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.     

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.     

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.     

A base substitution at the splice acceptor site of intron 5 of the COL1A2 gene activates a cryptic splice site within exon 6 and generates abnormal type I procollagen in a patient with Ehlers-Danlos syndrome type VII.

The dermal type I collagen of a patient with Ehlers-Danlos type VIIB (EDS-VIIB) contained normal alpha 2(I) chains and mutant pN-alpha 2(I)' chains in which the amino-terminal propeptide (N-propeptide) remained attached to the alpha 2(I) chain. Similar alpha 2(I) chains were produced by cultured dermal fibroblasts. Amino acid sequencing of tryptic peptides, prepared from the mutant amino-terminal pN-alpha 2(I) CB1' peptide, indicated that five amino acids, including the N-proteinase (the specific proteinase that cleaves the procollagen N-propeptide) cleavage site, had been deleted from the junction of the N-propeptide and the N-telopeptide (the nonhelical domain at the amino-terminus of the alpha chains of fully processed type I polypeptide chains) of the mutant pro-alpha 2(I)' chain. The corresponding 15 nucleotides, which were deleted from approximately half of the alpha 2(I) cDNA polymerase chain reaction products, of the alpha 2(I) cDNA polymerase chain reaction products, were encoded by the +1 to +15 nucleotides of exon 6 of the normal alpha 2(I) gene (COL1A2). These 15 nucleotides were deleted in the splicing of alpha 2(I) pre-mRNA to mRNA as a result of inactivation of the 3' splice site of intron 5 by an AG to AC mutation and the activation of a cryptic AG splice acceptor site corresponding to positions +14 and +15 of exon 6. Loss of the N-proteinase cleavage site explained the persistence of the pN-alpha 2(I)' chains in the dermis and in fibroblast cultures. Collagen production by cultured dermal fibroblasts was doubled, possibly due to reduced feedback inhibition by the N-propeptides. In contrast to previously reported cases of EDS-VIIB, Lys5 of the N-telopeptide was not deleted and appeared to take part in the formation of intramolecular cross-linkages. However, increased collagen solubility and abnormal extraction profiles of the mutant type I collagen molecules indicated that collagen cross-linking was abnormal in the dermis. The proband and her son were heterozygous for the mutation. It is likely that the heterozygous loss of the N-proteinase cleavage site, with persistence of a shortened N-propeptide, was the major factor responsible for the EDS-VIIB 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.     

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.     

Deletion of 24 amino acids from the pro-alpha 1(I) chain of type I procollagen in a patient with the Ehlers-Danlos syndrome type VII

A child with the type VII form of the Ehlers-Danlos syndrome was shown to have a structural defect in the amino terminus of the pro-alpha 1(I) chain of type I procollagen. Normal and mutant amino-terminal cyanogen bromide peptides (pN-alpha 1(I) CB0,1 peptides) were purified from the medium of the patient's cultured fibroblasts. Amino acid sequencing of tryptic peptides derived from the mutant pN-alpha 1(I) CB0,1 peptide showed that an expected sequence of 24 amino acids (positions 136-159 of the normal pN-alpha 1(I) CB0,1 peptide) was deleted. The segment deleted from the mutant pro-alpha 1(I) chain contains the small globular region of the NH2-propeptide, the procollagen N-proteinase cleavage site, the NH2-telopeptide, and first triplet of the helix of the alpha I(I) collagen chain (Chu, M.-L., de Wet, W., Bernard, M., Ding, J.F., Morabito, M., Myers, J., Williams, C., and Ramirez, F. (1984) Nature 310, 337-340). Loss of the procollagen N-proteinase cleavage site from the mutant pro-alpha 1(I) chain accounted for the persistence of its NH2-propeptide despite normal production of the N-proteinase by cultured mutant fibroblasts. Collagen production by mutant fibroblasts was doubled possibly due to reduced feedback inhibition by the NH2-propeptides. The child appeared to be heterozygous for the peptide deletion and, as the parents did not show any evidence of the deletion, it is likely that the child had a new mutation of one allele of the pro-alpha 1(I) gene. The deleted peptide corresponds precisely to the sequence coded by exon 46 of the normal pro-alpha 1(I) gene (Chu, M.-L., de Wet, W., Bernard, M., Ding, J.F., Morabito, M., Myers, J., Williams, C., and Ramirez, F. (1984) Nature 310, 337-340).     

