Pro Alpha Research Articles

Chick pro alpha 2 (I) collagen gene: exon location and coding potential for the prepropeptide.

We report the DNA sequence of a cDNA clone complementary to the 5' end of the chick pro alpha 2(I) mRNA. The sequence enables us to deduce the amino acid sequence of this region, which has been refractory to conventional protein sequencing techniques. Its importance lies in the role of the prepropeptide in secretion, triple helix formation of the mature protein and initiation of fibrillogenesis. We have also located four of the five exons which code for this region on the genome. One exon is only 11bp in size and appears to code exclusively for the signal propeptidase cleavage site. This is an extreme example of an exon defining a functional unit.

Binding of soluble type I collagen to fibroblasts: specificities for native collagen types, triple helical structure, telopeptides, propeptides, and cyanogen bromide-derived peptides.

Unlabeled collagenous proteins were quantified as inhibitors of binding of native, soluble, radioiodinated type I collagen to the fibroblast surface. Collagen types IV, V a minor cartilage isotype (1 alpha 2 alpha 3 alpha), and the collagenlike tail of acetylcholinesterase did not inhibit binding. Collagen types II and III behaved as competitive inhibitors of type I binding. Denaturation of native collagenous molecules exposed cryptic inhibitory determinants in the separated constituent alpha chains. Inhibition of binding by unlabeled type I collagen was not changed by enzymatic removal of the telopeptides. Inhibitory determinants were detected in cyanogen bromide-derived peptides from various regions of helical alpha 1 (I) and alpha 1(III) chains. The aminoterminal propeptide of chick pro alpha 1(I) was inhibitory for binding, whereas the carboxyterminal three-chain propeptide fragment of human type I procollagen was not. The data are discussed in terms of the proposal that binding to surface receptors initiates the assembly of periodic collagen fibrils in vivo.

DNA sequence analysis of a mouse pro alpha 1 (I) procollagen gene: evidence for a mouse B1 element within the gene.

In a 3.8-kilobase mouse DNA sequence encoding amino acid sequences for the pro alpha 1(I) chain of type I procollagen, 14 coding sequences were identified which specify a sequence 95% homologous to amino acid residues 568 to 963 of the bovine alpha 1(I) chain. All of these coding sequences were flanked by appropriate splice junctions following the GT/AG rule. These observations suggest, but do not prove, that this pro alpha 1(I) gene is transcriptionally active. Of the 14 coding sequences, 7 were 54 base pairs in length, whereas the remainder were higher multiples of 54 base pairs. Nonrandom utilization of codons pertained throughout all of the coding sequences showing a preference (56%) for U in the wobble position. Two of the intervening sequences encoded imperfect vestiges of coding sequences which exhibited a codon preference different from that of the pro alpha 1(I) gene proper and were not flanked by splice junctions. One intervening sequence encoded a member of the mouse B1 family of middle repetitive sequences. It was flanked by 8-base-pair direct repeats and had a truncated A-rich region, suggesting that it may be a mobile element. Within this element were sequences which could function as a RNA polymerase III split promoter.

DNA sequence analysis of a mouse pro alpha 1 (I) procollagen gene: evidence for a mouse B1 element within the gene.

In a 3.8-kilobase mouse DNA sequence encoding amino acid sequences for the pro alpha 1(I) chain of type I procollagen, 14 coding sequences were identified which specify a sequence 95% homologous to amino acid residues 568 to 963 of the bovine alpha 1(I) chain. All of these coding sequences were flanked by appropriate splice junctions following the GT/AG rule. These observations suggest, but do not prove, that this pro alpha 1(I) gene is transcriptionally active. Of the 14 coding sequences, 7 were 54 base pairs in length, whereas the remainder were higher multiples of 54 base pairs. Nonrandom utilization of codons pertained throughout all of the coding sequences showing a preference (56%) for U in the wobble position. Two of the intervening sequences encoded imperfect vestiges of coding sequences which exhibited a codon preference different from that of the pro alpha 1(I) gene proper and were not flanked by splice junctions. One intervening sequence encoded a member of the mouse B1 family of middle repetitive sequences. It was flanked by 8-base-pair direct repeats and had a truncated A-rich region, suggesting that it may be a mobile element. Within this element were sequences which could function as a RNA polymerase III split promoter.

Biosynthesis of type V procollagen by A204, a human rhabdomyosarcoma cell line.

