A tripeptide deletion in the triple-helical domain of the pro alpha 1(I) chain of type I procollagen in a patient with lethal osteogenesis imperfecta does not alter cleavage of the molecule by N-proteinase.

Dermal fibroblasts from a fetus with perinatal lethal osteogenesis imperfecta synthesized normal and abnormal type I procollagen molecules. The abnormal molecules contained one or two pro alpha 1(I) chains in which glycine, alanine, and hydroxyproline at positions 874, 875, and 876 in the triple-helical region were deleted as the result of a 9-base pair genomic deletion. Molecules that contained abnormal chains were overmodified from the site of the deletion toward the amino-terminal region of the molecule. Secretion of the overmodified molecules was impaired. The thermal stability of molecules containing abnormal chains was lower than that of normally modified molecules. After cleavage of molecules with vertebrate collagenase, the temperature of thermal denaturation of the overmodified A fragments was greater than that of the fragments from the normal molecules. The rates of cleavage of the normal and the abnormal molecules by N-proteinase were indistinguishable. Our findings suggest that the tripeptide deletion introduces a shift in the phase of the chains in the triple helix. This structural change is propagated from the site of the deletion toward the amino terminus of the molecule, but the subsequent alteration in the structure of the N-proteinase cleavage site is not sufficient to cause a decrease in the rate of cleavage by the enzyme.     

A single base mutation that converts glycine 907 of the alpha 2(I) chain of type I procollagen to aspartate in a lethal variant of osteogenesis imperfecta. The single amino acid substitution near the carboxyl terminus destabilizes the whole triple helix

Type I procollagen was examined in cultured skin fibroblasts from a patient with a lethal variant of osteogenesis imperfecta. About half of the pro-alpha chains were post-translationally overmodified and had a decreased thermal stability. The vertebrate collagenase A fragment had a normal thermal stability, but the B fragment had a decreased thermal stability. Therefore, there was a change in primary structure in amino acids 776-1014 of either the alpha 1(I) or alpha 2(I) chain. Three of five cDNA clones for the alpha 2(I) chain contained a single-base substitution of an A for a G that converted the codon for glycine at amino acid position 907 to aspartate. Complete nucleotide sequencing of bases coding for amino acids 776 to 1014 of the alpha 2(I) chain was carried out in one cDNA clone that contained the mutation in the glycine codon and in one that did not. Also, nucleotide sequencing was performed of bases coding for amino acids 776-1014 of the alpha 1(I) chain in seven independent cDNA clones. No other mutations were found. Therefore, the single base substitution that converts glycine 907 in the alpha 2(I) chain to aspartate is solely responsible for the decreased thermal stability of the type I procollagen synthesized by the proband's fibroblasts. Also, glycine 907 of the alpha 2(I) chain is an important component of a cooperative block that determines the melting temperature of the whole molecule.     

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 novel Gly to Arg substitution at position 388 of the alpha1 chain of type I collagen in lethal form of osteogenesis imperfecta

Cultured skin fibroblasts from a proband with a lethal form of osteogenesis imperfecta produce two forms of type I collagen chains, with normal and delayed electrophoretic migration; collagen of the proband's mother was normal. Peptide mapping experiments localized the structural defect in the proband to alpha1(I) CB8 peptide in which residues 123 to 402 are spaned. Direct sequencing of amplified cDNA covering this region revealed a G to A single base change in one allele of the alpha1(I) chain, that converted glycine 388 to arginine. Restriction enzyme digestion of the RT-PCR product was consistent with a heterozygous COL1A1 mutation. The novel mutation conforms to the linear gradient of clinical severity for the alpha1(I) chain and results in reduced thermal stability by 3 degrees C and intracellular retention of abnormal molecules.     

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 novel mutation causes a perinatal lethal form of osteogenesis imperfecta. An insertion in one alpha 1(I) collagen allele (COL1A1)

We have identified an infant with the perinatal lethal form of osteogenesis imperfecta (type II) whose cells synthesize in equal amounts two different pro alpha 1(I) chains of type I procollagen: one chain is normal in length, the other contains an insertion of approximately 50-70 amino acid residues within the triple helical domain defined by amino acids 123-220. The structure of the insertion is consistent with duplication of an approximately 600-base pair segment in one allele of the alpha 1(I) gene (COL1A1). These cells synthesize normal type I procollagen molecules as well as molecules that contain one or two mutant chains. Unlike type I procollagen molecules synthesized by cells from most other infants with osteogenesis imperfecta type II which contain increased lysyl hydroxylation and hydroxylysyl glycosylation along the triple helical domain, the abnormal molecules synthesized by these cells are not overmodified. The lethal effect of this mutation may result from secretion of about one-quarter the normal amount of normal type I procollagen and secretion of a large amount of a molecule which has a lowered melting temperature, is extended asymmetrically, and which has altered structure in domains important for cross-link formation and bone mineralization.     

