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.     

Complete primary structure of the human alpha 2 type V procollagen COOH-terminal propeptide

Recently we presented the partial covalent structure of a type V collagen chain. Analysis of amino acids 796-1020 in the human alpha 2(V) Gly-X-Y region showed strong conservation of charged positions with the interstitial collagens but also revealed substitutions unique to type V. To gain more information about this procollagen and primarily to resolve the ambiguous nature of the 3' noncollagenous propeptide, we sequenced several cDNA clones coding for amino acids adjacent to the carboxyl end of the alpha chain. Here we report the complete primary structure of the alpha 2(V) COOH-terminal propeptide. In general, the latter sequence (270 residues) bears a greater degree of similarity to those of the interstitial rather than the basement membrane procollagens. Compared to the interstitial procollagens, however, more divergence has occurred in alpha 2(V) surrounding the conserved N-asparaginyl-linked carbohydrate attachment site at residues 171-173, and alpha 2(V) possesses an additional potential glycosylation site (Asn-Lys-Thr) located in a hypervariable region near the NH2 terminus. Although certainly premature to form any rigid hypothesis, a pattern emerges that may be characteristic of alpha 2 versus alpha 1 chains. Both the alpha 2(I) and alpha 2(V) telopeptides are devoid of a lysine, which in alpha 1 chains forms an interchain cross-link with residue 87 of the collagenous region. Also in contrast to the interstitial alpha 1 carboxyl propeptides is the absence in alpha 2(I) and alpha 2(V) of a cysteine that probably participates in an interchain disulfide bond. Therefore, one can speculate that those alpha 2 chains, represented only once in procollagen trimers, may not be under the same selective pressure as alpha 1 chains to maintain certain residues responsible for stabilizing the triple helical molecules.     

Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-alpha 2(I) chain

A patient with Ehlers-Danlos syndrome Type VIIB was found to have an interstitial deletion of 18 amino acids in approximately half of the pro-alpha 2(I) chains of Type I procollagen. Analysis of pepsin-solubilized tissue and fibroblast collagen revealed an abnormal additional chain, alpha 2(I)', which migrated in sodium dodecyl sulfate-5% polyacrylamide gel electrophoresis between the normal alpha 1(I) and alpha 2(I) chains. The apparent ratio of normal alpha 1(I):mutant alpha 2(I)':normal alpha 2(I) was 4:1:1. Procollagen studies and enzyme digestion studies of native mutant collagen suggested defective removal of the amino propeptide. Sieve chromatography of CNBr peptides from purified alpha 2(I)' chains revealed the absence of the normal amino telopeptide fragment CB 1 and the appearance of a larger new peptide of approximately 60 residues (CB X). Compositional and sequencing studies of this peptide identified normal amino propeptide sequences. However, the most carboxyl-terminal tryptic peptide of CB X differed substantially in composition and sequence from the expected and was found to have an interstitial deletion of 18 amino acids corresponding to the N-telopeptide of the pro-alpha 2(I) chain. This deletion removes the normal sites of cleavage of the N-proteinase and also removes a critical cross-linking lysine residue. The 18 amino acids deleted correspond exactly to the residues encoded by exon 6 of the pro-alpha 2(I) collagen gene (COL 1 A2), and, therefore, the protein defect may be due to a genomic deletion, or alternatively, an RNA splicing defect.     

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.     

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.     