The A204 cell line, derived from a human rhabdomyosarcoma, was studied in culture for its capacity to synthesize collagen types and other extracellular matrix proteins. The cells synthesized and secreted into the culture medium collagenous proteins with apparent molecular weights of 220,000 and 150,000. These were identified as the pro alpha 1 and pro alpha 2 chains of type V collagen by immunoprecipitation and by peptide mapping. The pro alpha 1(V) chain was made in excess of a 2:1 ratio for pro alpha 1(V) to pro alpha 2(V), and a fraction of the pro alpha 1(V) chains together with all of the pro alpha 2(V) chains participated in intermolecular disulfide bonding. The chains were extensively glycosylated at hydroxylysyl residues in ascorbate-supplemented cultures. A fraction of the secreted pro alpha 1(V) chains was processed to the p alpha 1(V) form, but further processing in the culture medium was very slow and the type V collagen molecules deposits in the extracellular matrix apparently retained large non-triple helical domains. Since the A204 cell line does not produce other collagen types, it may prove useful in further studies of the biosynthesis of type V procollagen.

Identification of the carboxyl peptides of mouse procollagen IV and its implications for the assembly and structure of basement membrane procollagen.

Clusters of mouse PF-HR9 endoderm cells derived from teratocarcinoma PCC4-F cells were incubated with [3H]proline and [35S]methionine. The synthesis of pro alpha 1 IV and pro alpha 2 IV chains and their association into triple helically folded disulfide-linked molecules were followed. Short incubations and incubations with pactamycin showed that approximately 30,000 molecular weight collagenase-resistant peptides, which are destroyed by pepsin, form the carboxyl end of the pro alpha IV chains. While disulfide links bridge parts of individual peptides, the carboxyl peptides of the three chains of a molecule are not disulfide linked to each other. We propose that these peptides form the knob protrusion seen in electron micrographs of rotary shadowed procollagen IV molecules. The implications of these findings, especially for the relatively slow assembly of procollagen IV, are discussed.

Mouse procollagen IV. Characterization and supramolecular association.

The endodermal cell line PF-HR9, derived from the murine teratocarcinoma cell line PCC4-F, was grown as monolayers and as cell clusters called embryoid bodies. Procollagen IV and laminin were isolated from both kinds of culture media. Antibodies specific to collagen IV and to laminin demonstrated these materials in association with the cells and in the culture media. The procollagen IV consisted of pro alpha 1 IV and pro alpha 2 IV chains and gave a circular dichroic spectrum characteristic for collagen helices, with thermal transitions at 40, 44, and 51 degrees C. The molecules were visualized electron microscopically after rotary shadowing. Laminin showed the characteristic beaded cross-appearance, and procollagen IV was a 434 +/- 12-nm long linear thread containing a 17-nm carboxyl-terminal knob. The 7% of collagen helix with Tm = 51 degrees C corresponds to about a 30-nm length of the molecule and is probably that section of the amino end through which several procollagen IV molecules form a junctional complex. Several noncovalent associations of procollagen IV molecules were demonstrated by velocity sedimentation and electron microscopy of concentrated culture media, specifically associations of two and four procollagen IV molecules through their amino ends and dimers linked at their carboxyl ends. The results show that procollagen IV molecules associate noncovalently into the components which others have isolated from basement membranes and strongly support a network model of these supramolecular assemblies.

Inhibition of protein synthesis in vitro by procollagen-derived fragments is associated with changes in protein phosphorylation.

The NH2-terminal extension fragment (Col 1) of the pro alpha 1(I) procollagen chain selectively inhibits the translation of procollagen mRNA in a reticulocyte lysate system, whereas the reduced and alkylated fragment (AE-Col 1) and its proteolytically derived peptides inhibit the translation of all mRNAs (Hörlein, D., McPherson, J., Goh, S. H., and Bornstein, P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6163-6167). The latter inhibitory function, which occurs at the level of polypeptide chain initiation, has now been shown to be associated with an increase in phosphorylation of an Mr = 94,000 protein. The time span required for observation of changes in phosphorylation and in inhibition of protein synthesis is similar. Since AE-Col 1 can serve as a substrate for casein kinase II, we suggest that phosphorylation of AE-Col 1 and its derivatives may be required for their activity.

Structure and function of basement membrane.

