Identification of the molecular recognition sequence which determines the type-specific assembly of procollagen.

A key question relating to procollagen biosynthesis is the way in which closely related procollagen chains discriminate between each other to assemble in a type-specific manner. Intracellular assembly of procollagen occurs via an initial interaction between the C-propeptides followed by vectorial propagation of the triple-helical domain in the C to N direction. Recognition signals within the C-propeptides must, therefore, determine the selective association of individual procollagen chains. We have used the pro alpha1 chain of type III procollagen [pro alpha1(III)] and the pro alpha2 chain of type I procollagen [pro alpha2(I)] as examples of procollagen chains that are either capable or incapable of self-assembly. When we exchanged the C-propeptides of the pro alpha1(III) chain and the pro alpha(I) chain we demonstrated that this domain is both necessary and sufficient to direct the assembly of homotrimers with correctly aligned triple-helices. To identify the sequences within this domain that determine selective association we constructed a series of chimeric procollagen chains in which we exchanged specific sequences from the pro alpha1(III) C-propeptide with the corresponding region within the pro alpha2(I) C-propeptide (and vice versa) and assayed for the ability of these molecules to form homotrimers. Using this approach we have identified a discontinuous sequence of 15 amino acids which directs procollagen self-association. By exchanging this sequence between different procollagen chains we can direct chain association and, potentially, assemble molecules with defined chain compositions.     

Translation of chick calvarial procollagen messenger RNA'S by a messenger RNA dependent reticulocyte lysate.

A method was developed for the extraction of RNA from chick embryo calvaria which should be generally applicable to other connective tissues. Total RNA prepared by this method was translated by a mRNA-dependent reticulocyte lysate into discrete pro alpha chains. Several criteria were used to identify these translation products, including (1) preferential labeling with [3H]proline, (2) appropriate migration on sodium dodecyl sulfate-polyacrylamide gels, (3) selective sensitivity to collagenase digestion, and (4) specific precipitability by two different antisera against procollagen. Data from the immunoprecipitation experiments indicated that the majority of the pro alpha chains contained the carboxy-terminal antigenic determinants. These results demonstrate that this translation system can be used as an assay for intact procollagen mRNAs and as a source of in vitro synthesized pro alpha chains for future structural analysis.     

Chain assembly intermediate in the biosynthesis of type III procollagen in chick embryo blood vessels

A general mechanism for the assembly of procollagens is proposed from a biosynthetic study of procollagen III. This was shown to proceed by a stepwise process punctuated by disulfide bond formation and an assembly intermediate was recovered. The biosynthesis of type III procollagen in excised chick embryo blood vessels was studied by radioactive labeling for 30 min. Velocity sedimentation under denaturing conditions and purified antibodies specific against bovine amino propeptide III were used to identify and characterize monomeric pro alpha 1 III chains and a type III procollagen intermediate which is interchain disulfide-linked only at the carboxyl end but not at the amino end. The monomeric chains presumably have intrachain disulfide bonds within the propeptides. The monomeric pro alpha 1 III chains were also found when alpha, alpha'-dipyridyl was present during incubation. Pulse-chase experiments show that the monomeric chains and the intermediate are biosynthetic precursors of type III procollagen. Furthermore, it is shown that monomeric pro alpha 1 chains are not triple helical when extracted under nondenaturing conditions. The results indicate that the assembly of pro alpha 1 III chains into type III procollagen starts with the association of the folded carboxyl propeptides and is followed by formation of disulfide bonds between carboxyl propeptides, folding of the triple helix, and formation of disulfide bonds between amino propeptides. All procollagens may follow a similar assembly sequence.     

