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.     

Increased expression of the gene for the pro alpha 1(IV) chain of basement-membrane procollagen in cultured skin fibroblasts from two variants of osteogenesis imperfecta.

Fibroblasts from two lethal variants of osteogenesis imperfecta were shown to synthesize increased amounts of type IV procollagen. Previous studies established that one of these variants had a non-functional allele for the pro alpha 2 chain of type I procollagen, whereas the other pro alpha 2(I) allele contained a mutation leading to synthesis of shortened pro alpha 2(I) chains. In the two variants, the relative level of mRNA for pro alpha 1(IV) was 31 and 42% of the level of mRNA for pro alpha 1(I) chains. A value of less than 2% was found for a third lethal and four non-lethal variants of osteogenesis imperfecta. Immunofluorescent staining of fibroblasts from the two variants synthesizing increased amounts of type IV procollagen indicated that a homogeneous population of cells synthesized both type IV and type I procollagen. The results suggest that mutations in the type I procollagen genes that result in osteogenesis imperfecta can be associated with increased expression of the genes for type IV procollagen.     

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.     

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 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.     

RNA sequence analysis of a perinatal lethal osteogenesis imperfecta mutation

The perinatal lethal form of osteogenesis imperfecta often results from mutations which disrupt stable assembly, delay secretion, and cause excessive posttranslational modification of type I procollagen molecules. One such mutation was efficiently characterized by an indirect method of RNA sequence analysis. The mutation initially was localized in procollagen by mapping the distribution of abnormal posttranslational modification within the triple helical domain of mutant molecules. Total RNA was isolated from osteogenesis imperfecta cells in culture, cDNA was synthesized using alpha 1(I) and alpha 2(I) specific primers, and fragments of cDNA suspected to harbor the mutation were amplified by the polymerase chain reaction technique and then cloned in M13 vectors. Sequence analysis of the amplified cDNA revealed a new, heterozygous Gly----Val substitution at residue 256 of the triple helical domain of alpha 1(I) chains produced by the perinatal lethal osteogenesis imperfecta cells. The nature and location of the mutation were confirmed by sequence analysis of amplified genomic DNA. A Gly----Val substitution has not previously been associated with the lethal form of osteogenesis imperfecta, and this mutation has the most amino-terminal location within the alpha 1(I) chain triple helical domain reported to date.     

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.     

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.     

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.     

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.     

Defects in the processing of procollagen to collagen are demonstrable in cultured fibroblasts from patients with the Ehlers-Danlos and osteogenesis imperfecta syndromes

This is a study of the processing of procollagen to collagen in cultures of skin and tendon fibroblasts. Processing was markedly increased by growing cells for 2-4 days postconfluence and then adding ascorbate to the medium for 2 days prior to labeling with [3H] proline. With this system, more than two-thirds of the pro-alpha chains of type I procollagen in the culture medium, and more than 90% of those in the cell layer, were rapidly processed to pC-alpha, pN-alpha, or alpha chains. Purified, exogenous procollagen was also rapidly processed in cell-free culture medium. The results showed for the first time that exogenous procollagen can be processed in conditioned cell-free medium. The system was then used to compare the processing of procollagen in the medium of normal fibroblasts, cells from one bovine and four human variants of osteogenesis imperfecta, and those from eight human variants of the Ehlers-Danlos syndrome. The cells could be divided into three groups, based on their ability to process type I procollagen: normal, consistently slow, and very slow. The cause of the decreased processing was shown to be associated with either a mutation causing a shortening of an alpha chain or decreased activity of procollagen N-proteinase in cell-free culture medium. Decreased processing of procollagen to collagen occurred with cultured fibroblasts from patients with different forms of both osteogenesis imperfecta and Ehlers-Danlos syndrome. Both of these disease syndromes are associated with abnormalities in the structure or metabolism of procollagen in fibrous connective tissues, bones, and teeth. The results show that defects in the structure, synthesis, or processing of procollagen are readily demonstrated with cultured fibroblasts.     

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.     

Osteogenesis imperfecta type IV. Detection of a point mutation in one alpha 1(I) collagen allele (COL1A1) by RNA/RNA hybrid analysis

We have identified a point mutation in one alpha 1(I) collagen allele (COL1A1) of a child with the type IV osteogenesis imperfecta phenotype. When compared to parental and control samples, skin fibroblasts of the proband synthesized two populations of type I collagen molecules. One population was normal; the other was delayed in secretion and electrophoretic migration due to post-translational overmodification. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated a gradient of overmodification beginning near the carboxyl-terminal CB peptides. This predicts that the mutation delaying helix formation is near the carboxyl-terminal end of one of the component chains of type I collagen. The mRNA of the patient was probed with overlapping antisense riboprobes to type I collagen cDNA. Cleavage of a mismatch in RNA/RNA hybrids of RNase A allowed the location of the mutation to a 225-base pair region of alpha 1(I) cDNA. The mismatch was not present in RNA/RNA hybrids from either parent. This region of both alpha 1(I) alleles of the patient was isolated by screening a lambda ZAP cDNA library. Sequence determination of both alleles demonstrated a single nucleotide change, G----A, resulting in the substitution of a serine for a glycine at amino acid residue 832. This point mutation occurs in the coding region for alpha 1(I) CB6 and is concordant with the protein data. The finding of a glycine substitution in an alpha 1(I) chain of a patient with the milder type IV osteogenesis imperfecta phenotype requires modification of current molecular models for types II and IV osteogenesis imperfecta.     