Progress has been made in identifying and characterizing basement membrane macromolecules, including type IV collagen, laminin, a heparan sulfate proteoglycan and bullous pemphigoid antigen. Basement membrane contains a unique collagen, type IV collagen, which is formed of pro alpha 1(IV) (Mr = 185,000) and pro alpha 2(IV) (Mr = 170,000) chains. As opposed to the fibrillar pattern seen with other collagens, the type IV collagen molecules are thought to be arranged in a honey-comb or reticular pattern which provides the major structural element of the basement membrane. Consistent with this model, type IV collagen has been localized to the basement membrane lamina densa, a nonfibrillar structure. Laminin is a large (Mr = 1,000,000) noncollagenous glycoprotein with chains of 200,000 and 400,000 daltons. It has been localized to the basement membrane lamina lucida and functions to bind epithelial cells to the basement membrane. A heparan sulfate proteoglycan has also been identified in the basement membrane. Its biological function may be to restrict the penetration of anionic macromolecules through the basement membrane. In contrast to the above-mentioned components which are found in all tissue basement membranes, bullous pemphigoid antigen is only found in certain basement membranes, mostly those of stratified squamous epithelia. Bullous pemphigoid antigen is a protein, synthesized by keratinocytes in culture, with disulfide-linked chains (Mr = 220,000). By immunoelectron microscopy, it is localized in the lamina lucida of epidermal basement membrane and is closely associated with the basal cell surface. Its biological function is not known, but could involve epidermal basal cell-substrate interactions which occur when basal cells re-epithelialize wounds.

How Does a Skin Fibroblast Make Type I Collagen Fibers?

We now have an impressive amount of information about several steps in the synthesis of type I collagen fibers by skin fibroblasts: (a) the transcription of the genes for pro alpha 1(I) and pro alpha 2(I) polypeptide chains; (b) the processing of the initial RNA transcripts of the genes to mature mRNAs; (c) translation of the mRNAs to prepro alpha 1(I) and prepro alpha 2(I) chains; (d) processing of the prepro alpha 1(I) and prepro alpha 2(I) chains to procollagen; and (e) the further processing of procollagen in collagen fibers. The speed with which we are currently developing this information about collagen and comparable information about other constituents of skin suggests that skin may well be the first human organ which we fully understand.

How does a skin fibroblast make type I collagen fibers?

We now have an impressive amount of information about several steps in the synthesis of type I collagen fibers by skin fibroblasts: (a) the transcription of the genes for pro alpha 1(I) and pro alpha 2(I) polypeptide chains; (b) the processing of the initial RNA transcripts of the genes to mature mRNAs; (c) translation of the mRNAs to prepro alpha 1(I) and prepro alpha 2(I) chains; (d) processing of the prepro alpha 1(I) and prepro alpha 2(I) chains to procollagen; and (e) the further processing of procollagen in collagen fibers. The speed with which we are currently developing this information about collagen and comparable information about other constituents of skin suggests that skin may well be the first human organ which we fully understand.

Molecular defects in the Ehlers-Danlos syndrome.

Several abnormalities in collagen biosynthesis have been described in patients with Ehlers-Danlos syndrome. Examples of collagen structural mutations as well as post-translational enzymatic defects have been detected. Patients with hydroxylysine-deficient collagen disease (Ehlers-Danlos type VI) have diminished lysyl hydroxylase activity. One mutant enzyme has been characterized which is thermally labile and had an altered affinity for ascorbate. Another mutant enzyme had a normal requirement for cofactors but activity was diminished. Type VII Ehlers-Danlos syndrome is associated with altered processing of procollagen to collagen. Most often the disorder is associated with deficient procollagen aminoprotease activity. One patient appears to represent a structural mutation of pro alpha 2 (I) resulting in incomplete cleavage of the amino terminal propeptide. One family with x-linked Ehlers-Danlos syndrome (type V) has been described with altered lysyl oxidase activity. Other patients with this disorder have had normal lysyl oxidase activity. The ecchymotic form of Ehlers-Danlos syndrome (type IV) has defective type III collagen-synthesis. Patients have been described with absent synthesis, diminished synthesis and diminished secretion.

Type I osteogenesis imperfecta: a nonfunctional allele for pro alpha 1 (I) chains of type I procollagen.

Type I osteogenesis imperfecta (OI) is a dominantly inherited disease characterized clinically by bone fractures during childhood, blue sclerae, and frequent hearing loss accompanied by a decreased content of type I collagen in bone and skin. Cultured skin fibroblasts from three individuals affected with the disease produce half-normal levels of type I procollagen, a disulfide-bonded trimer that contains two pro alpha 1(I) chains and one pro alpha 2(I) chain. In normal cells, pro alpha 1(I) and pro alpha 2(I) are synthesized in a 2:1 ratio and only assembled molecules are secreted. In contrast, the OI cells contain equimolar amounts of pro alpha 1(I) and pro alpha 2(I), which suggests that trimer assembly and secretion are limited by the level of pro alpha 1(I) synthesis. The "extra" pro alpha 2(I) in the OI cells is in a nondisulfide-bonded configuration and is not secreted but apparently contributes to an increased level of intracellular degradation. Thus, decreased production of type I procollagen in these patients is the result of decreased synthesis of pro alpha 1(I). These results suggest that the stoichiometry of pro alpha chains in type I procollagen is determined by the conformation of the chains rather than the ratio in which they are synthesized, that molecules containing more than a single pro alpha 2(I) chain are not assembled, and that the production of this heteropolymeric molecule may be effectively regulated by controlling the synthesis of only one of the subunits.

