Molecular defects of type III procollagen in Ehlers-Danlos syndrome type IV

Summary Fibroblasts from most patients with Ehlers-Danlos syndrome (EDS) type IV, a disorder characterized by fragility of skin, blood vessels, and internal organs, secrete reduced amounts of type III procollagen. In 7 of 8 cell strains analyzed, we found evidence of structural defects in half of the type III procollagen chains synthesized, such as deletions or bona fide amino acid substitutions, which cause delayed formation and destabilization of the collagen triple helix and, as a consequence, reduced secretion of the molecule. The data suggest that EDS type IV is often caused by heterozygosity for mutations at the COL3A1 locus, which affect the structure of type III procollagen. The triple-helical region of the molecule, like the homologous region of type I procollagen, appears to be particularly vulnerable.Parts of this work have been presented at the 2nd International Conference on Molecular Biology and Pathology of Matrix, Philadelphia, June 15–18, 1988     

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

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

Multiexon deletion in the procollagen III gene is associated with mild Ehlers-Danlos syndrome type IV

We have characterized a deletion of approximately 9 kilobases which spans from intron 33 to exon 48 of one pro-alpha 1 (III) collagen allele in a patient with Ehlers-Danlos syndrome type IV. The mutation results in the production of an in-frame species of mRNA which lacks the sequences corresponding to residues 595-1,008 of the triple-helical domain. Thus, half of the pro-alpha 1 (III) chains synthesized by the patient's fibroblasts are nearly 30% shorter than normal. The procollagen III molecules composed of either three normal length or three shortened chains are thermally stable and efficiently secreted. In contrast, the procollagen III molecules that contain one or two shortened chains are unstable and are not secreted. Failure to secrete unstable molecules and a residual functional role of the shortened but stable homotrimers may explain the somewhat milder phenotype of this individual compared with that of another Ehlers-Danlos type IV patient bearing a deletion of similar size in the amino-terminal portion of the alpha 1 (III) collagen chain.     

Synthesis of structurally unstable type III procollagen in patients with cerebral artery aneurysm.

The biosynthesis of collagen was studied in skin fibroblast cultures established from 11 patients with cerebral artery aneurysms. Six patients had familial subarachnoid hemorrhage (SAH), while five patients were considered as sporadic cases. The structural stability of the triple-helical medium procollagen was studied by measuring the thermal denaturation temperature (Tm) of type I and type III procollagen molecules. Structural instability of type III procollagen was demonstrated in two patients with familial SAH. The Tm of type III procollagen was 39.0 degrees C and 39.5 degrees C in two of the cell lines, while the control value was 40.3 degrees C. The stability of type I procollagen did not differ from that of the controls, and the main features of the biosynthesis of collagen were similar in the aneurysm patient cell lines and in the controls. The results suggest that a structural defect of type III procollagen may serve as an etiological factor in the formation of cerebral artery aneurysms.     

Ehlers-Danlos syndrome type IV: a multi-exon deletion in one of the two COL3A1 alleles affecting structure, stability, and processing of type III procollagen

We have studied a patient with severe, dominantly inherited Ehlers-Danlos syndrome type IV. The results indicate that this patient carries a deletion of 3.3 kilo-base pairs in the triple helical coding domain of one of the two alleles for the pro-alpha-chains of type III collagen (COL3A1). His cultured skin fibroblasts contain equal amounts of normal length mRNA and of mRNA shortened by approximately 600 bases, and synthesize both normal and shortened pro-alpha 1(III)-chains. In procollagen molecules containing one or more shortened chains, a triple helix is formed with a length of only about 780 amino acids. The mutant procollagen molecules have decreased thermal stability, are less efficiently secreted, and are not processed as their normal counterpart. The deletion in this family is the first mutation to be described in COL3A1.     

Procollagen is more stable in cellulo than in vitro

The thermal denaturation of both intracellular and freshly secreted chick embryo tendon type I procollagen was investigated using susceptibility to proteolysis by trypsin and chymotrypsin as a probe for triple-helical conformation. Freshly secreted procollagen from the medium of matrix-free tendon cells in suspension or procollagen within the cells and in the pericellular environment melted at 45 degrees C. In contrast, if freshly secreted procollagen was subjected to the melting procedure after dialysis of the medium against 0.4 M NaCl, 0.1 M Tris HCl, pH 7.4 the protein melted at 42 degrees C, the melting temperature of purified procollagen dissolved in the same buffer. In each of these cases, the thermal denaturation profile was narrow, with a width of 1.0-1.5 degrees C. These results demonstrate that, in situ, procollagen is more stable toward thermal denaturation than was previously thought. This extra margin of thermal stability partially resolves the dilemma of how tissues are able to assemble triple-helical procollagen molecules at body temperatures that closely approach the melting temperature of the purified protein.     

