title: Procollagen with skipping of alpha 1(I) exon 41 has lower binding affinity for alpha 1(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 alpha1(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) --> 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 alpha1(I) cDNA. The mutant transcript is translated; structurally abnormal alpha chains are demonstrated in the cell layer of proband fibroblasts by SDS-urea-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 alpha1(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.in
authors.affiliation: Section on Connective Tissue Disorders, Heritable Disorders Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Cabral
authors.forename: Andrzej
authors.initials: A
authors.lastname: Fertala
authors.forename: Laura K
authors.initials: LK
authors.lastname: Green
authors.forename: Jarmo
authors.initials: J
authors.lastname: Korkko
authors.forename: Antonella
authors.initials: A
authors.lastname: Forlino
authors.forename: Joan C
authors.initials: JC
authors.lastname: Marini
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002648:Child; D003094:Collagen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D024042:Collagen Type I; D017931:DNA Primers; D005091:Exons; D006801:Humans; D008297:Male; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011347:Procollagen / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D024042:Collagen Type I; D017931:DNA Primers; D010455:Peptides; D011347:Procollagen; D012333:RNA, Messenger; C052929:collagen type I trimeric cross-linked peptide; D003094:Collagen