistance to digestion by chymotrypsin and trypsin, the type Is and Iss trimers denatured at a temperature at least 3 degrees C lower than normal type I procollagen. Procollagen containing the shortened pro-alpha 1(I) chains was slowly secreted by the cells but was degraded by extracellular proteinases within 6 h of chase into the medium. The results indicated that the presence of the shortened pro-alpha 1(I) chains in procollagen trimers produces a delay in rate of helix formation, overmodification of the polypeptides by post-translational enzymes, a decrease in the thermal stability of the trimers, and increased susceptibility of the protein to endogenous proteinases. Additionally, the fibroblasts of this patient synthesized and secreted a type III-like species of procollagen with unusual chromatographic properties.     

Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae

The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heat-stable heterotrimers in Saccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.     

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.     

Preferential hydroxylation of type IV collagen by lysyl hydroxylase from Ehlers-Danlos syndrome type VI fibroblasts

Normal and Ehlers-Danlos syndrome type VI human skin and cornea fibroblasts were assayed for lysyl hydroxylase activity using two different collagen types as substrates. The enzyme from normal fibroblasts hydroxylated type I collagen more readily than type IV collagen. In the diseased cells the enzyme activity was significantly reduced, and the residual activity was preferentially directed towards type IV collagen. This suggests the existence of isoenzymes of lysyl hydroxylase or an alteration in the Ehlers-Danlos syndrome type VI that affects the binding of type I collagen more than that of type IV collagen.     

Immunochemical and immunocytochemical studies of the C-propeptide of type II procollagen in chondrocytes of the growth plate.

The C-propeptide of type II procollagen has previously been implicated in cartilage calcification. To further characterize this propeptide, we have investigated its molecular status and intracellular distribution in bovine fetal growth plate chondrocytes, particularly within the calcifying zone, using cell isolation, Western blotting, and localization with immunofluorescence and immunogold techniques. We found that in all cells freshly isolated by collagenase digestion the C-propeptide was a component of type II pro-alpha chains. No free C-propeptide was detected intracellularly. In situ localization of the C-propeptide by immunostaining employing immunofluorescence revealed the presence of procollagen in most growth plate cells, staining being most intense in hypertrophic cells. In the latter, large dilations of the rough endoplasmic reticulum were observed. These were not found in proliferating cells and had an approximate diameter of 5 microns. With immunogold localization these, together with Golgi-derived secretory granules, stained for the C-propeptide. These combined results suggest that in all cells of the growth plate the C-propeptide is a constituent part of type II collagen pro-alpha chains, and that it is usually segregated in the rough endoplasmic reticulum at a time when, according to other studies, collagen synthesis ceases in the lower hypertrophic zone and calcification of the extracellular matrix ensues. This suggests that the intracellular translocation of type II collagen pro-alpha chains may change in hypertrophic cells at this time.     

Biosynthesis and proteolytic processing of type XI collagen in embryonic chick sterna

The biosynthesis and proteolytic processing of type XI procollagen was examined using pulse-chase labelling of 17-day embryonic chick sterna in organ culture with [3H]proline. Products of biosynthesis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with and without prior reduction of disulfide bonds. Pro-alpha chains, intermediates, and matrix forms were identified by cyanogen bromide or Staphylococcus aureus V8 protease digestion. The results show that type XI pro-alpha chains assemble into trimeric molecules with interchain disulfide bonds. Proteolytic processing begins at least 40 min after the start of labeling which is later than that of type II procollagen (25 min). This first processing step involves the loss of the domain containing the interchain disulfide bonds which most likely is the carboxyl propeptide. In the case of the pro-alpha 3 chain, this generates the matrix form, m alpha 3, which retains its amino propeptide. For the pro-alpha 1 and pro-alpha 2 chains, this step generates intermediate forms, p alpha 1 and p alpha 2, which undergo a second proteolytic conversion to m alpha 1 and m alpha 2, and yet retain a pepsin-labile domain. The conversion of p alpha 2 to m alpha 2 is largely complete 2 h after labeling. p alpha 1 is converted to m alpha 1 very slowly and is 50% complete after 18 h of chase in organ culture. The apparent proteolytic processing within the amino propeptide, and the differential rate of processing between two chains in the same molecule are unusual and distinguish type XI from collagen types I, II, and III. It is possible that the extremely slow processing of p alpha 1 affects the formation of the heterotypic cartilage collagen fibrils and may be related to the function of type XI collagen.     

Regulation of collagen post-translational modification in transformed human and chick-embryo cells.