Inheritance of an RNA splicing mutation (G+ 1 IVS20) in the type III procollagen gene (COL3A1) in a family having aortic aneurysms and easy bruisability: phenotypic overlap between familial arterial aneurysms and Ehlers-Danlos syndrome type IV.

Inheritance of a single base mutation in the type III procollagen gene (COL3A1) was studied in a family with aortic aneurysms and easy bruisability. The mutation was a substitution of A for G+ 1 of intron 20 of the gene and caused aberrant splicing of RNA transcribed from the mutated allele. The phenotype in the family included aortic aneurysms that ruptured and produced death. It also included easy bruisability, but it did not include other characteristic features of Ehlers-Danlos syndrome type IV, such as ecchymoses, abnormal scarring, or prominent subcutaneous blood vessels. The data from the family, together with a review of other probands with mutations in the type III procollagen gene, indicated that there is phenotypic overlap between Ehlers-Danlos syndrome type IV and familial arterial aneurysms not associated with any overlap between Ehlers-Danlos syndrome type IV and familial arterial aneurysms not associated with any of the striking changes in skin originally cited as a characteristic feature of Ehlers-Danlos syndrome type IV. In addition, the results suggested that DNA tests for mutations in the type III procollagen gene may be useful to identify individuals predisposed to developing arterial aneurysms.     

A single base mutation that substitutes serine for glycine 790 of the alpha 1 (III) chain of type III procollagen exposes an arginine and causes Ehlers-Danlos syndrome IV

Previous observations (Stolle, C.A., Pyeritz, R.E., Myers, J.C., and Prockop, D.J. (1985) J. Biol. Chem. 260, 1937-1944) indicated that fibroblasts from a proband with dominantly inherited Ehlers-Danlos syndrome type IV synthesized type III procollagen with a structural defect near the collagenase cleavage site at amino acid 781 and near the trypsin-sensitive site at 789. The type III procollagen was unusually sensitive to proteinases and cleaved by trypsin into a three-quarter fragment at 0 degrees C. Here we demonstrate that the mutation in the type III procollagen gene is a single base mutation that converts the codon for glycine at amino acid 790 of the alpha 1(III) chain to a codon for serine. The mutation probably makes the procollagen molecule unusually sensitive to proteases because it causes local unfolding of the triple helix and exposes the adjacent arginine residue. The results provide the first indication that not all glycine substitutions in the triple helices of fibrillar collagens are equivalent in terms of their effects of the biological function of the molecule.     

Ehlers-Danlos syndrome type IV: cosegregation of the phenotype to a COL3A1 allele of type III procollagen

Summary Ehlers-Danlos syndrome (EDS) type IV is a rare and catastrophic genetic disorder of the connective tissue. Individuals from two families with this disorder were studied for a restriction fragment length polymorphism (RFLP) associated with the COL3A1 gene. Our results suggested cosegregation of the EDS type IV phenotype with a COL3A1 RFLP allele. Biochemical studies in cultured skin fibroblasts indicated the presence of different mutations affecting the stability and secretion of the pro1(III) chains of type III procollagen in the two families, thus suggesting that EDS type IV is biochemically heterogeneous. Our data demonstrated the feasibility of molecular diagnosis in this condition using COL3A1 gene related RFLPs.     

Substitution of aspartate for glycine 1018 in the type III procollagen (COL3A1) gene causes type IV Ehlers-Danlos syndrome: the mutated allele is present in most blood leukocytes of the asymptomatic and mosaic mother.

A proband with arterial ruptures and skin changes characteristic of the type IV variant of Ehlers-Danlos syndrome was found to have a single-base mutation in the type III procollagen gene, which converted the codon for glycine at amino acid position 1018 to a codon for aspartate. (Amino acid positions are numbered by the standard convention in which the first glycine of the triple-helical domain of an alpha chain is number 1. The numbers of positions in the alpha 1(III) chains can be converted to positions in the human pro alpha(III) chain by adding 167.) Nucleotide sequencing of overlapping PCR products in which the two alleles were distinguished demonstrated that the mutation of glycine 1018 was the only mutation that changed the primary structure of type III procollagen. The glycine substitution markedly decreased the amount of type III procollagen secreted into the medium by cultured skin fibroblasts from the proband. It is surprising that the same mutation was found in about 94% of the peripheral blood leukocytes from the proband's asymptomatic 72-year-old mother. Other tissues from the mother contained the mutated allele; it was present in 0%-100% of different samples of hair cells and in about 40% of cells from the oral epithelium. Therefore, the mother was a mosaic for the mutation. Since the mutated allele was present in cells derived from all three germ layers, the results indicated that the mutation arose by the late blastocyst stage of development. The results also indicate that assays of blood leukocytes do not always reveal mosaicism or predict phenotypic involvement of tissues, such as blood vessels, that are derived from the same embryonic cells as are leukocytes.     