Assembly and processing of procollagen type III in chick embryo blood vessels

The processing of [3H]proline-labeled procollagen III in excised chick embryo blood vessels was found to differ significantly from that of procollagen I in the same tissue. While first the amino propeptides and then the carboxyl propeptides were fairly rapidly cleaved from procollagen I, only the carboxyl propeptides were split off procollagen III, leaving pN-collagen III. This intermediate, which is only slowly converted to collagen III by loss of amino propeptides, was characterized by its sedimentation properties, isolation of the amino propeptide, and reaction with purified antibodies that are specific against bovine amino propeptide III. It is interchain disulfide-linked, both through the amino propeptide and the carboxyl ends of the collagen chains. The conversion of procollagen III to pN-collagen III either in blood vessels, or after isolation by a carboxyl procollagen peptidase obtained from chick tendon fibroblast cultures, is inhibited by 50 mM arginine. Underhydroxylated procollagen III was isolated from blood vessels treated with alpha, alpha'-dipyridyl. Its amino propeptides reacted with the above antibodies but were not linked to each other. In contrast, its carboxyl propeptides were interchain disulfide-bridged, supporting previous suggestions that the carboxyl propeptides play a role in the assembly of procollagen trimer.     

The molecular defect in a nonlethal variant of osteogenesis imperfecta. Synthesis of pro-alpha 2(I) chains which are not incorporated into trimers of type I procollagen

Cultured fibroblasts were examined from a patient with a nonlethal form of osteogenesis imperfecta. As reported previously (Nicholls, A. C., Pope, F. M., and Schloon, H. (1979) Lancet 1, 1193), the cells synthesized and secreted a type I procollagen which lacked pro-alpha 2(I) chains and consisted of a trimer of pro-alpha 1(I) chains. No pro-alpha 2(I) chains were recovered from the medium under conditions in which nonhelical pro-alpha 1(I) and pro-alpha 2(I) chains were readily detected in the medium of normal fibroblasts incubated with the hydroxylase inhibitor, alpha, alpha'-dipyridyl. Examination of cellular proteins demonstrated that the fibroblasts synthesized both pro-alpha 1(I) and pro-alpha 2(I) chains. The cellular pro-alpha 2(I) chains did not, however, become disulfide-linked into dimers or trimers of pro-alpha chains. Since the association of pro-alpha chains during the biosynthesis of type I procollagen is directed by the conformation of the COOH-terminal propeptides, the data suggest that the pro-alpha 2(I) chains synthesized by the fibroblasts have a mutated structure in the COOH-terminal propeptides which markedly reduces their affinity for pro-alpha 1(I) chains. A further observation was that the ratio of newly synthesized pro-alpha (I):pro-alpha 2(I) chains in the patient's fibroblasts was 7.18 +/- 0.58 S.E. instead of the value of 2.25 +/- 0.16 S.E. seen in control fibroblasts. One possible explanation for the high ratio is that the proband is homozygous for a mutation altering the structure of the pro-alpha 2(I) chain and that a secondary effect of the structural mutation is a decreased rate of synthesis of pro-alpha 2(I) chains.     

Type I procollagen carboxyl-terminal proteinase from chick embryo tendons. Purification and characterization

Procollagen carboxyl-terminal proteinase, the enzyme which cleaves the carboxyl-terminal propeptides from type I procollagen, was extensively purified in a yield of 25% from pooled culture media of 17-day-old chick embryo tendons using a procedure which involved chromatography on Green A Dye matrix gel, concanavalin A-Sepharose and heparin-Sepharose, and filtration gels of Sephacryl S-300 and S-200. The purified enzyme is a neutral, Ca2+-dependent proteinase which is inhibited by metal chelators, but not by inhibitors for serine and cysteine proteinases. Calcium in a concentration of 5-10 mM is required for optimal activity. The molecular weight of the enzyme was determined to be 97,000-110,000 by gel filtration and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Other properties of the carboxyl-terminal proteinase are: 1) the Km for the type I procollagen is 96 nM at pH 7.5 and 35 degrees C; 2) the activation energy for the reaction with type I procollagen is 21,000 cal mol-1; 3) amino acid sequencing of the released carboxyl-terminal propeptide indicated the enzyme specifically cleaves an -Ala-Asp- bond in both the pro-alpha 1(I) and pro-alpha 2(I) chains; 4) the enzyme specifically cleaves the carboxyl-terminal propeptides of a homotrimer of pro-alpha 1(I) chains and type II and III procollagens, but it does not cleave type IV procollagen. The results suggest that the enzyme is involved in the processing of type I procollagen in vivo.     

