Substitution of serine for alpha 1(I)-glycine 844 in a severe variant of osteogenesis imperfecta minimally destabilizes the triple helix of type I procollagen. The effects of glycine substitutions on thermal stability are either position of amino acid specific

Recent reports have demonstrated that a series of probands with severe osteogenesis imperfecta had single base mutations in one of the two structural genes for type I procollagen that substituted amino acids with bulkier side chains for glycine residues and decreased the melting temperature of the triple helix. Here we demonstrate that the type I procollagen synthesized by cultured fibroblasts from a proband with a severe form of osteogenesis imperfecta consisted of normal molecules and molecules over-modified by post-translational reactions. The thermal stability of the intact type I collagen was normal as assayed by protease digestion under conditions in which a decrease in thermal stability was previously observed with eight other substitutions for glycine in the alpha 1(I) chain. In contrast, the thermal stability of the one-quarter length B fragment generated by digestion with vertebrate collagenase was decreased by 2-3 degrees C under the same conditions. Nucleotide sequencing of cDNAs and genomic DNA established that the proband had a substitution of A for G in one allele of the pro alpha 1(I) gene that converted the codon for alpha 1-glycine 844 to a codon for serine. The results also established that the alpha 1-serine 844 was the only mutation that could account for the decrease in thermal stability of the collagenase B fragment. There are at least two possible explanations for the failure of the alpha 1-serine 844 substitution to decrease the thermal stability of the collagen molecule whereas eight similar mutations decreased the melting temperature. One possibility is that the effects of glycine substitutions are position specific because not all glycine residues make equivalent contributions to cooperative blocks of the triple helix that unfold in the predenaturation range of temperatures. A second possible explanation is that substitutions of glycine by serine have much less effect on the stability of protein than the substitutions by arginine, cysteine, and aspartate previously studied.     

A point mutation in a type I procollagen gene converts glycine 748 of the alpha 1 chain to cysteine and destabilizes the triple helix in a lethal variant of osteogenesis imperfecta

Synthesis of type I procollagen was examined in fibroblasts from a proband with a lethal perinatal variant of osteogenesis imperfecta. After trypsin digestion of the type I procollagen, a portion of the alpha 1 (I) chains was recovered as disulfide-linked dimers. Digestion of the protein with vertebrate collagenase and mapping of cyanogen bromide peptides suggested that a new cysteine residue was present between residues 551 and 775 of the alpha 1 (I) chain. Sequencing of cloned cDNAs prepared using mRNA from the proband's fibroblasts demonstrated that some of the clones contained a single base mutation that converted the glycine codon in amino acid position 748 of the alpha 1 (I) chain to a cysteine codon. About 80% of the type I procollagen synthesized by the proband's fibroblasts had a decreased thermal stability. The results, therefore, were consistent with the conclusion that normal pro-alpha 1 (I) chains and pro-alpha 1 (I) chains containing a cysteine residue in the alpha chain domain were synthesized in about equal amounts and incorporated randomly into type I procollagen. However, only about 10% of the alpha 1 (I) chains generated by trypsin digestion were disulfide-linked. Further studies demonstrated a decreased rate of secretion of type I procollagen containing the new cysteine residue and decreased processing of the protein by procollagen N-proteinase in cultures of postconfluent fibroblasts. Both parents were phenotypically normal and their fibroblasts synthesized only normal type I procollagen. Therefore, the mutation in the proband was a sporadic one and is very likely to have caused the connective tissue fragility that produced the lethal phenotype.     

A single base mutation in type I procollagen (COL1A1) that converts glycine alpha 1-541 to aspartate in a lethal variant of osteogenesis imperfecta: detection of the mutation with a carbodiimide reaction of DNA heteroduplexes and direct sequencing of products of the PCR.

Skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta synthesized both apparently normal type I procollagen and a type I procollagen that had slow electrophoretic mobility because of posttranslational overmodifications. The thermal unfolding of the collagen molecules as assayed by protease digestion was about 2 degrees C lower than normal. It is surprising, however, that collagenase A and B fragments showed an essentially normal melting profile. Assay of cDNA heteroduplexes with a new technique involving carbodiimide modification indicated a mutation at about the codon for amino acid 550 of the alpha 1(I) chain. Subsequent amplification of the cDNA by the PCR and nucleotide sequencing revealed a single-base mutation that substituted an aspartate codon for glycine at position alpha 1-541 in the COL1A1 gene. The results here confirm previous indications that the effects of glycine substitutions in type I procollagen are highly position specific. They also demonstrate that a recently described technique for detecting single-base differences by carbodiimide modification of DNA heteroduplexes can be effectively employed to locate mutations in large genes.     