Haploinsufficiency for one COL3A1 allele of type III procollagen results in a phenotype similar to the vascular form of Ehlers-Danlos syndrome, Ehlers-Danlos syndrome type IV

Mutations in the COL3A1 gene that encodes the chains of type III procollagen result in the vascular form of Ehlers-Danlos syndrome (EDS), EDS type IV, if they alter the sequence in the triple-helical domain. Although other fibrillar collagen-gene mutations that lead to allele instability or failure to incorporate proalpha-chains into trimers-and that thus reduce the amount of mature molecules produced-result in clinically apparent phenotypes, no such mutations have been identified in COL3A1. Furthermore, mice heterozygous for Col3a1 "null" alleles have no identified phenotype. We have now found three frameshift mutations (1832delAA, 413delC, and 555delT) that lead to premature termination codons (PTCs) in exons 27, 6, and 9, respectively, and to allele-product instability. The mRNA from each mutant allele was transcribed efficiently but rapidly degraded, presumably by the mechanisms of nonsense-mediated decay. In a fourth patient, we identified a point mutation, in the final exon, that resulted in a PTC (4294C-->T [Arg1432Ter]). In this last instance, the mRNA was stable but led to synthesis of a truncated protein that was not incorporated into mature type III procollagen molecules. In all probands, the presenting feature was vascular aneurysm or rupture. Thus, in contrast to mutations in genes that encode the dominant protein of a tissue (e.g., COL1A1 and COL2A1), in which "null" mutations result in phenotypes milder than those caused by mutations that alter protein sequence, the phenotypes produced by these mutations in COL3A1 overlap with those of the vascular form of EDS. This suggests that the major effect of many of these dominant mutations in the "minor" collagen genes may be expressed through protein deficiency rather than through incorporation of structurally altered molecules into fibrils.     

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.     

G to T transversion at position +5 of a splice donor site causes skipping of the preceding exon in the type III procollagen transcripts of a patient with Ehlers-Danlos syndrome type IV.

We identified a splicing mutation in a patient with Ehlers-Danlos syndrome type IV, a heritable connective tissue disorder associated with dysfunctions of type III collagen. The mutation was first localized in the patient's type III procollagen mRNA by amplifying the reverse transcribed product in several overlapping fragments using the polymerase chain reaction. Amplified products spanning exon 24-26 sequences displayed two distinct fragments, one of normal size and the other lacking the 99 base pairs of exon 25. Sequencing of amplified genomic products identified a G to T transversion at position +5 of the splice donor site of intron 25 in one of the patient's procollagen III genes. Expression of allelic minigene constructs correlated the T for G substitution with skipping of exon 25 sequences. Like previously characterized splicing mutations in other collagen genes, lowering the temperature at which the patient's fibroblasts were incubated nearly abolished exon skipping. As a part of this study, we also identified a highly polymorphic, intronic DNA sequence whose different allelic forms can be detected easily by the polymerase chain reaction technique.     

Synthesis of an altered type III procollagen in a patient with type IV Ehlers-Danlos syndrome. A structural change in the alpha 1(III) chain which makes the protein more susceptible to proteinases

The synthesis of type III procollagen was examined in cultured fibroblasts from ten patients with type IV Ehlers-Danlos syndrome, a heritable disorder of connective tissue. With fibroblasts from nine patients, a decreased amount of labeled type III procollagen was recovered in the medium after the cells were incubated with radioactive amino acids for 24 h. The results were compatible with undefined defects in type III procollagen. The culture medium from one patient contained apparently normal amounts of type III procollagen after a 24-h labeling. However, the pro-alpha 1(III) chains from the medium of the patient's fibroblasts appeared as an abnormally broad band when examined by gel electrophoresis in sodium dodecyl sulfate. Analysis of fragments generated by vertebrate collagenase and cyanogen bromide located a structural defect between amino acid residues 555 and 775 in half of the alpha 1(III) chains. Most of the patient's type III procollagen was susceptible to digestion by pepsin or a mixture of chymotrypsin and trypsin at temperatures at which normal type III procollagen resisted digestion. Cyanogen bromide digestion of samples of the patient's skin revealed that the amount of type III was reduced more than 4-fold. The results support the hypothesis that both normal and structurally altered pro-alpha 1(III) chains are being incorporated into type III procollagen synthesized by the patient's fibroblasts and that type III procollagen molecules containing one, two, or three structurally altered pro-alpha 1(III) chains are rapidly degraded by proteinases in the tissues.     

