Structure of a full-length cDNA clone for the prepro alpha 1(I) chain of human type I procollagen.

A full-length cDNA clone for the human prepro alpha 1(I) chain of type I procollagen was characterized. Nucleotide sequencing of the first 1500 nucleotide residues of the 5'-end of the cDNA clone provided 729 nucleotide residues and the codons for 243 amino acid residues not previously defined from any species. The data made it possible, for the first time, to compare completely codon usage for the human alpha 1(I) and alpha 2(I) chains.     

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 a full-length cDNA clone for the prepro alpha 2(I) chain of human type I procollagen. Comparison with the chicken gene confirms unusual patterns of gene conservation.

A cDNA clone from a human placental library was found to consist of an essentially full-length cDNA of 4.6 kb for the prepro alpha 2(I) chain of type I procollagen. Nucleotide sequencing of the 5'-end of the cDNA provided a sequence of 1617 nucleotide residues and codons for 539 amino acid residues not previously defined. Comparison of the complete structure of the prepro alpha 2(I) cDNA with previously reported sequences for the chicken pro alpha 2(I) gene indicated that 83% of 1366 total amino acid residues were conserved. In the alpha-chain domain 84% of 1014 amino acid residues were conserved. Also, there was conservation of the previously noted preference for U and C in the third position of codons for glycine, proline and alanine. One major difference between the human and the chicken prepro alpha 2(I) chain was that the human chain contained 21 fewer proline residues, an observation that probably explains why the triple helix of human type I procollagen unfolds at temperatures that are 1-2 degrees C lower. In parallel experiments, sequencing of intron-exon boundaries for nine exons of genomic subclones confirmed and extended previous observations that the pro alpha 2(I) gene, like other genes from fibrillar collagens, has an unusual 54-base pattern of exon sizes that is highly conserved through evolution.     

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.     

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)     

Analysis of cDNA and genomic clones coding for the pro alpha 1 chain of calf type II collagen.

A bovine cDNA library constructed from fetal cartilage RNA was screened with a pro alpha 1(II) collagen specific chicken cDNA. A recombinant clone (Bc 7), with an insert of 1 kb, was identified and shown to contain sequences exhibiting 85% homology with the chicken pro alpha 1(II) collagen C-propeptide. Interspecies comparison strongly suggested that one potential glycosylation site present in the avian C-propeptide is not utilized, since this site is absent in the bovine chain. In addition, two overlapping genomic clones (Pal 3 and Pal 4) were isolated and partially characterized. These clones span 23 kb of DNA and contain approximately 17 kb of the pro alpha 1(II) calf gene. Sequencing of exon 1 has determined the length of the 3' untranslated region and the exact location of the polyadenylation attachment site.     

cDNA cloning and characterization of Type I procollagen alpha1 chain in the skate Raja kenojei

A full-length cDNA of the Type I procollagen alpha1 [pro-alpha1(I)] chain (4388 bp), coding for 1463 amino acid residues in the total length, was determined by RACE PCR using a cDNA library constructed from 4-week embryo of the skate Raja kenojei. The helical region of the skate pro-alpha1(I) chain consisted of 1014 amino acid residues - the same as other fibrillar collagen alpha chains from higher vertebrates. Comparison on denaturation temperatures of Type I collagens from the skate, rainbow trout (Oncorhynchus mykiss) and rat (Rattus norvegicus) revealed that the number of Gly-Pro-Pro and Gly-Gly in the alpha1(I) chains could be directly related to the thermal stability of the helix. The expression property of the skate pro-alpha1(I) chain mRNA and phylogenetic analysis with other vertebrate pro-alpha1(I) chains suggested that skate pro-alpha1(I) chain could be a precursor form of the skate Type I collagen alpha1 chain. The present study is the first evidence for the primary structure of full-length pro-alpha1(I) chain in an elasmobranch.     

Procollagen alpha2 mRNA is significantly different from procollagen alpha1(I) mRNA in size or secondary structure.