Structure of cDNA clones coding for the entire prepro alpha 1 (III) chain of human type III procollagen. Differences in protein structure from type I procollagen and conservation of codon preferences.

Two overlapping cDNA clones that cover the complete length of the mRNA for human type III procollagen were characterized. The data provided about 2500 base pairs of sequence not previously defined for human type III procollagen. Two tripeptide sequences of -Gly-Xaa-Yaa- were identified that were not detected previously by amino acid sequencing of human type III collagen. The two additional tripeptide units, together with three previously detected, establish that the alpha 1 (III) chain is 15 amino acids longer than either the alpha 1 (I) or alpha 2 (I) chains of type I collagen. The additional tripeptide units made hydropathy plots of the N-terminal and C-terminal regions of type III collagen distinctly different from those of type I collagen. The data also demonstrated that human type III procollagen has the same third base preference in codons for glycine, proline and alanine that was previously found with human and chick type I procollagen. In addition, comparison of two cDNA clones from the same individual revealed a variation in structure in that the codon for amino acid 880 of the alpha 1 (III) chain was -CTT- for leucine in one clone and -TTT- for phenylalanine in the other.     

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.     

Single base mutation in the type III procollagen gene that converts the codon for glycine 883 to aspartate in a mild variant of Ehlers-Danlos syndrome IV

Experiments were carried out to test the hypothesis that a 19-year-old proband with a mild variant of Ehlers-Danlos syndrome type IV had a mutation in the gene for type III procollagen. cDNA and genomic DNA were analyzed by using the polymerase chain reaction and cloning of the products into M13 filamentous phage. A mutation was found that converted the codon for glycine 883 of the triple-helical domain in one allele for type III procollagen to a codon for aspartate. The polymerase chain reaction introduced a few artifactual single base substitutions. Also, it was difficult to distinguish copies from the two alleles in many of the M13 clones. Therefore, several different strategies and analyses of about 50,000 nucleotide sequences in a series of clones were used to demonstrate that the mutation in the codon for glycine 883 was the only mutation in coding sequences for the triple-helical domain of type III procollagen that could have contributed to the phenotype. The same mutation in the codon for glycine 883 in one allele for type III procollagen was found in the proband's 52-year-old father who also had a mild variant of Ehlers-Danlos syndrome type IV. The type III procollagen synthesized by the proband's fibroblasts was analyzed by polyacrylamide gel electrophoresis. Less type III procollagen was secreted by the proband's fibroblasts than by control fibroblasts. Also, the thermal stability of the type III procollagen synthesized by the proband's fibroblasts was lower than the thermal stability of normal type III procollagen as assayed by brief protease digestion. The results, therefore, demonstrated that the single base mutation that converted the codon of glycine 883 to a codon for aspartate destabilized the entire triple helix of type III procollagen and probably accounted for the mild phenotype of Ehlers-Danlos syndrome type IV seen in the proband and her father.     

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.     

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.     

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.     

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.     

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 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.     

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.     

Type I procollagens containing substitutions of aspartate, arginine, and cysteine for glycine in the pro alpha 1 (I) chain are cleaved slowly by N-proteinase, but only the cysteine substitution introduces a kink in the molecule.