A base substitution at a splice site in the COL3A1 gene causes exon skipping and generates abnormal type III procollagen in a patient with Ehlers-Danlos syndrome type IV

The dermis of a child with Ehlers-Danlos syndrome type IV (EDS-IV) contained about 11% of the normal amount of type III collagen and cultured dermal fibroblasts produced a reduced amount of type III procollagen which was secreted poorly. Type III collagen produced by these cells contained normal and abnormal alpha-chains and cyanogen bromide peptides. The site of the structural defect in the abnormal alpha 1 (III) chains was localized to the region of Met797, which is at the junction of the two carboxyl-terminal CB5 and CB9 cyanogen bromide peptides. Chemical cleavage of heteroduplexes formed between EDS-IV mRNA and a normal cDNA clone covering the CB5 and CB9 region showed that about 100 nucleotides were mismatched. Sequencing of amplified and cloned cDNA spanning the mutant region revealed a 108 nucleotide deletion corresponding to amino acid residues Gly775 to Lys810. The deleted nucleotide sequence corresponded to sequences that, by analogy to the organization of the type I collagen genes, should be precisely encoded by exon 41 of the COL3A1 gene. Sequencing of amplified genomic DNA, prepared using disimilar amounts of primers specific for exons 41 and 42, displayed a base substitution (G-to-A) in the highly conserved GT dinucleotide of the 5' splice site of intron 41. Normal sequences were also obtained from the normal allele. It is likely that the GT-to-AT transition at the splice donor site of intron 41 generated an abnormally spliced mRNA in which sequences of exon 40 and 42 were joined together with maintenance of the reading frame. The corresponding peptide deletion included the cyanogen bromide cleavage site Met797-Pro798 and the mammalian collagenase cleavage site at Gly781-Ile782. These losses account for the resistance of EDS-IV collagen to cyanogen bromide and mammalian collagenase digestion. Cultured fibroblasts produced normal homotrimer, mutant homotrimer, and mixed heterotrimer type III collagen molecules. The mutant homotrimer molecules were the major pepsin-resistant species and about 69% of the alpha 1(III) mRNA was in the mutant form.     

Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-alpha 2(I) chain

A patient with Ehlers-Danlos syndrome Type VIIB was found to have an interstitial deletion of 18 amino acids in approximately half of the pro-alpha 2(I) chains of Type I procollagen. Analysis of pepsin-solubilized tissue and fibroblast collagen revealed an abnormal additional chain, alpha 2(I)', which migrated in sodium dodecyl sulfate-5% polyacrylamide gel electrophoresis between the normal alpha 1(I) and alpha 2(I) chains. The apparent ratio of normal alpha 1(I):mutant alpha 2(I)':normal alpha 2(I) was 4:1:1. Procollagen studies and enzyme digestion studies of native mutant collagen suggested defective removal of the amino propeptide. Sieve chromatography of CNBr peptides from purified alpha 2(I)' chains revealed the absence of the normal amino telopeptide fragment CB 1 and the appearance of a larger new peptide of approximately 60 residues (CB X). Compositional and sequencing studies of this peptide identified normal amino propeptide sequences. However, the most carboxyl-terminal tryptic peptide of CB X differed substantially in composition and sequence from the expected and was found to have an interstitial deletion of 18 amino acids corresponding to the N-telopeptide of the pro-alpha 2(I) chain. This deletion removes the normal sites of cleavage of the N-proteinase and also removes a critical cross-linking lysine residue. The 18 amino acids deleted correspond exactly to the residues encoded by exon 6 of the pro-alpha 2(I) collagen gene (COL 1 A2), and, therefore, the protein defect may be due to a genomic deletion, or alternatively, an RNA splicing defect.     

Cleavage of type I and type II procollagens by type I/II procollagen N-proteinase. Correlation of kinetic constants with the predicted conformations of procollagen substrates