Type I procollagen in the severe non-lethal form of osteogenesis imperfecta

Summary We have screened type I procollagen synthesized in vitro by skin fibroblasts from several patients with the severe non-lethal form of osteogenesis imperfecta. Cells from one patient synthesized and secreted both normal and a larger amount of abnormal type I procollagen. The abnormal alpha chains are larger in size due to post-translational overmodifications involving the whole triple helical domain. Abnormal collagen heterotrimers had a melting temperature 2.5°–3°C lower than normal ones or from controls. Chemical analysis of collagen in the medium showed a greater degree of both lysyl hydroxylation and hydroxylysyl glycosylation, the major increase in molecular mass of overmodified alpha chains being due to the higher hydroxylysine-bound hexose content. The proband's cells modify proteoglycan metabolism and mineral proband's cells modify proteoglycan metabolism and mineral crystals form in the dermis, possibly a response to abnormal collagen-proteoglycan interactions. These findings can be explained by a small defect in the product of one allele for pro-1(I) chains: three-quarters of the synthesized type I procollagen molecules are composed of trimers containing one or two chains defective near the C-terminus of the triple helix or in the C-propeptide. The data obtained for this patient confirmed that the severity of clinical manifestations in osteogenesis imperfecta strongly depends on the location and nature of the mutations, and that the phenotype could be a consequence of a collagen defect(s) and its influence on collagen-collagen interactions and collagen interactions with other connective tissue components.     

Cysteine in the triple-helical domain of one allelic product of the alpha 1(I) gene of type I collagen produces a lethal form of osteogenesis imperfecta

We studied tissue and cultured skin fibroblasts from a newborn with the lethal perinatal form of osteogenesis imperfecta born to a mother with the Marfan syndrome and her unrelated husband. Dermis from the infant was thinner and fibril diameter smaller than control; dermal fibroblastic cells had dilated endoplasmic reticulum. His fibroblasts in culture synthesized two different species of pro alpha 1(I) chains in about equal quantity. One chain was normal, the other contained cysteine within the triple-helical portion of the COOH-terminal cyanogen bromide peptide alpha 1(I)CB6. Molecules which contained two copies of the mutant chain formed alpha 1(I)-dimers linked through interchain disulfide bonds. Molecules which contained either one or two mutant chains were delayed in secretion and underwent excessive lysyl hydroxylation and hydroxylysyl glycosylation of all chains in the molecule, probably as a result of delayed triple-helix formation. Molecules containing either one or two copies of the mutant chain melted at 38 degrees C instead of 41 degrees C. The most likely explanation for these findings is that a cysteine is substituted for a glycine in the triple-helical domain of the products of one of the alpha 1(I) alleles. Such a substitution would interfere with triple-helix formation and stability and thus explain 1) the decreased melting temperature, 2) the increased post-translational modification, 3) the altered rate of secretion and accumulation of intracellular material, 4) the increased intracellular degradation of newly synthesized collagen, and 5) the decreased collagen production. Since neither parental cell strain produced the same mutant chain, the findings are best explained by a new mutation in one of the alpha 1(I) genes. The role of the uncharacterized "Marfan" gene in modifying the phenotype in this patient is unclear.     

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.     

Decreased thermal denaturation temperature of osteogenesis imperfecta mutant collagen is independent of post-translational overmodifications of lysine and hydroxylysine

Fibroblasts from many patients with osteogenesis imperfecta (OI) synthesize and secrete Type I collagen which is both overmodified and exhibits a decreased thermal denaturation temperature. We have examined the relationship between overmodification and decreased melting temperature in several favorable OI mutants by selectively inhibiting lysyl hydroxylase activity with the drug Minoxidil and comparing the melting profiles of the resultant undermodified collagen with untreated control. Minoxidil treatment causes an appreciable decrease in hydroxylysine with compensatory increases in lysine content, and the delayed sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobility of the overmodified collagen chains becomes normal. However, the decreased melting temperature was unchanged from untreated OI control. When unhydroxylated collagen produced by normal control and OI fibroblasts incubated with alpha,alpha'-dipyridyl was examined, mutant OI molecules melted at a lower temperature than control. These data indicate that the decreased thermal denaturation temperature of OI mutant collagen is independent of post-translational overmodification of lysine or hydroxylysine. Presumably, substitutions for glycine in the Gly-X-Y structural motif distort the helix and produce lower melting temperatures by presently unknown mechanisms.     

