Procollagen with Skipping of α1(I) Exon 41 Has Lower Binding Affinity for α1(I) C-telopeptide, Impaired in Vitro Fibrillogenesis, and Altered Fibril Morphology

Abstract

Previous in vitro data on type I collagen self-assembly into fibrils suggested that the amino acid 776 -796 region of the ␣1(I) chain is crucial for fibril formation because it serves as the recognition site for the telopeptide of a docking collagen monomer. We used a natural collagen mutation with a deletion of amino acids 766 -801 to confirm the importance of this region for collagen fibril formation. The proband has type III osteogenesis imperfecta and is heterozygous for a COL1A1 IVS 41 A ؉4 3 C substitution. The intronic mutation causes splicing of exon 41, confirmed by sequencing of normal and shorter reverse transcriptase-PCR products. Reverse transcriptase-PCR using RNA from proband dermal fibroblasts and clonal cell lines showed the mutant cDNA was about 15% of total ␣1(I) cDNA. The mutant transcript is translated; structurally abnormal ␣ chains are demonstrated in the cell layer of proband fibroblasts by SDSurea-PAGE. The proportion of mutant chains in the secreted procollagen was determined to be 10% by resistance to digestion with MMP-1, since chains lacking exon 41 are missing the vertebral collagenase cleavage site. Secreted proband collagen was used for analysis of kinetics of binding of ␣1(I) C-telopeptide using an optical biosensor. Telopeptide had slower association and faster dissociation from proband than from normal collagen. Purified proband pC-collagen was used to study fibril formation. The presence of the mutant molecules decreases the rate of fibril formation. The fibrils formed in the presence of 10 -15% mutant molecules have strikingly increased length compared with normal collagen, but are well organized, as demonstrated by D-periodicity. These results suggest that some collagen molecules containing the mutant chain are incorporated into fibrils and that the absence of the telopeptide binding region from even a small portion of the monomers interferes with fibril growth. Both abnormal fibrils and slower remodeling may contribute to the severe phenotype.Type I collagen is the predominant protein in the extracellular matrix of skin, bone, and tendon. In tissue, the type I collagen heterotrimer is organized into heterotypic fibrils along with types III and V collagen and non-collagenous proteins of matrix, such as decorin (1-4). Collagen fibrils in a wide range of different tissues have distinct diameter distributions (5), supporting a process for strict control over fibril growth and assembly. Structural abnormalities of type I collagen are the cause of significant medical disorders such as osteogenesis imperfecta, a genetic disorder of connective tissue characterized by bone fragility, and two of the types of Ehlers-Danlos

Correction of a mineralization defect by overexpression of a wild-type cDNA for COL1A1 in marrow stromal cells (MSCs) from a patient with osteogenesis imperfecta: a strategy for rescuing mutations that produce dominant-negative protein defects

“…No distinctive band of abnormal proa1 chains was detected (Figure 4). The failure to detect the abnormal protein band in the MSCs was consistent with the previous failure to detect abnormal proa1 chains in cultures of the proband's osteoblasts 9 and is probably explained by the previous observation that there are discrepancies in the levels at which mutated proa chains were detected in different cells from the same patients. 15 To assay the synthesis of type I procollagen by the MSCs, the levels of 3 H-proa chains secreted into the medium were compared (Figure 4).…”

“…The low level of the abnormally spliced mRNA was consistent with the previous report that the abnormally spliced mRNA was only about 15% of the COL1A1 transcripts in fibroblasts from the patient. 9 With RNA from the patient's transfected MSCs, the RT-PCR assays generated the same two bands, but there was an increase in the ratio of the 384 bp to the 276 bp band, indicating that there was overexpression of COL1A1 transcripts for the hybrid genomic/cDNA construct ( Figure 1). Correction of a mineralization defect by overexpression of cDNA RR Pochampally et al…”

“…One set of primers corresponded to the coding sequences found in exons 40 and 43 and were designed to amplify the small amount of aberrantly spliced mRNA lacking the sequences of exon 41 that was detected in the patient's fibroblasts. 9 With RNA from the patient's untransfected MSCs, the primers generated a major fragment of 384 bp from mRNA transcribed from the normal allele of the patient and the transfected cDNA ( Figure 3). There was also a minor fragment of 276 bp corresponding to the low levels of an mRNA lacking the coding sequence of exon 41.…”

“…[4][5][6]9 The protocol for obtaining MSCs from healthy donors was approved by the Institutional Review Board of the Tulane University Health Sciences Center and the protocol for obtaining MSCs from the patient was approved by the Institutional Review Board of the St Jude Children's Research Hospital. The MSCs were obtained from the patient before the patient was transplanted with cells transfected with a neomycin resistance gene.…”

“…[4][5][6] The patient's mutation altered an RNA splice site IVS 41A +4 C in the COL1A1 gene and generated a small amount of abnormal mRNA and proa1 chains lacking the coding sequences of exon 41. 9 The mutation was unusual among mutations producing severe phenotypes because only low levels of abnormal proa chains were detected in cultured fibroblasts and no abnormal proa chains were detected in cultures of osteoblasts from the same patient.…”