Changes in the regulation of collagen post-translational modification in transformed cells were studied in three established human sarcoma cell lines and in chick-embryo fibroblasts freshly transformed by Rous sarcoma virus. The collagens synthesized by all but one of these and by all the control human and chick-embryo cell lines were almost exclusively of types I and/or III. The relative rate of collagen synthesis and the amounts of prolyl hydroxylase activity and immunoreactive protein were markedly low in all the transformed human cell lines. The other enzymes studied, lysyl hydroxylase, hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase, never showed as large a decrease in activity as did prolyl hydroxylase, suggesting a more efficient regulation of the last enzyme than of the three others. The chick-embryo fibroblasts freshly transformed by Rous sarcoma virus differed from the human sarcoma cells in that prolyl hydroxylase activity was distinctly increased, whereas the decreases in immunoreactive prolyl hydroxylase protein and the three other enzyme activities were very similar to those in the simian-virus-40-transformed human fibroblasts. It seems possible that this increased prolyl hydroxylase activity is only a temporary phenomenon occurring shortly after the transformation, and may be followed by a decrease in activity later. The newly synthesized collagens of all the transformed cells that produced almost exclusively collagen types I and/or III had high extents of lysyl hydroxylation, and there was also an increase in the ratio of glycosylated to non-glycosylated hydroxylysine. The data suggest that one critical factor affecting modification is the rate of collagen synthesis, which affects the ratio of enzyme to substrate in the cell.     

Immunological characterization of lysyl hydroxylase, an enzyme of collagen synthesis

Antibodies to pure lysyl hydroxylase from whole chick embryos were prepared in rabbits and used for immunological characterization of this enzyme of collagen biosynthesis. In double immunodiffusion a single precipitation line was seen between the antiserum and crude or pure chick-embryo lysyl hydroxylase. The antiserum effectively inhibited chick-embryo lysyl hydroxylase activity, whether measured with the biologically prepared protocollagen substrate or a synthetic peptide consisting of only 12 amino acids. This suggests that the antigenic determinant was located near the active site of the enzyme molecule. Essentially identical amounts of the antiserum were required for 40% inhibition of the same amount of lysyl hydroxylase activity units from different chick-embryo tissues synthesizing various genetically distinct collagen types. In double immunodiffusion a single precipitation line of complete identity was found between the antiserum and the purified enzyme from whole chick embryos and the crude enzymes from chick-embryo tendon, cartilage and kidneys. These results do not support the hypothesis that lysyl hydroxylase has collagen-type-specific or tissue-specific isoenzymes with markedly different specific activities or immunological properties. The antibodies to chick-embryo lysyl hydroxylase showed a considerable degree of species specificity when examined either by activity-inhibition assay or by double immuno-diffusion. Nevertheless, a distinct, although weak, cross-reactivity was found between the chick-embryo enzyme and those from all mammalian tissues tested. The antiserum showed no cross-reactivity against prolyl 3-hydroxylase, hydroxylysyl galactosyl-transferase or galactosylhydroxylysyl glucosyltransferase in activity-inhibition assays, whereas a distinct cross-reactivity was found against prolyl 4-hydroxylase. Furthermore, antiserum to pure prolyl 4-hydroxylase inhibited lysyl hydroxylase activity. These findings suggest that there are structural similarities between these two enzymes, possibly close to or at their active sites.     

Characterization of collagen hydroxylysyl glycosyltransferases as mainly intramembranous microsomal enzymes

The localization of collagen hydroxylysine galactosyl- and galactosyl-hydroxylysine glucosyltransferases in purified chick embryo bone microsomes was studied by differential solubilization with nonionic detergents. Brij-35 (polyoxyethylene 25-lauryl ether) which selectively releases intracisternal proteins, and Triton X-100, whose specificity varies with its concentration, were used in the presence or absence of high ionic strength NaCl. These methods were used previously to characterize prolyl hydroxylase as intracisternal and lysyl hydroxylase as mainly intramembranous. The distribution of both glycosyltransferases within microsomes was similar to that of lysyl hydroxylase; approximately 70-80% of their activities are intramembranous with the remainder intracisternal. Collagen hydroxylysine glucosyltransferase differed from prolyl and lysyl hydroxylase and the galactosyltransferase in that its activity in vitro was apparently inhibited by membrane vesicles, even in the presence of detergents at concentrations which permeabilize the membrane. Accurate measurement of its activity could be achieved only by its separation from vesicles after detergent treatment. The common location of the major portion of lysyl hydroxylase and the glycosyltransferase activities suggests that they may act as a multienzyme complex to preferentially modify certain lysyl residues in nascent procollagen chains as they traverse the membrane of the endoplasmic reticulum. Since these enzymes do not act on helical collagen, their physical separation from prolyl hydroxylase may ensure that modifications of lysine residues occur prior to formation of hydroxyproline, which stabilizes the helical form.     