Variable expression of osteogenesis imperfecta in a nuclear family is explained by somatic mosaicism for a lethal point mutation in the alpha 1(I) gene (COL1A1) of type I collagen in a parent.

Fibroblasts from a man with a mild form of osteogenesis imperfecta (OI) and from his son with perinatal lethal OI (OI type II) produced normal and abnormal type I procollagen molecules. The abnormal molecules synthesized by both cell strains contained one or two pro alpha 1(I) chains in which the glycine at position 550 of the triple-helical domain was substituted by arginine as the result of a G-to-A transition in the first base of the glycine codon. Cells from the mother produced only normal type I procollagen molecules. By allele-specific oligonucleotide hybridization to amplified genomic sequences from paternal tissues we determined that the mutant allele accounted for approximately 50% of the COL1A1 alleles in fibroblasts, 27% of those in blood, and 37% of those in sperm. These findings demonstrate that the father is mosaic for the potentially lethal mutation and suggest that the OI phenotype is determined by the nature of the mutation and the relative abundance of the normal and mutant alleles in different tissues. Furthermore, the findings make it clear that some individuals with mild to moderate forms of OI are mosaic for mutations that will be lethal in their offspring.     

Collagen defects in lethal perinatal osteogenesis imperfecta.

Quantitative and qualitative abnormalities of collagen were observed in tissues and fibroblast cultures from 17 consecutive cases of lethal perinatal osteogenesis imperfecta (OI). The content of type I collagen was reduced in OI dermis and bone and the content of type III collagen was also reduced in the dermis. Normal bone contained 99.3% type I and 0.7% type V collagen whereas OI bone contained a lower proportion of type I, a greater proportion of type V and a significant amount of type III collagen. The type III and V collagens appeared to be structurally normal. In contrast, abnormal type I collagen chains, which migrated slowly on electrophoresis, were observed in all babies with OI. Cultured fibroblasts from five babies produced a mixture of normal and abnormal type I collagens; the abnormal collagen was not secreted in two cases and was slowly secreted in the others. Fibroblasts from 12 babies produced only abnormal type I collagens and they were also secreted slowly. The slower electrophoretic migration of the abnormal chains was due to enzymic overmodification of the lysine residues. The distribution of the cyanogen bromide peptides containing the overmodified residues was used to localize the underlying structural abnormalities to three regions of the type I procollagen chains. These regions included the carboxy-propeptide of the pro alpha 1(I)-chain, the helical alpha 1(I) CB7 peptide and the helical alpha 1(I) CB8 and CB3 peptides. In one baby a basic charge mutation was observed in the alpha 1(I) CB7 peptide and in another baby a basic charge mutation was observed in the alpha 1(I) CB8 peptide. The primary defects in lethal perinatal OI appear to reside in the type I collagen chains. Type III and V collagens did not appear to compensate for the deficiency of type I collagen in the tissues.     

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.     

Altered triple helical structure of type I procollagen in lethal perinatal osteogenesis imperfecta

Cultured dermal fibroblasts from an infant with the lethal perinatal form of osteogenesis imperfecta (type II) synthesize normal and abnormal forms of type I procollagen. The abnormal type I procollagen molecules are excessively modified during their intracellular stay, have a lower than normal melting transition temperature, are secreted at a reduced rate, and form abnormally thin collagen fibrils in the extracellular matrix in vitro. Overmodification of the abnormal type I procollagen molecules was limited to the NH2-terminal three-fourths of the triple helical domain. Two-dimensional mapping of modified and unmodified alpha chains of type I collagen demonstrated neither charge alterations nor large insertions or deletions in the region of alpha 1(I) and alpha 2(I) in which overmodification begins. Both the structure and function of type I procollagen synthesized by cells from the parents of this infant were normal. The simplest interpretation of the results of this study is that the osteogenesis imperfecta phenotype arose from a new dominant mutation in one of the genes encoding the chains of type I procollagen. Given the requirement for glycine in every third position of the triple helical domain, the mutation may represent a single amino acid substitution for a glycine residue. These findings demonstrate further heterogeneity in the biochemical basis of osteogenesis imperfecta type II and suggest that the nature and location of mutations in type I procollagen may determine phenotypic variation.     

