Abstract
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.
Highlights
A Substitution of Cysteine for Glycine748 of the 01 Chain Produces a Kink at This Site inthe ProcollagenI Molecule and an Altered N-Proteinase Cleavage Site over 225 nm Away*
14737-14744), we identified a single-base mutation six others the in-frame deletions of amino acids were caused that converted the glycine at position 748 of the a1 by exon skipping during RNA splicing (8, 9, 12)
An unexplained finding was that procollagen was poorly processed to pccollagen in postconfluentcultures of skin fibroblasts
Summary
The torsional backbone angles 4 (N-Cor) and P (Ca-CO) in the regular triple-helical regionwere adjusted to standard values of Fraser et al (28). Coordinates of this standard triple helix were kindly provided by A. All amide bonds were fixed in a planartrans-configuration.Torsional angles in the region of the defect were measured on the Kendrew model. Proteinase-Procollagen I was purified from the medium of cultures of t h e proband's fibroblasts. T h e procollagen and purified procollagen N-proteinase was used to test the cleavage rate of procollagen I synthesized by the proband's fibroblasts Becausoef differences in electrophoretic mobility, we were able to discriminate betweenthe overmodified speciesof proal(1) chains and the normal proal(1) chains in the same sample (Fig. 1)
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