Incorporation of sulphate into type III procollagen by cultured human fibroblasts. Identification of tyrosine O-sulphate

Confluent cultures of normal human skin fibroblasts were labelled overnight with [35S]sulphate, and the incorporation of the isotope into type III procollagen, secreted into the medium, was verified by radioimmunoassay and immunoprecipitation after removing the heavily sulphated proteoglycans by anion-exchange chromatography. Type III procollagen and its pro and pN alpha chains were visualized in fluorographs of the immunoprecipitates. The labelled procollagen could be isolated by a combination of ion-exchange chromatography and gel filtration and was found to contain tyrosine O-sulphate, which was identified by thin-layer electrophoresis after Ba(OH)2 hydrolysis. The regions sulphated in the type III procollagen molecule were susceptible to pepsin digestion. Digestion with purified bacterial collagenase at +37 degrees C produced a labelled fragment that was recognized by antibodies against the aminoterminal propeptide of type III procollagen, indicating that the sulphated tyrosine residues are located either in this propeptide or in the non-helical telopeptide region of the type III collagen molecule proper. Sulphation of tyrosine residues is a new post-translational modification in procollagen, which could be involved in the regulation of the processing of type III procollagen into collagen and thus affect the formation of collagen fibres.     

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.     

Shape and assembly of type IV procollagen obtained from cell culture.

Type IV procollagen was isolated from the culture medium of the teratocarcinoma cell line PYS-2 by affinity chromatography on heparin-Sepharose. Immunological studies showed that type IV procollagen is composed of pro-alpha 1(IV) and pro-alpha 2(IV) chains and contains two potential cross-linking sites which are located in the short triple-helical 7S domain and the globular domain NC1 . The 7S domain was also identified as the heparin binding site. Rotary shadowing visualized type IV procollagen as a single triple-helical rod (length 388 nm) with a globule at one end. Some of the procollagen in the medium, however, had formed aggregates by alignment of 2-4 molecules along their 7S domains. After deposition in the cell matrix, non-reducible cross-links between the 7S domains are formed while the globules of two procollagen molecules connect to each other. The latter may require a slight proteolytic processing of the globular domains NC1 . The shape of type IV procollagen and the initial steps in its assembly are compatible with a recently proposed network of type IV collagen molecules in basement membranes. Since both type IV collagen and laminin bind to heparin, the formation of higher ordered structures by interaction of both proteins with heparan-sulfate proteoglycan may occur in situ.     

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.     

Vascular Ehlers-Danlos syndrome

Vascular Ehlers-Danlos syndrome, also known as Ehlers-Danlos syndrome type IV, is a life-threatening inherited disorder of connective tissue, resulting from mutations in the COL3A1 gene coding for type III procollagen. Vascular EDS causes severe fragility of connective tissues with arterial and gastrointestinal rupture, and complications of surgical and radiological interventions. As for many rare orphan diseases, delay in diagnosis is common, even when the clinical features are typical, leading to inadequate or inappropriate treatment and management. In childhood many individuals with vascular EDS are first thought to have coagulation disorders. In adulthood, four main clinical findings, including a striking facial appearance, easy bruising, translucent skin with visible veins and rupture of vessels, gravid uterus or intestines, contribute to the diagnosis, which can be confirmed by SDS-PAGE studies of type III procollagen molecules synthesis by cultured fibroblasts or by the identification of a mutation in the COL3A1 gene coding for type III procollagen. Vascular EDS is inherited as an autosomal dominant trait. Varied molecular mechanisms have been observed and, of the mutations described to date, most have been unique to each family or "private", with no correlation between genotype and phenotype. Vascular EDS is of particular importance to surgeons, radiologists, obstetricians and geneticists since, although there is currently no specific treatment for the condition, knowledge of the diagnosis may help in the management of visceral complications, pregnancy and genetic counseling.     

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.