The role of glycosylation in the enzymatic conversion of procollagen to collagen: studies using tunicamycin and concanavalin A

The enzymatic conversion of chick embryo cranial bone procollagen was studied in vitro using procollagen proteases isolated from the culture medium of chick tendon fibroblasts. During the normal conversion process, chains intermediate in length between proα and α chains, as well as the COOH-terminal extension peptides, can be identified. Underglycosylated procollagen, synthesized by bones treated with an inhibitor of protein glycosylation (tunicamycin), was processed by these proteases in a manner similar to that of intact procollagen. However, medium from cells cultured with tunicamycin lacked the COOH-terminal procollagen protease activity; this did not result from a direct inhibition of the protease by the drug. Concanavalin A also inhibited the conversion of procollagen to collagen by fibroblasts in culture. In an in vitro system, Concanavalin A inhibited the COOH-terminal procollagen protease, and this inhibition was reversed by methyl-α-d-glucopyranoside. These data suggest that the COOH-terminal procollagen protease contains oligosaccharide side chains that are recognized by concanavalin A and that tunicamycin affects the secretion, activity, or activation of the enzyme.     

Mutations that alter the primary structure of type I procollagen have long-range effects on its cleavage by procollagen N-proteinase

Type I/II procollagen N-proteinase was partially purified from chick embryos and used to examine the rate of cleavage of a series of purified type I procollagens synthesized by fibroblasts from probands with heritable disorders of connective tissue. The rate of cleavage was normal with procollagen from a proband with osteogenesis imperfecta that was overmodified by posttranslational enzymes. Therefore, posttranslational overmodification of the protein does not in itself alter the rate of cleavage under the conditions of the assay employed. Cleavage of the procollagen, however, was altered in several procollagens with known mutations in primary structure. Two of the procollagens had in-frame deletions of 18 amino acids encoded by exons 11 and 33 of the pro alpha 2(I) gene. In both procollagens, both the pro alpha 1(I) and the pro alpha 2(I) chains were totally resistant to cleavage. With a procollagen in which glycine-907 of the alpha 2(I) chain domain was substituted with aspartate, both pro alpha chains were cleaved but at a markedly decreased rate. The results, therefore, establish that mutations that alter the primary structure of the pro alpha chains of procollagen at sites far removed from the N-proteinase cleavage site can make the protein resistant to cleavage by the enzyme. The long-range effects of in-frame deletions or other changes in amino acid sequence are probably explained by their disruption of the hairpin structure that is formed by each of the three pro alpha chains in the region containing the cleavage site and that is essential for cleavage of the procollagen molecule by N-proteinase.     

Sequential cleavage of type I procollagen by procollagen N-proteinase. An intermediate containing an uncleaved pro alpha 1(I) chain

The conversion of type I procollagen to type I collagen was studied by cleaving the protein with partically purified type I procollagen N-proteinase from chick embryos. Examination of the reaction products after incubation for varying times at 30 degrees C indicated that, during the initial stages of the reaction, pro alpha 1(I) and pro alpha 2(I) chains were cleaved at about the same rate. As a result, all the pro alpha 2(I) chains were converted to pC alpha 2(I) chains well before all the pro alpha 1 chains were cleaved. When the reaction products were examined by gel electrophoresis without reduction of interchain disulfide bonds, a distinct band of an intermediate was detected. The same intermediate was seen when the reaction was carried out at 35, 37, and 40 degrees C. The data established that over two-thirds of the type I procollagen was converted to the intermediate and that this intermediate was then slowly converted to the final product of pCcollagen. The kinetics for the reaction, however, did not fit a simple model for precursor-product relationship among substrate, intermediate, and product. Examination of the reaction products with a two-step gel procedure demonstrated that the intermediate consisted of three polypeptide chains in which the N propeptide was cleaved from one pro alpha 1 chain and one pro alpha 2(I) chain but the N propeptide was still present on one of the pro alpha 1(I) chains. In further experiments it was demonstrated that a similar intermediate was seen when a homotrimer of pro alpha 1(I) chains was partially cleaved by the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the six zebrafish clade B fibrillar procollagen genes,with evidence for evolutionarily conserved alternative splicing within the pro-α1(V) C-propeptide