Isolation and partial characterization of the amino-terminal propeptide of type II procollagen from chick embryos

The amino-terminal propeptide from type II procollagen was isolated from organ cultures of sternal cartilages from 17-day-old chick embryos. The procedure provided the first isolation of the propeptide in amounts adequate for chemical characterization. The propeptide had an apparent molecular weight of 18000 as estimated by gel electrophoresis in sodium dodecyl sulfate. It contained a collagen-like domain as demonstrated by its amino acid composition, circular dichroism spectrum, and susceptibility to bacterial collagenase. One residue of hydroxylysine was present, the first time this amino acid has been detected in a propeptide. The peptide contained no methionine and only two residues of half-cystine. Antibodies were prepared to the propeptide and were used to establish its identity. The antibodies precipitated type II procollagen but did not precipitate type II procollagen from which the amino and carboxy propeptides were removed with pepsin. Also, they did not precipitate the carboxy propeptide of type II procollagen. The data demonstrated th at the type II amino propeptide was similar to the amino propeptides of type I and type III procollagens in that it contained a collagen-like domain. It differed, however, in that it lacked a globular domain as large as the globular domain of 77-86 residues found at the amino-terminal ends of the pro alpha 1 chains of type I and type III procollagens.     

In vivo and in vitro noncovalent association of excised alpha 1 (I) amino-terminal propeptides with mutant pN alpha 2(I) collagen chains in native mutant collagen in a case of Ehlers-Danlos syndrome, type VII

The cause of the Ehlers-Danlos syndrome Type VII (EDS VII) is considered to be defective removal of the amino-terminal propeptide (N-propeptide) of Type I procollagen due to deficiency of procollagen N-proteinase, the enzyme responsible for the normal proteolytic excision of this precursor-specific domain. Molecules retaining the N-propeptide (pN-collagen molecules) are thought to cause defective fibrillogenesis and cross-linking which eventuate in dramatic joint laxity and joint dislocations, the clinical hallmark of this variety of EDS. Recent studies demonstrate that some EDS VII patients harbor small deletions of either the pro-alpha 1(I) or pro-alpha 2(I) chain of Type I procollagen. We have found an 18-amino acid deletion (due to exon outsplicing) in a mutant pro-alpha 2(I) chain from such a patient. The deleted peptide is the junctional segment (N-telopeptide) linking the alpha 2(I) N-propeptide and major triple helical domains; loss of this short segment results in union of these latter domains and produces a shortened pN alpha 2(I) chain. Directly extracted tissue collagen and pepsin-digested fibroblast collagen contain this mutant pN alpha 2(I) chain and normal alpha 1(I) chains, but not pN alpha 1(I) chains, indicating that the relatively larger alpha 1(I) N-propeptide is excised from the related alpha 1(I) chains. The fate of this alpha 1(I) N-propeptide was unclear and therefore whether or not the intact N-propeptide was, in fact, retained in native mutant collagen was also unclear. In this paper, we describe morphologic, chemical, and immunochemical studies which indicate that the alpha 1(I) N-propeptide is retained in noncovalent association with the mutant pN alpha 2(I) chain in native mutant collagen molecules both in vivo and in vitro. In both instances, the alpha 1(I) N-propeptides are proteolytically cleaved from the related alpha 1(I) chains. These data suggest that retention of a partially cleaved, but essentially intact N-propeptide in mutant collagen may play a role in the pathogenesis of this disease.     