Type I procollagen was purified from the medium of dermal fibroblasts cultured from four individuals with osteogenesis imperfecta (OI) type II who had mutations in the COL1A1 gene of type I procollagen. The procollagens were mixtures of normal molecules and molecules that contained substitutions of aspartate for glycine 97, arginine for glycine 550, cysteine for glycine 718, and aspartate for glycine 883 in one or both of the alpha 1 (I) chains of the molecule. The procollagens were cleaved more slowly than control type I procollagen by procollagen N-proteinase. Double-reciprocal plots of initial relative velocities and initial substrate concentrations indicated that the OI procollagens were all cleaved slowly by N-proteinase because of decreased Vmax, rather than increased Km. This suggested that slow cleavage of the OI procollagens by N-proteinase was the result of slow conversion of the N-proteinase-procollagen complex. Further experiments showed that the vertebrate collagenase A fragment of the aspartate for glycine alpha 1(I) 883 OI procollagen that contained the N-proteinase cleavage site but not the site of the substitution was also cleaved more slowly by N-proteinase than the normal vertebrate collagenase A fragments in the samples. These data show, for the first time, that an altered triple-helical structure is propagated from the site of a substitution of a bulky residue for glycine to the amino-terminal end of the procollagen molecule and disrupts the conformation of the N-proteinase cleavage site. Rotary shadowing electron microscopy of molecules in the preparation of cysteine for glycine alpha 1(I)-718 showed the presence of a kink in approximately 5% of a population of molecules in which 60% were abnormal and 20% contained a disulfide bond. In contrast, procollagens containing aspartate and arginine for glycine were indistinguishable by rotary shadowing electron microscopy from those in control samples. The results here confirm previous suggestions that substitution of cysteine for glycine in the alpha 1(I) chain of type I collagen can introduce a kink near the site of the substitution. However, the presence of a kink is not a prerequisite for delayed cleavage of abnormal procollagens by N-proteinase.     

A substitution of cysteine for glycine 748 of the alpha 1 chain produces a kink at this site in the procollagen I molecule and an altered N-proteinase cleavage site over 225 nm away

In previous work (Vogel, B. E., Minor, R. R., Freund, M., and Prockop, D. J. (1987) J. Biol. Chem. 262, 14737-14744), we identified a single-base mutation that converted the glycine at position 748 of the alpha 1 chain of type I procollagen to a cysteine in a proband with a lethal variant of osteogenesis imperfecta. In addition to posttranslational overmodification, the abnormal molecules displayed decreased thermal stability and a decreased rate of secretion. An unexplained finding was that procollagen was poorly processed to pCcollagen in postconfluent cultures of skin fibroblasts. Here, we show that the procollagen synthesized by the proband's cells is resistant to cleavage by procollagen N-proteinase, a conformation-sensitive enzyme. Since the only detectable defect in the molecule was the cysteine for glycine substitution, we assembled several space-filling models to try to explain how the structure of the N-proteinase cleavage site can be affected by an amino acid substitution over 700 amino acid residues or 225 nm away. The models incorporated a phase shift of a tripeptide unit in one or both of the alpha 1 chains. The most satisfactory models produced a flexible kink of 30 degrees or 60 degrees at the site of the cysteine substitution. Therefore, we examined the procollagen by electron microscopy. About 25% of the molecules had a kink not seen in control samples, and the kink was at the site of the cysteine substitution.     

Substitution of arginine for glycine at position 847 in the triple-helical domain of the alpha 1 (I) chain of type I collagen produces lethal osteogenesis imperfecta. Molecules that contain one or two abnormal chains differ in stability and secretion

Dermal fibroblasts from a fetus with perinatal lethal OI synthesized normal and abnormal type I procollagen molecules. The abnormal molecules contained one or two pro alpha 1 (I) chains in which glycine at position 847 in the triple helical region was substituted by arginine as the result of a de novo G-to-A transition in the first base of the glycine codon. The substitution resulted in increased posttranslational modification amino-terminal of the mutation site of all chains in molecules that contained one or more abnormal chains. Secretion of the overmodified molecules was impaired, and intracellular retention of molecules which contained two abnormal chains was greater than that of molecules which contained one abnormal chain. The thermal stability of molecules that contained two abnormal chains was markedly lower than that of molecules containing one abnormal chain. After cleavage of molecules with vertebrate collagenase, the thermal stability of the overmodified A fragments was greater than that of the normal molecules. Our findings indicate that the cell distinguishes three classes of molecules and suggest that these molecules differ depending on the number of abnormal chains in the trimer.     

The substitution of arginine for glycine 85 of the alpha 1(I) procollagen chain results in mild osteogenesis imperfecta. The mutation provides direct evidence for three discrete domains of cooperative melting of intact type I collagen.