Genes for tetrapod fibrillar procollagen chains can be divided into two clades, A and B, based on sequence homologies and differences in protein domain and gene structures. Although the major fibrillar collagen types I–III comprise only clade A chains, the minor fibrillar collagen types V and XI comprise both clade A chains and the clade B chains pro-α1(V), pro-α3(V), pro-α1(XI) and pro-α2(XI), in which defects can underlie various genetic connective tissue disorders. Here we characterize the clade B procollagen chains of zebrafish. We demonstrate that in contrast to the four tetrapod clade B chains, zebrafish have six clade B chains, designated here as pro-α1(V), pro-α3(V)a and b, pro-α1(XI)a and b, and pro-α2(XI), based on synteny, sequence homologies, and features of protein domain and gene structures. Spatiotemporal expression patterns are described, as are conserved and non-conserved features that provide insights into the function and evolution of the clade B chain types. Such features include differential alternative splicing of NH₂-terminal globular sequences and the first case of a non-triple helical imperfection in the COL1 domain of a clade B, or clade A, fibrillar procollagen chain. Evidence is also provided for previously unknown and evolutionarily conserved alternative splicing within the pro-α1(V) C-propeptide, which may affect selectivity of collagen type V/XI chain associations in species ranging from zebrafish to human. Data presented herein provide insights into the nature of clade B procollagen chains and should facilitate their study in the zebrafish model system.     

Structure of cDNA clones coding for human type II procollagen. The alpha 1(II) chain is more similar to the alpha 1(I) chain than two other alpha chains of fibrillar collagens.

Overlapping cDNA clones were isolated for human type II procollagen. Nucleotide sequencing of the clones provided over 2.5 kb of new coding sequences for the human pro alpha 1(II) gene and the first complete amino acid sequence of type II procollagen from any species. Comparison with published data for cDNA clones covering the entire lengths of the human type I and type III procollagens made it possible to compare in detail the coding sequences and primary structures of the three most abundant human fibrillar collagens. The results indicated that the marked preference in the third base codons for glycine, proline and alanine previously seen in other fibrillar collagens was maintained in type II procollagen. The domains of the pro alpha 1(II) chain are about the same size as the same domains of the pro alpha chains of type I and type III procollagens. However, the major triple-helical domain is 15 amino acid residues less than the triple-helical domain of type III procollagen. Comparison of hydropathy profiles indicated that the alpha chain domain of type II procollagen is more similar to the alpha chain domain of the pro alpha 1(I) chain than to the pro alpha 2(I) chain or the pro alpha 1(III) chain. The results therefore suggest that selective pressure in the evolution of the pro alpha 1(II) and pro alpha 1(I) genes is more similar than the selective pressure in the evolution of the pro alpha 2(I) and pro alpha 1(III) genes.     

Structure of antigenic determinants in the N-terminal region of dermatosparactic sheep procollagen type I.

About half of the rabbit antisera raised against type-I procollagen, p alpha 1(I) chain or nonreduced procollagen peptides reacted in a radioimmunoassay with the reduced form of peptide Col 1, which comprises the whole non-collagenous region at the N-terminus of procollagen. Proteolytic fragments prepared from reduced peptide Col 1 were still effective inhibitors of the antibodies and allowed the localization of two antigenic determinants. The antigenically active regions have the sequences less than Glu-Glu-Glu-Gly-Gln-Gln-Glu and Gly-Asp-Thr-Gly-Pro-Arg, and are located at the N- and C-termini of the peptide respectively. Antibodies raised against reduced peptide Col 1 bind to a determinant localized in a different region of the peptide.     

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.     

Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species.

Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.     