Amino-terminal propeptide of human pro-alpha 2(V) collagen conforms to the structural criteria of a fibrillar procollagen molecule

We have determined the nucleotide sequence of a cDNA clone encoding the amino-terminal portion of human alpha 2(V) procollagen and found that the structure of the 186-residue amino-terminal propeptide closely resembles those of the fibril-forming procollagens. Juxtaposed to a 26-residue leader peptide, pro-alpha 2(V) exhibits a characteristic cysteine-rich globular region followed by 24 Gly-X-Y repeats which are interrupted by two short non-collagenous sequences. Upon closer examination, each of these two sequences was noted to display structural motifs characteristic of either pro-alpha 1(I) and pro-alpha 1(III) collagens or pro-alpha 1(II) collagen, respectively. Finally, within the amino-terminal telopeptide, a putative amino-terminal proteinase cleavage site, Ala-Gln, was identified. This latter finding strongly suggests that the alpha 2(V) amino-terminal propeptide can be potentially processed and thus leaves unresolved the issue pertaining to the nature of the collagenase-resistant sequence that is retained by mature type V collagen molecules.     

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.     

A heterozygous defect for structurally altered pro-alpha 2 chain of type I procollagen in a mild variant of osteogenesis imperfecta. The altered structure decreases the thermal stability of procollagen and makes it resistant to procollagen N-proteinase

Cultured skin fibroblasts from a proband with an autosomal dominant variant of osteogenesis inperfecta were found to synthesize approximately equal amounts of normal pro-alpha 2(I) chains of type I procollagen and pro-alpha 2(I) chains which migrated more rapidly when examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The structural alteration was present in alpha 2(I)-CB4, a cyanogen bromide fragment containing amino acid residues 7-327 of the alpha 2 chain, and it appeared to be a deletion of about 30 amino acids. The pro-alpha 2(I) chains with the apparent deletion associated with normal pro-alpha 1(I) chains synthesized by the same fibroblasts and formed triple-helical type I procollagen. The presence of the altered pro-alpha 2 chains in trimers of procollagen had two consequences in terms of the physical properties of the molecule. One was to decrease the thermal stability of the protein as judged by resistance to proteolysis at 37 degrees C and by the helix to coil transition as assayed by circular dichroism. The second consequence was to make type I procollagen containing the shortened pro-alpha 2(I) chains resistant to digestion by procollagen N-proteinase. The simplest explanation for the data is that the apparent deletion in half the pro-alpha 2(I) chains produced a partial unfolding of the N-terminal region of type I procollagen which prevented processing of the protein by procollagen N-proteinase.     

Synthesis of an altered type III procollagen in a patient with type IV Ehlers-Danlos syndrome. A structural change in the alpha 1(III) chain which makes the protein more susceptible to proteinases

The synthesis of type III procollagen was examined in cultured fibroblasts from ten patients with type IV Ehlers-Danlos syndrome, a heritable disorder of connective tissue. With fibroblasts from nine patients, a decreased amount of labeled type III procollagen was recovered in the medium after the cells were incubated with radioactive amino acids for 24 h. The results were compatible with undefined defects in type III procollagen. The culture medium from one patient contained apparently normal amounts of type III procollagen after a 24-h labeling. However, the pro-alpha 1(III) chains from the medium of the patient's fibroblasts appeared as an abnormally broad band when examined by gel electrophoresis in sodium dodecyl sulfate. Analysis of fragments generated by vertebrate collagenase and cyanogen bromide located a structural defect between amino acid residues 555 and 775 in half of the alpha 1(III) chains. Most of the patient's type III procollagen was susceptible to digestion by pepsin or a mixture of chymotrypsin and trypsin at temperatures at which normal type III procollagen resisted digestion. Cyanogen bromide digestion of samples of the patient's skin revealed that the amount of type III was reduced more than 4-fold. The results support the hypothesis that both normal and structurally altered pro-alpha 1(III) chains are being incorporated into type III procollagen synthesized by the patient's fibroblasts and that type III procollagen molecules containing one, two, or three structurally altered pro-alpha 1(III) chains are rapidly degraded by proteinases in the tissues.     

Pro-alpha 1(XI) collagen. Structure of the amino-terminal propeptide and expression of the gene in tumor cell lines.