Heterozygosity for a large deletion in the alpha 2(I) collagen gene has a dramatic effect on type I collagen secretion and produces perinatal lethal osteogenesis imperfecta

We characterized a de novo 4.5 kilobase pair deletion in the paternally derived alpha 2(I) collagen allele (COL1A2) from a patient with perinatal lethal osteogenesis imperfecta. The intron-to-intron deletion removed the seven exons which encode residues 586-765 of the triple helical domain of the chain. Type I procollagen molecules that contain the mutant pro-alpha 2(I) chain have a lower than normal thermal stability, undergo increased post-translational modification amino-terminal to the deletion junction, and are retained within the rough endoplasmic reticulum. The block to secretion appears to result from improper assembly of the triple helix, apparently a consequence of a disruption of charge-charge interactions between the shortened pro-alpha 2(I) chain and normal pro-alpha 1(I) chains. The lethal effect may be due to decreased secretion of normal collagen and secretion of a small amount of abnormal collagen that disrupts matrix formation.     

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 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.     

Structure, stability and interactions of type I collagen with GLY349-CYS substitution in alpha 1(I) chain in a murine Osteogenesis Imperfecta model.

Here we report the structural and functional studies of collagen from the Brtl mouse, a heterozygous knock-in model for Osteogenesis Imperfecta, which has a G349C substitution introduced in one col1a1 allele. We observed that 25+/-5% of alpha 1(I) chains in different tissues and in different extracts from matrix deposited by cultured cells were S-S-linked mutant dimers. Apparently mutant and normal molecules are equally well incorporated into the matrix and they form mature covalent crosslinks with the same efficiency. We found different extents of post-translational overmodification of mutant molecules in different tissues, but we found no consistent differences between lethal and non-lethal animals. We did not detect any changes in the thermal stability or rate of thermal denaturation of mutant collagen. We also did not detect any changes in collagen-collagen recognition and interactions except for disruption of quasi-crystalline lateral packing of molecules in tendons from some, mostly prepubertal, mutant animals. In contrast, alpha 1(I)(3) collagen from the oim mouse--the only other non-lethal murine OI model studied by similar techniques--has altered stability, fibrillogenesis, collagen-collagen interactions and produces a more consistent and more pronounced disruption of tendon crystallinity. Nevertheless, while the G349C substitution causes moderate or lethal OI, heterozygous oim mice are much less affected. Overall, our results suggest that OI symptoms and phenotype variation in G349C animals are related to abnormal interactions of mutant collagen helices with other matrix molecules or abnormal function of osteoblasts rather than to abnormal structure, physical properties or interactions between mutant collagen helices.     

A type I collagen with substitution of a cysteine for glycine-748 in the alpha 1(I) chain copolymerizes with normal type I collagen and can generate fractallike structures

Type I procollagen was purified from cultured fibroblasts of a proband with a lethal variant of osteogenesis imperfecta. The protein was a mixture of normal procollagen and mutated procollagens containing a substitution of cysteine for glycine in either one pro alpha 1(I) chain or both pro alpha 1(I) chains, some or all of which were disulfide-linked through the cysteine at position alpha 1-748. The procollagen was then examined in a system for generating collagen fibrils de novo by cleavage of the pCcollagen to collagen with procollagen C-proteinase [Kadler et al. (1987) J. Biol. Chem. 262, 15696-15701]. The mutated collagens and normal collagens were found to form copolymers under a variety of experimental conditions. With two preparations of the protein that had a high content of alpha 1(I) chains disulfide-linked through the cysteine alpha 1-748, all the large structures formed had a distinctive, highly branched morphology that met one of the formal criteria for a fractal. Preparations with a lower content of disulfide-linked alpha 1(I) chains formed fibrils that were 4 times the diameter of control fibrils. The formation of copolymers was also demonstrated by the observation that the presence of mutated collagens decreased the rate of incorporation of normal collagen into fibrils. In addition, the solution-phase concentration at equilibrium of mixtures of mutated and normal collagens was 5-10-fold greater than that of normal collagen.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of different cysteine for glycine substitutions within alpha 2(I) chains. Evidence of distinct structural domains within the type I collagen triple helix

Affected individuals from two apparently distinct, mild osteogenesis imperfecta families were heterozygous for a G to T transition in the COL1A2 gene that resulted in cysteine for glycine substitutions at position 646 in the alpha 2(I) chain of type I collagen. A child with a moderately severe form of osteogenesis imperfecta was heterozygous for a G to T transition that resulted in a substitution of cysteine for glycine at position 259 in the COL1A2 gene. Type I collagen molecules containing an alpha 2(I) chain with cysteine at position 259 denaturated at a lower temperature than molecules containing an alpha 2(I) chain with cysteine at position 646. In contrast to cysteine for glycine substitutions in the alpha 1(I) chain, the severity of the osteogenesis imperfecta phenotype is not directly proportional to the distance of the mutation from the amino-terminal end of the triple helix. These findings could be explained if the type I collagen triple helix contains discontinuous domains that differ in their contributions to maintaining helix stability.