The kinetic constants were examined for the cleavage of several types of procollagen by type I/II procollagen N-proteinase. The Km values were essentially the same (0.2 microM) for chick type I procollagen, human type I procollagen, and chick type II procollagen. However, the Vmax values differed over a 14-fold range. As reported previously, the enzyme did not cleave denatured type I or II procollagen. Also, it did not cleave human type III procollagen which contains the same scissle -Pro-Gln- bond as the pro-alpha 1(I) chain of type I procollagen. To explain the observations, Chou-Fasman rules were used to compare the secondary structures of the cleavage sites in the procollagens. The results supported a previous suggestion (Helseth, D. L., Jr., Lechner, J. L., and Veis, A. (1979) Biopolymers 18, 3005-3014) that the region carboxyl-terminal to cleavage site in the pro-alpha 1(I) chain of type I procollagen was in a hairpin conformation consisting of a beta-sheet, beta-turn, and beta-sheet. In both chick and human type I procollagen, the hairpin loop in the pro-alpha 1(I) chain consisted of about 18 amino acids. The cleavage site itself was in a short alpha-helical structure of four or five amino acids. The pro-alpha 2(I) chains had a similar hairpin loop of about 14 amino acids and alpha-helix of four or five amino acids containing the cleavage site. Chick type II procollagen, which had the highest Vmax value, had a longer hairpin structure of 22 amino acids, and the cleavage site was in a longer alpha-helical domain of 10 amino acids. In contrast, type III procollagen had a random-coil conformation in the same region. The results help to explain the unusual substrate requirements of type I/II N-proteinase. They also help explain why mutations that produce in-frame deletions of amino acids 84 or more residues carboxyl-terminal to the cleavage site make the protein resistant to the enzyme.     

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 N-proteinase from whole chick embryos. Cleavage of a homotrimer of pro-alpha 1(I) chains and the requirement for procollagen with a triple-helical conformation

Procollagen N-proteinase, the enzyme which cleaves the NH2-terminal propeptides from type I procollagen, was purified over 15,000-fold from extracts of chick embryos by chromatography on columns of DEAE-cellulose, concanavalin A-agarose, heparin-agarose, pN-collagen-agarose, and a filtration gel. The purified enzyme had an apparent molecular weight of 320,000 as estimated by gel filtration and a pH optimum for activity of 7.4 to 9.0. The enzyme was inhibited by metal chelators and the thiol reagent dithiothreitol. Addition of calcium was required for maximal activity under the standard assay conditions, and the presence of calcium decreased thermal inactivation at 37 degrees C. The purified enzyme cleaved a homotrimer of pro-alpha 1(I) chains, an observation which indicated that the presence of pro-alpha 2(I) chain is not essential for the enzymic cleavage of NH2-terminal propeptides. Previous observations suggesting that the enzyme requires a substrate with a native conformation were explored further by reacting the enzyme with type I procollagen at different temperatures. Type I procollagen from chick embryo fibroblasts became resistant to cleavage at about 43 degrees C. Type I procollagen from human skin fibroblasts, which was previously shown to have a slightly lower thermal stability than chick embryo type I procollagen, became resistant to cleavage at temperatures that were about 2 degrees C lower. The results suggested that the enzyme is a sensitive probe for the three-dimensional structure of the NH2-terminal region of the procollagen molecule and that it requires the protein substrate to be triple helical.     

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.     

Ehlers Danlos syndrome type VIIB. Incomplete cleavage of abnormal type I procollagen by N-proteinase in vitro results in the formation of copolymers of collagen and partially cleaved pNcollagen that are near circular in cross-section.

We have shown that a child with Ehlers Danlos syndrome (EDS) type VII has a G to A transition at the first nucleotide of intron 6 in one of her COL1A2 alleles. Half of the cDNA clones prepared from the proband's pro alpha 2(I) mRNA lacked exon 6. The type I procollagen secreted by the proband's dermal fibroblasts in culture was purified, and collagen fibrils were generated in vitro by cleavage of the procollagen with the procollagen N- and C-proteinases. Incubation of the procollagen with N-proteinase resulted in a 1:1 mixture of pCcollagen and uncleaved procollagen. Incubation of this mixture with C-proteinase generated collagen and abnormal pNcollagen (pNcollagen-ex6) that readily copolymerized into fibrils. By electron microscopy these fibrils resembled the hieroglyphic fibrils seen in the N-proteinase-deficient skin of dermatosparactic animals and humans and were distinct from the near circular cross-section fibrils seen in the tissues of individuals with EDS type VII. Further incubation of the hieroglyphic fibrils with N-proteinase resulted in partial cleavage of the pNcollagen-ex6 in which the abnormal pN alpha 2(I) chains remained intact. These fibrils were not hieroglyphic but were near circular in cross-section. Fibrils formed from collagen and pNcollagen-ex6 that had been partially cleaved with elevated amounts of N-proteinase prior to fibril formation were also near circular in cross-section. The results are consistent with a model of collagen fibril formation in which the intact N-propeptides are located exclusively at the surface of the hieroglyphic fibrils. Partial cleavage of the pNcollagen-ex6 by N-proteinase allows the N-propeptides to be incorporated within the body of the fibrils. The model provides an explanation for the morphology and molecular composition of collagen fibrils in the tissues of patients with EDS type VII.     