We report a case of mild osteogenesis imperfecta in a 56-year-old male undergoing aortic valve replacement surgery. The primary defect in this patient was the substitution of arginine for glycine 85 in one of the two chains of alpha 1(I) procollagen. The thermal stability of the type I collagen synthesized by the patient's cultured skin fibroblasts was examined by enzymatic digestion. Digestion of the mutant type I collagen with trypsin and chymotrypsin at increasing temperatures sequentially generated three discrete collagenous fragments, approximately 90, 170, and 230 amino acids shorter than normal type I collagen. This incremental thermal denaturation is indicative of cooperative melting blocks within the type I collagen. This is the first demonstration of such cooperative blocks of melting in intact, essentially normal post-translationally modified type I collagen. This direct evidence for cooperative melting domains of uncut type I collagen suggests that discrete blocks of amino acids function as core sites stabilizing the collagen helix. The location of mutations of the alpha chains of type I collagen relative to these discrete blocks of amino acids may influence the severity of the disease phenotype.     

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.     

Lethal perinatal osteogenesis imperfecta due to the substitution of arginine for glycine at residue 391 of the alpha 1(I) chain of type I collagen

A baby with the lethal perinatal form of osteogenesis imperfecta was shown to have a structural defect in the alpha 1(I) chain of type I procollagen. Normal and mutant alpha 1(I) CB8 cyanogen bromide peptides, from the helical part of the alpha 1(I) chains, were purified from bone. Amino acid sequencing of tryptic peptides derived from the mutant alpha 1(I) CB8 peptide showed that the glycine residue at position 391 of the alpha 1(I) chain had been replaced by an arginine residue. This substitution accounted for the more basic charged form of this peptide that was observed on two-dimensional electrophoresis of the collagen peptides obtained from the tissues. The substitution was associated with increased enzymatic hydroxylation of lysine residues in the alpha 1(I) CB8 and the adjoining CB3 peptides but not in the carboxyl-terminal CB6 and CB7 peptides. This finding suggested that the sequence abnormality had interfered with the propagation of the triple helix across the mutant region. The abnormal collagen was not incorporated into the more insoluble fraction of bone collagen. The baby appeared to be heterozygous for the sequence abnormality and as the parents did not show any evidence of the defect it is likely that the baby had a new mutation of one allele of the pro-alpha 1(I) gene. The amino acid substitution could result from a single nucleotide mutation in the codon GGC (glycine) to produce the codon CGC (arginine).     

A de novo G to T transversion in a pro-alpha 1 (I) collagen gene for a moderate case of osteogenesis imperfecta. Substitution of cysteine for glycine 178 in the triple helical domain

Cultured fibroblasts from a patient affected with a moderate form of osteogenesis imperfecta were defective for the synthesis of type I collagen molecules; about half of the alpha 1(I) chains contained a cysteine residue in the triple helical domain and a disulfide link formed when two mutant alpha 1(I) chains were incorporated into a type I collagen heterotrimer. The proband's parents were clinically and biochemically normal. The cysteine was localized within peptide alpha 1(I)CB8 between residues 170 and 200 of the triple helical domain using a chemical procedure with 2-nitro-5-thiocyanobenzoic acid (Tenni, R., Rossi, A., Valli, M., Mottes, M., Pignatti, P. F., and Cetta, G. (1990) Matrix 10, 20-26). Type I procollagen heterotrimers containing either one or two mutant chains showed (i) a slight abnormality in secretion from cells; (ii) a low degree of post-translational overmodifications; (iii) the same, but lower than normal, thermal stability. Total RNA was isolated from the proband's dermal fibroblast cultures, and cDNAs for pro-alpha 1(I) were prepared d using total RNA. A portion of cDNA, coding for the region encompassing residues 119-193 of alpha 1(I) triple helical domain, was amplified by polymerase chain reaction. A single base pair mismatch was identified by chemical cleavage of DNA.DNA heteroduplexes, indicating a possible substitution of a guanine in the triplet coding for glycine 178 or 181. The same unique mismatch was detected by chemical cleavage in about one-half of the molecules in heteroduplexes formed between patient's pro-alpha 1(I) mRNAs and a normal cDNA probe. The amplified products were cloned and sequenced, confirming the heterozygous nature of the patient and demonstrating the presence and the location of a missense mutation; a single T for G substitution was found in the first base of the triplet coding for residue 178 of alpha 1(I) triple helical domain, leading to a cysteine for glycine substitution. Allele-specific oligonucleotide hybridization to amplified DNA confirmed a de novo point mutation in the proband's genome. The findings in this patient are in accord with the phenotypic gradient model, which correlates the localization of the structural defect with the clinical outcome of osteogenesis imperfecta. The mutant protein has some properties that differ from the caused by the cysteine for glycine 175 substitution, suggesting a direct influence of the neighboring amino acids on the effects of the mutation.