Immunological characterization of the major chick cartilage proteoglycan and its intracellular localization in cultured chondroblasts: a comparison with Type II procollagen

Polyclonal antibodies were raised in a rabbit against the major proteoglycan of chick sternal cartilage. A total of six antisera was obtained, three after the first booster injection (A1, A2, and A3) and three after the second booster injection (A4, A5, and A6). The A1 antiserum, which was characterized in most detail, immunoprecipitated native as well as chondroitinase ABC-digested or chondroitinase ABC/keratanase-digested cartilage proteoglycan synthesized by cultured chick chondroblasts, but failed to immunoprecipitate the major proteoglycan synthesized by chick skin fibroblasts. This antiserum was also able to immunoprecipitate the cartilage proteoglycan core protein newly synthesized by cultured chondroblasts, but no other major cell protein. However, the late bleed antisera obtained from the same rabbit after a second booster injection reacted with a new chondroblast- specific polypeptide(s) of approximately 60,000 mol wt in addition to the cartilage proteoglycan. By immunofluorescence procedures, the A1 antiserum stained the extracellular proteoglycan matrix of cultured chondroblasts but not that of skin fibroblasts. Following enzymatic removal of the extracellular matrix and cell membrane permeabilization, this antiserum stained primarily a large, juxtanuclear structure. Additional radioautographic evidence suggests that this structure represents the Golgi complex. Similar immunofluorescent staining with antibodies to the cartilage-characteristic Type II collagen revealed that type II procollagen was localized in numerous cytoplasmic, vacuole- like structures which were scattered throughout most of the chondroblast cytoplasm but were notably scanty in the Golgi complex area. In conclusion, our data suggest the transit of the major cartilage proteoglycan through the Golgi complex of cultured chondroblasts and possible differences in the intracellular distribution of newly synthesized cartilage proteoglycan and Type II procollagen.     

Intermediates in the conversion of procollagen to collagen. Evidence for stepwise limited proteolysis of the COOH-terminal peptide extensions.

Intermediates in the conversion of procollagen to collagen were isolated from radioactively labeled chick cranial bones by ion-exchange chromatography. Cleavage of these proteins with vertebrate collagenase revealed that each of the several forms of these intermediates lacked NH2-terminal but retained COOH-terminal extensions. The chain composition of each intermediate was resolved by two-dimensional slab gel electrophoresis. The intermediates differed from each other in having sustained cleavages in zero, one or two pcalpha chains. The relative proportions of intermediates with different intact pcalpha chains, observed in conversion of procollagen, have enabled us to construct a detailed model of the stepwise limited proteolysis of procollagen.     

Structure of a cDNA for the pro alpha 2 chain of human type I procollagen. Comparison with chick cDNA for pro alpha 2(I) identifies structurally conserved features of the protein and the gene

Nucleotide sequences were determined for cloned cDNAs encoding for more than half of the pro alpha 2 chain of type I procollagen from man. Comparisons with previously published data on homologous cDNAs from chick embryos made it possible to examine evolution of the gene in two species which have diverged for 250-300 million years. The amino acid sequence of the alpha-chain domain supported previous indications that there is a strong selective pressure to maintain glycine as every third amino acid and to maintain a prescribed distribution of charged amino acids. However, there is little apparent selective pressure on other amino acids. The amino acid sequence of the C-propeptide domain showed less divergence than the alpha-chain domain. The 5' end or N terminus of the human C-propeptide, however, contained an insert of 12 bases coding for 4 amino acids not found in the chick C-propeptide. About 100 amino acid residues from the N terminus, two residues found in the chick sequence were missing from the human. In the second half of the C-propeptide, there was complete conservation of a 37 amino acid sequence and conservation of 50 out of 51 amino acids in the same region, an observation which suggested that the region serves some special purpose such as directing the association of one pro alpha 2(I) C-propeptide with two pro alpha 1(I) C-propeptides so as to produce the heteropolymeric structure of type I procollagen. In addition, comparison of human and chick DNAs for pro alpha 2(I) revealed three different classes of conservation of nucleotide sequence which have no apparent effect on the structure of the protein: a preference for U on the third base position of codons for glycine, proline, and alanine; a high degree of nucleotide conservation in the 51 amino acid highly conserved region of the C-propeptide; a high degree of nucleotide conservation in the 3'-noncoding region. These three classes of nucleotide conservation may reflect unusual features of collagen genes, such as their high GC content or their highly repetitive coding sequences.     