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.

Expression and novel structure of a collagen gene in Drosophila.

We report the structure and developmental expression of collagen gene sequences in Drosophila melanogaster. Collagen-like genomic clones were isolated by screening a Drosophila genomic library with a chicken pro alpha 2(I) cDNA clone as a hybridization probe. A 1.5-kilobase (kb) DNA sequence from a 9.2-kb DNA clone (pDCg1) is presented. Unlike the highly fragmented genes for vertebrate type I collagen, there is no evidence of a 54-base-pair primordial unit within this gene segment. Instead, the fragment is composed of two large coding sequences. Together they specify a sequence of 469 amino acids. This collagen product is composed almost entirely of the Gly-X-Y repeat characteristic of peptides involved in triple helix formation. Within the polypeptide there are four minor discontinuities in the Gly-X-Y pattern. Similar interruptions have been observed in a mouse basement membrane collagen protein sequence. Therefore, the Drosophila collagen gene may encode a nonfibrous collagen such as a basement membrane or cuticle collagen or a novel collagenous protein. By using the DNA segment of known sequence as a hybridization probe, a developmental sequence of polyadenylylated RNA samples was screened for the presence of homologous sequences. A RNA species 6.4 kb in length was detected as a prominent band only in the first- and second-instar larval stages. This pattern of developmental hybridization correlates with the production of the cuticle and basement membranes, and the large size of the RNA is consistent with its identification as a collagen-encoding RNA.

Cloning and characterization of five overlapping cDNAs specific for the human pro alpha 1(I) collagen chain.

We report the cloning of five overlapping cDNAs bearing sequences specific for the human pro alpha 1(I) collagen chain. Poly-A RNA enriched for collagen sequences was purified from normal human fibroblasts and used as template to synthesize double stranded cDNA. The cDNA was inserted into the Eco RI site of pBR 322 by blunt-ending and dG:dC tailing. The clones were screened by colony hybridization using the original RNA population and the resulting five positive clones subjected to restriction endonuclease mapping analysis and DNA sequencing. These overlapping clones cover from residue 247 in the alpha chain to part of the 3' end untranslated region of the pro alpha 1(I) mRNA for a total of 3400 nucleotides.

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.

Structure of the pro alpha 2 (I) collagen gene.

Fifty-four kilobase pairs (kbp) of cloned chicken DNA containing the entire 38-kbp pro alpha 2 (I) collagen gene have been isolated and characterized. DNA sequence analysis of a select 4 kbp of the gene has precisely described 14 exons which comprise one-third of the sequences encoding the triple-helical domain of the collagen protein. These exons range in size from 45 to 108 base pairs (bp), are all multiples of the 9 bp that code for the repeating triplet, Gly-X-Y, and have an average size of 70 bp. About 50 introns interrupt this gene. Nevertheless, introns do not separate the coding sequences for the ends of the central triple-helical structural domain and the ends of the propeptide domains.

Biosynthesis of procollagen [(pro alpha 1 V)2 (pro alpha 2 V)] by chick tendon fibroblasts and procollagen (pro alpha 1 V)3 by hamster lung cell cultures.

Biosynthesis of procollagen [(pro alpha 1 V)2 (pro alpha 2 V)] by chick tendon fibroblasts and procollagen (pro alpha 1 V)3 by hamster lung cell cultures.

Assembly and processing of procollagen V (AB) in chick blood vessels and other tissues.

The biosynthesis and processing of type V procollagens was investigated in chick embryo blood vessels labeled with radioactive amino acids. Monomeric, pepsin-sensitive pro alpha 1 V and pro alpha 2 V chains are slowly assembled into triple helically folded molecules. A small proportion of these procollagen V molecules contain interchain disulfide bridges, and the disulfide-linked heterodimer and heterotrimer (pro alpha 1 V)2pro alpha 2 V were found. A relatively fast conversion of procollagen V to p-collagen V is followed by slow change to collagen V. This time course is similar to the processing of procollagen III and is much slower than the rate of appearance of type I collagen in blood vessels. A combination of sedimentation and electrophoretic analyses was used to measure the relative size of the type V chains and to demonstrate attachment of noncollagenous peptides (Mr = 33,000) to p alpha 2 V by disulfide linkage. Similar quantitative pulse-chase studies were made with calvaria and crop. As the same unusual features of assembly and processing of type V chains were seen in muscle, bone, and blood vessels, we conclude that these are characteristic of type V collagen biosynthesis.