We have determined the nucleotide sequence of several overlapping cDNA clones encoding the amino-terminal portion of human alpha 1(XI) procollagen. These experiments have revealed that this domain of the pro-alpha(XI) chain displays structural features common to other fibrillar procollagen molecules, such as a putative amino-terminal proteinase cleavage site and an interrupted collagenous segment. In the latter, structural similarities were noted when alpha 1(XI) was compared with alpha 1(II) and alpha 2(V) procollagens. Overall, however, the amino-terminal region of pro-alpha 1(XI) differs greatly in composition and size from that of other fibrillar chains. Nearly three-fourths of this domain is in fact composed of a 383-amino acid globular region in which a 3-cysteine cluster signals the transition to a long and highly acidic carboxyl-terminal segment. Finally, the unrestricted expression of this cartilage-specific collagen gene has been confirmed by the finding of high levels of pro-alpha 1(XI) mRNA in two human rhabdomyosarcoma cell lines.     

Procollagen intermediates during tendon fibrillogenesis

The purpose of this study was to correlate ultrastructural features of tendon collagen fibrils at various stages of development with the presence of procollagen, pN-collagen, pC-collagen, and the free amino propeptides and carboxyl propeptide of type I procollagen. Tendons from 10-, 14-, and 18-day chicken embryos reveal small, well-defined intercellular compartments containing collagen fibrils with diameters showing a unimodal distribution. At 21 days (hatching) and 9 days (post hatching) and at 5 weeks (post hatching), the compartments are larger, less well-defined, and there is multimodal distribution of tendon fibril diameters. Procollagen and the intermediates pN-collagen and pC-collagen are present in tendons up to 18 days. Thereafter there is a marked reduction in procollagen, whereas the intermediates persist throughout all stages of development. Similarly, free amino propeptides and carboxyl propeptides of type I procollagen were found at all stages. The amino propeptide of type III procollagen was restricted to the peritendineum until 7 weeks post hatching. At that time, a network of fibrils containing the amino propeptide of type III procollagen was seen delineating well-circumscribed compartments of collagen fibrils throughout the entire tendon. This study supports the notion that pN- and pC-collagen have an extracellular role and participate in collagen fibrillogenesis.     

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.     

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'.     

Suppression of synthesis of pro-alpha 1(I) and production of altered pro-alpha 2(I) procollagen subunits in 4-nitroquinoline-1-oxide-transformed fibroblasts

The collagen phenotype of a 4-nitroquinoline-1-oxide-transformed line of Syrian hamster embryo fibroblasts, NQT-SHE, was markedly altered from that of normal Syrian hamster embryo cells, which synthesized mainly type I procollagen [pro-alpha 1(I)]2 pro-alpha 2(I). Total collagen synthesis in the transformant was reduced to about 30% of the control level primarily because synthesis of the pro-alpha 1(I) subunit was completely suppressed. The major collagenous products synthesized consisted of two polypeptides, designated as N-33 and N-50, which could be completely separated by precipitation with ammonium sulfate at 33 and 50% saturation, respectively. N-33 migrated similarly to pro-alpha 2(I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and N-50 migrated slightly more slowly. The collagenous regions of these chains were more sensitive to protease than the analogous region of procollagen I, but alpha-chains could be obtained by digestion for 2 h at 4 degrees C with high ratios of protein:pepsin. Staphylococcus V8 protease and cyanogen bromide peptide maps of N-33 alpha and N-50 alpha chains indicated that the chains were homologous with, but different than, alpha 2(I) chains and that they differed from each other. Considering their similarity to pro-alpha 2(I), it was surprising to find that the N-collagens were secreted to the same extent as was type I procollagen from Syrian hamster embryo cells and that there were no disulfide bonds between N-collagen chains. Intrachain disulfides were present. One possible explanation for the unusual collagen phenotype of NQT-SHE cells is that transformation induced one or more mutations in the pro-alpha 2(I) structural gene while suppression of synthesis of the pro-alpha 1(I) subunit may be due to a mutation in the regulatory region of its gene or in a general regulatory gene.