Biosynthesis of type I procollagen. Characterization of the distribution of chain sizes and extent of hydroxylation of polysome-associated pro-alpha-chains

[3H]Proline-labeled nascent procollagen chains were isolated from chick tendon polysome preparations as peptidyl-tRNA complexes by ion exchange chromatography. Proline hydroxylation of the nascent chains was at least 40% complete, based on radioactive hydroxyproline/proline ratios. These data provide the first direct evidence that hydroxylation of procollagen proline residues does occur on nascent chains. The electrophoretic profiles of [3H]proline-labeled nascent chains and of unlabeled nascent chains visualized by Western blotting with 35S-labeled monoclonal antibodies to the alpha 1(I) N-propeptide or the C-propeptides indicate that there are pauses in the translation of procollagen alpha-chains in the intact cells. Approximately 25% of the radioactivity associated with [3H]proline-labeled polysomes was in fully elongated but underhydroxylated (relative to secreted procollagen) pro-alpha-chains. The association of these completely elongated but only partially modified procollagen chains with the polysome complex may facilitate the carboxyl-terminal interactions which lead to triple helix formation.     

Increased expression of the gene for the pro alpha 1(IV) chain of basement-membrane procollagen in cultured skin fibroblasts from two variants of osteogenesis imperfecta.

Fibroblasts from two lethal variants of osteogenesis imperfecta were shown to synthesize increased amounts of type IV procollagen. Previous studies established that one of these variants had a non-functional allele for the pro alpha 2 chain of type I procollagen, whereas the other pro alpha 2(I) allele contained a mutation leading to synthesis of shortened pro alpha 2(I) chains. In the two variants, the relative level of mRNA for pro alpha 1(IV) was 31 and 42% of the level of mRNA for pro alpha 1(I) chains. A value of less than 2% was found for a third lethal and four non-lethal variants of osteogenesis imperfecta. Immunofluorescent staining of fibroblasts from the two variants synthesizing increased amounts of type IV procollagen indicated that a homogeneous population of cells synthesized both type IV and type I procollagen. The results suggest that mutations in the type I procollagen genes that result in osteogenesis imperfecta can be associated with increased expression of the genes for type IV procollagen.     

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.     

Inhibition of the assembly and secretion of procollagen by incorporation of a threonine analogue, hydroxynorvaline.

Hydroxynorvaline (DL-alpha-amino-beta-hydroxyvaleric acid) was shown to competitively inhibit the activation of threonine and valine when tested with tRNA and synthetases prepared from whole chick embryos. However, the hydroxynorvaline was transferred only to threonyl-tRNA and not valyl-tRNA. The hydroxynorvaline had no effect when tested with other amino acids. The Km for threonine was 25 muM and the Ki for hydroxynorvaline was 181 muM. When fibroblasts from embryonic chick tendons were incubated with [3H]threonine and increasing concentrations of hydroxynorvaline, there was a progressive decrease in the incorporation of [3H]threonine so that 1 mM hydroxynorvaline the incorporation into nondialyzable protein was 26% of the control value. A much smaller decrease in the incorporation of other radioactive amino acids was observed. When the cells were incubated hith [14C]proline and 1 mM hydroxynorvaline, the labeled procollagen containing hydroxynorvaline accumulated intracellularly and very little was secreted. Control experiments demonstrated that free hydroxynorvaline did not inhibit the secretion of unsubstituted procollagen. Although the individual pro alpha chains containing hydroxynorvaline were of normal molecular weight (125,000) and hydroxyproline content, only about 50% of this intracellularly retained procollagen was triple helical within the cell as 37 degrees as measured by sensitivity to pepsin digestion. Also only approximately 50% of the pro alpha chains were disulfide-linked to form triple stranded molecules as compared to greater than 85% linkage in unsubstituted procollagen. We postulate that incorporation of hydroxynorvaline alters the conformation of the propeptide extension sufficiently so that: (a) normal assembly of disulfide-linked, triple helical molecules is reduced and (b) assembled triple helical molecules are not properly recognized by the secretory mechanism.