The fractionation of fetal calf tendon messenger RNA in 85 percent formamide sucrose gradients shows a separation of the mRNAs coding for pro α1(I) and pro α2 chains of type I collagen. This difference in sedimentation in denaturing gradients suggests that pro α2 mRNA is approxmately 1000 bases shorter than pro α1(I) mRNA. However, such a size difference is significantly greater than would be predicted from consideration of the size of the polypeptide chains coded for by these mRNAs, and thus, residual secondary structure in the mRNAs may contribute to these apparent size differences.     

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.     

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.     

cDNA clones completing the nucleotide and derived amino acid sequence of the alpha 1 chain of basement membrane (type IV) collagen from mouse

Six cDNA clones add 3549 nucleotides to the DNA sequence of the alpha 1 chain of basement membrane (type IV) collagen. Thus the complete nucleotide and derived amino acid sequence of the alpha 1 type IV collagen with 5007 nucleotides coding for 1669 amino acids with a calculated Mr of 160,827 is known. The six cDNA clones cover the putative N-terminal signal peptide, the 7 S region and two thirds of the helical region extending into the previously published murine nucleotide sequence [(1986) Gene 43, 301]. The protein sequence for 289 amino acids of the helical region adjacent to the 7 S region has not previously been published for any species.     

Complete primary structure of human collagen alpha 1 (V) chain

Several cDNA clones, encoding prepropeptide of human collagen alpha 1(V) chain, have been isolated. The prepropeptide (1838 amino acids length) of the alpha 1(V) chain was composed of a putative signal peptide, a large NH2-terminal noncollagenous region, a main collagenous region, and a COOH-terminal noncollagenous region. The signal peptide contained many leucine residues. The NH2-terminal noncollagenous region was much larger than those of the other collagens and had a region homologous to the COOH-terminal domain of laminin A chain, but it did not contain a cysteine-rich region that was maintained in the region of the other collagens. This region also contained probable tyrosine sulfation sites, and short collagenous sequences that were interrupted by three noncollagenous segments. The main collagenous region of the alpha 1(V) chain consisted of 338 repeats of Gly-X-Y-triplet. This region had a high degree (82%) of homology with the amino acids of the collagen alpha 1(XI) chain. The COOH-terminal noncollagenous region resembled that of the alpha 1(XI) chain, too, and 8 residues of cysteine that were important for the formation of the triple helix structure of collagens were observed. These results suggest that the alpha 1(V) chain belongs to the fibrillar collagen relative to the alpha 1(XI) chain, but codon usage of the alpha 1(V) cDNA was clearly different from those of the other fibrillar collagens including the alpha 1(XI), while it was similar to type IV collagen. This result supposes a different evolution of the alpha 1(V) gene from those of the other fibrillar collagens.     

Construction of a cDNA clone corresponding to mouse alpha 1(IV) procollagen

A new procedure for the synthesis of double stranded cDNA, based upon release of mRNA by "in vitro" translation, was used to clone type IV collagen. Collagen synthesizing polysomes selectively isolated from a mouse parietal yolk sac carcinoma (PYS-2) were used for translation in an heterologous cell-free system. Translation products were collagenase-sensitive and displayed an electrophoretic mobility correspondent to type IV collagen. Translation released mRNA was employed to construct a 100 base pairs long cDNA clone which hybridized to a 7,800 nucleotides long mRNA. Peptides synthesized by "in vitro" translation of hybrid selected mRNA displayed an electrophoretic mobility compatible with that of alpha 1 (IV) collagen, were sensitive to collagenase and were immunoprecipitated by anti-type IV collagen antibody.     

The covalent structure of calf skin type III collagen. I. The amino acid sequence of the amino terminal region of the alpha 1(III) chain (position 1--222).

The amino terminal 227 amino acid residues of the alpha 1(III) chain contain four CNBr peptides: alpha 1(III)CB3A (79 residues), CB3B, CB3C (6 residues each), CB7 (37 residues) and CB6 (99 residues). Fragmentation of the CNBr peptides was carried out using trypsin, chymotrypsin and the protease from Staphylococcus aureus V8. The fragments obtained were isolated by a combination of molecular sieve and ion exchange chromatography. The sequence analysis was performed according to the automated Edman degradation procedure.