Cleavage of type I and type II procollagens by type I/II procollagen N-proteinase. Correlation of kinetic constants with the predicted conformations of procollagen substrates

The kinetic constants were examined for the cleavage of several types of procollagen by type I/II procollagen N-proteinase. The Km values were essentially the same (0.2 microM) for chick type I procollagen, human type I procollagen, and chick type II procollagen. However, the Vmax values differed over a 14-fold range. As reported previously, the enzyme did not cleave denatured type I or II procollagen. Also, it did not cleave human type III procollagen which contains the same scissle -Pro-Gln- bond as the pro-alpha 1(I) chain of type I procollagen. To explain the observations, Chou-Fasman rules were used to compare the secondary structures of the cleavage sites in the procollagens. The results supported a previous suggestion (Helseth, D. L., Jr., Lechner, J. L., and Veis, A. (1979) Biopolymers 18, 3005-3014) that the region carboxyl-terminal to cleavage site in the pro-alpha 1(I) chain of type I procollagen was in a hairpin conformation consisting of a beta-sheet, beta-turn, and beta-sheet. In both chick and human type I procollagen, the hairpin loop in the pro-alpha 1(I) chain consisted of about 18 amino acids. The cleavage site itself was in a short alpha-helical structure of four or five amino acids. The pro-alpha 2(I) chains had a similar hairpin loop of about 14 amino acids and alpha-helix of four or five amino acids containing the cleavage site. Chick type II procollagen, which had the highest Vmax value, had a longer hairpin structure of 22 amino acids, and the cleavage site was in a longer alpha-helical domain of 10 amino acids. In contrast, type III procollagen had a random-coil conformation in the same region. The results help to explain the unusual substrate requirements of type I/II N-proteinase. They also help explain why mutations that produce in-frame deletions of amino acids 84 or more residues carboxyl-terminal to the cleavage site make the protein resistant to the enzyme.     

Biosynthetic processing of the pro-alpha 1(V)2pro-alpha 2(V) collagen heterotrimer by bone morphogenetic protein-1 and furin-like proprotein convertases.

The low abundance fibrillar collagen type V is incorporated into and regulates the diameters of type I collagen fibrils. Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays key roles in regulating formation of vertebrate extracellular matrix; it cleaves the C-propeptides of the major fibrillar procollagens I-III and processes precursors to produce the mature forms of the cross-linking enzyme prolysyl oxidase, the proteoglycan biglycan, and the basement membrane protein laminin 5. Here we have successfully produced recombinant pro-alpha1(V)(2)pro-alpha2(V) heterotrimers, and we have used these to characterize biosynthetic processing of the most prevalent in vivo form of type V procollagen. In addition, we have compared the processing of endogenous pro-alpha1(V) chains by wild type mouse embryo fibroblasts and by fibroblasts derived from embryos doubly homozygous null for the Bmp-1 gene and for a gene encoding the closely related metalloprotease mammalian Tolloid-like 1. Together, results presented herein indicate that within pro-alpha1(V)(2)pro-alpha2(V) heterotrimers, pro-alpha1(V) N-propeptides and pro-alpha2(V) C-propeptides are processed by BMP-1-like enzymes, and pro-alpha1(V) C-propeptides are processed by furin-like proprotein convertases in vivo.     

Isolation and characterization of a cDNA clone for the amino-terminal portion of the pro-alpha 1(II) chain of cartilage collagen

We have isolated a cDNA clone (pRcol 2) which is complementary to the 5'-terminal portion of the rat pro-alpha 1(II) chain mRNA. A synthetic oligonucleotide was used both as a primer for cDNA synthesis and as a probe for screening a cDNA library. The probe was a mixture of sixteen 14-mers deduced from an amino acid sequence present in the amino-terminal telopeptide of the rat cartilage alpha 1(II) chain. This primer was chosen so that the resulting cDNA would contain the sequence of the 5' end of the mRNA. The nucleotide sequences of the cDNA were determined and compared with that of three other interstitial procollagen chain mRNAs (pro-alpha 1(I), pro-alpha 2(I), and pro-alpha 1(III) chain mRNA). pRcol 2 contains a 521-base pair (bp) insert, including 153 bp of the 5' untranslated region plus 368 bp coding for the signal peptide, the amino-terminal propeptide, and a part of the telopeptide. The signal peptide of the type II collagen chain is composed of about 20 amino acids. There is little homology between the amino acid sequence of the signal peptide in the pro-alpha 1(II) chain and that of three other interstitial procollagen chains. The NH2-terminal propeptide is deduced to contain short nonhelical sequences at its amino and carboxyl ends and an internal helical collagenous domain comprising 25 repeats of Gly-X-Y with one interruption. There is a strong conservation of the amino acid sequence of the carboxyl-terminal part of the NH2-terminal propeptide in the pro-alpha 1(II), pro-alpha 1(I), and pro-alpha 2(I) chains. Type II collagen mRNA does not contain a sequence corresponding to a uniquely conserved nucleotide sequence around the translation initiation site which occurs in mRNA for other procollagen chains.