A mutation in the pro alpha 2(I) gene (COL1A2) for type I procollagen in Ehlers-Danlos syndrome type VII: evidence suggesting that skipping of exon 6 in RNA splicing may be a common cause of the phenotype.

Fibroblasts from a proband with Ehlers-Danlos syndrome type VII synthesized approximately equal amounts of normal and shortened pro alpha 2(I) chains of type I procollagen. Nuclease S1 probe protection experiments with mRNA demonstrated that the pro alpha 2(I) chains were shortened because of a deletion of most or all of the 54 nucleotides in exon 6, the exon that contains codons for the cleavage site for procollagen N-proteinase. Sequencing of genomic clones revealed a single-base mutation that converted the first nucleotide of intron 6 from G to A. Therefore, the mutation was a change, in the -GT-consensus splice site, that produced efficient exon skipping. Allele-specific oligonucleotide hybridizations demonstrated that the proband's mother, father, and brother did not have the mutation. Therefore, the mutation was a sporadic one. Analysis of potential 5' splice sites in the 5' end of intron 6 indicated that none had favorable values by the two commonly employed techniques for evaluating such sites. The proband is the fourth reported proband with Ehlers-Danlos syndrome VII with a single-base mutation that causes skipping of exon 6 in the splicing of RNA from either the COL1A1 gene or COL1A2 gene. No other mutations in the two type I procollagen genes have been found in the syndrome. Therefore, such mutations may be a common cause of the phenotype. The primers developed should be useful in screening for the same or similar mutations causing the disease.     

Defective folding and stable association with protein disulfide isomerase/prolyl hydroxylase of type I procollagen with a deletion in the pro alpha 2(I) chain that preserves the Gly-X-Y repeat pattern.

We have studied the folding, processing, and association with two endoplasmic reticulum (ER) resident proteins of the abnormal type I procollagen molecules produced by a strain of fibroblasts harboring a 4.5 kilobase deletion in an allele of COL1A2 (Willing, M. C., Cohn, D.H., Starman, B. Holbrook, K.A., Greenberg, C.R., and Byers, P.H. (1988) J. Biol. Chem. 263, 8398-8404). By sequencing cDNA, we found that the mutant allele encodes pro alpha 2(I) chains that are shortened by 180 amino acids but retain the Gly-X-Y repeat pattern crucial for collagen triple helix formation. The type I procollagen molecules that incorporated the shortened chain were retained intracellularly and were stable. The triple helical domain in these molecules did not attain a normal conformation and remained accessible to posttranslational modifying enzymes amino-terminal to the deletion site for a prolonged period. The abnormal molecules folded into a triple helical conformation more slowly than the normal molecules, and the amino-terminal ends of the pro alpha 1(I) chains failed to become protease-resistant. While the abnormal procollagen molecules were not bound by the ER-resident protein BiP, they stably associated with protein disulfide isomerase, the beta-subunit of prolyl-4-hydroxylase. These results indicate that some mutations in type I collagen genes both transiently delay folding and permanently disrupt the structure of the triple helix and suggest that binding to prolyl-4-hydroxylase helps to retain certain abnormal procollagen molecules within the ER.     

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.     

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.     

Defects of type I procollagen metabolism correlated with decrease of prolidase activity in a case of lethal osteogenesis imperfecta.

We have studied the structure and metabolism of type I procollagen in a case of perinatal lethal osteogenesis imperfecta (OI) type II. Cultured skin fibroblasts from the proband synthesized both normal and abnormal forms of type I procollagen. Some abnormal, overmodified molecules were secreted by OI cells, although less efficiently than normal molecules from control cells. The OI fibroblasts accumulated large amounts of abnormal proalpha1(I) and proalpha2(I) chains intracellularly. The extracellular collagenolytic activity was decreased compared to control cells. Furthermore, OI cells produced less type I procollagen and demonstrated lower capacity to synthesize DNA than control cells. We have found that in contrast to prolinase activity, the activity of prolidase (an enzyme essential for collagen synthesis and cell growth) is also significantly reduced in OI cells. No differences were found in the amount of the enzyme protein recovered from both the OI and control cells. However, we found that expressions of beta1 integrin and insulin-like growth factor-I receptor (receptors known to play an important role in up regulation of prolidase activity) were decreased in OI cells compared to control cells. The decrease in prolidase activity may provide an important mechanism of altered cell growth and collagen metabolism involved in producing the perinatal lethal form of the OI phenotype.