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.     

A frameshift mutation results in a truncated nonfunctional carboxyl-terminal pro alpha 1(I) propeptide of type I collagen in osteogenesis imperfecta

A codon frameshift mutation caused by a single base (U) insertion after base pair 4088 of prepro alpha 1(I) mRNA of type I procollagen was identified in a baby with lethal perinatal osteogenesis imperfecta. The mutation was identified in fibroblast RNA by a new method that allows the direct detection of mismatched bases by chemical modification and cleavage in heteroduplexes formed between mRNA and control cDNA probes. The region of mismatches was specifically amplified by the polymerase chain reaction and sequenced. The heterozygous mutation in the amplified cDNA most likely resulted from a T insertion in exon 49 of COL1A1. The frameshift resulted in a truncated pro alpha 1(I) carboxyl-terminal propeptide in which the amino acid sequence was abnormal from Val1146 to the carboxyl terminus. The propeptide lacked Asn1187, which normally carries an N-linked oligosaccharide unit, and was more basic than the normal propeptide. The distribution of cysteines was altered and the mutant propeptide was unable to form normal interchain disulfide bonds. Some of the mutant pro alpha 1(I)' chains were incorporated into type I procollagen molecules but resulted in abnormal helix formation with over-hydroxylation of lysine residues, increased degradation, and poor secretion. Only normal type I collagen was incorporated into the extracellular matrix in vivo resulting in a tissue type I collagen content approximately 20% of that of control (Bateman, J. F., Chan, D., Mascara, T., Rogers, J. G., and Cole, W. G. (1986) Biochem. J. 240, 699-708).     

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.     

Various characteristics of the structure and synthesis of procollagens produced by cultured skin fibroblasts from patients with Danlos-Ehlers syndrome type I

The electrophoretic mobilities of the collagen and procollagen type I and III chains synthesized by the fibroblasts isolated from patients with type I Ehlers-Danlos syndrome as well as a set of peptides obtained by splitting of pro alpha 1(I) and pro alpha 2(I) type I procollagens by cyanbromide are not different from the normal ones. The fact demonstrates the absence of long insertions or deletions, or the sufficient defects in intracellular chain modifications. The changes were also nor registered for the ratio of type I and III collagens from the digested by pepsin preparations of protein accumulating in the culture media of the cultured skin fibroblasts from patients. The studied strains of cultured fibroblasts from patients suffering the Ehlers-Danlos syndrome have the trend to increased accumulation of partially processed chains of proc alpha 1(I) and proc alpha 2(I) type I procollagen and to the increased ratio of pro alpha 1(I) to pro alpha 2(I).     

Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the alpha 1 chain of type I collagen produces mild osteogenesis imperfecta

We have characterized a mutation that produces mild, dominantly inherited osteogenesis imperfecta. Half of the alpha 1 (I) chains of type I collagen synthesized by cells from an affected individual contain a cysteine residue in the 196-residue carboxyl-terminal cyanogen bromide peptide of the triple-helical domain (Steinmann, B., Nicholls, A., and Pope, F. M. (1986) J. Biol. Chem. 261, 8958-8964). Unexpectedly, sequence determined from a proteolytic fragment of the alpha 1 (I) chain derived from procollagen molecules synthesized in the presence of both [3H]proline and [35S]cysteine indicated that the cysteine is located at the third residue carboxyl-terminal to the triple-helical domain, normally a glycine. The nucleotide sequence of a fragment amplified from genomic DNA confirmed the location of the cysteine residue and showed that the mutation was a single nucleotide change in one COL1A1 allele. This represents a new class of mutations, point mutations outside the triple-helical domain of the chains of type I collagen, that produce the osteogenesis imperfecta phenotype.     

Studies on type I collagen in skin fibroblasts cultured from twins with lethal osteogenesis imperfecta

Studies on type I procollagen produced by skin fibroblasts cultured from twins with lethal type II of osteogenesis imperfecta (OI) showed that biosynthesis of collagen (measured by L-[5-(3)H]proline incorporation into proteins susceptible to the action of bacterial collagenase) was slightly increased as compared to the control healthy infant. SDS/PAGE showed that the fibroblasts synthesized and secreted only normal type I procollagen. Electrophoretic analysis of collagen chains and CNBr peptides showed the same pattern of electrophoretic migration as in the controls. The lack of posttranslational overmodification of the collagen molecule suggested a molecular defect near the amino terminus of the collagen helix. Digestion of OI type I collagen with trypsin at 30 degrees C for 5 min generated a shorter than normal alpha2 chain which melted at 36 degrees C. Direct sequencing of an asymmetric PCR product revealed a heterozygous single nucleotide change C-->G causing a substitution of histidine by aspartic acid in the alpha2 chain at position 92. Pericellular processing of type I procollagen by the twin's fibroblasts yielded a later appearance of the intermediate pC-alpha1(I) form as compared with control cells.