Endoplasmic Reticulum-mediated Quality Control of Type I Collagen Production by Cells from Osteogenesis Imperfecta Patients with Mutations in the proα1(I) Chain Carboxyl-terminal Propeptide which Impair Subunit Assembly

Abstract

A heterozygous single base change in exon 49 of COL1A1, which converted the codon for pro alpha 1(I) carboxyl-terminal propeptide residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a baby with lethal osteogenesis imperfecta (OI64). The C-propeptide mutations in OI64 and in another lethal osteogenesis imperfecta cell strain (OI26), which has a frameshift mutation altering the sequence of the carboxyl-terminal half of the propeptide (Bateman, J. F., Lamande, S. R., Dahl, H.-H. M., Chan, D., Mascara, T. and Cole, W. G. (1989) J. Biol. Chem. 264, 10960-10964), disturbed procollagen folding and retarded the formation of disulfide-linked trimers. Although assembly was delayed, the presence of slowly migrating, overmodified alpha 1(I) and alpha 2(I) chains indicated that mutant pro alpha 1(I) could associate with normal pro alpha 1(I) and pro alpha 2(I) to form pepsin-resistant triple-helical molecules, a proportion of which were secreted. Further evidence of the aberrant folding of mutant procollagen in OI64 and OI26 was provided by experiments demonstrating that the endoplasmic reticulum resident molecular chaperone BiP, which binds to malfolded proteins, was specifically bound to type I procollagen and was coimmunoprecipitated in the osteogenesis imperfecta cells but not control cells. Experiments with brefeldin A, which inhibits protein export from the endoplasmic reticulum, demonstrated that unassembled mutant pro alpha 1(I) chains were selectively degraded within the endoplasmic reticulum resulting in reduced collagen production by the osteogenesis imperfecta cells. This biosynthetic deficiency was reflected in the inability of OI64 and OI26 cells to produce a substantial in vitro collagenous matrix when grown in the continuous presence of ascorbic acid to allow collagen matrix formation. Both these carboxyl-terminal propeptide mutants showed a marked reduction in collagen accumulation to 20% (or less) of control cultures, comparable to the reduced collagen content of tissues from OI26.

Trimerization of the Amino Propeptide of Type IIA Procollagen Using a 14-Amino Acid Sequence Derived from the Coiled-Coil Neck Domain of Surfactant Protein D

“…Trimerization was also more efficient, requiring less cross-linker than for the other truncation mutants. We speculate that this "all-or-none" cross-linking of mIIA-237 results from the presence of two contiguous sites for BS 3 crosslinking at Lys 229 -Lys 230 within the fourth heptad repeat. Although this seems at odds with the observation that crosslinking of IIA/SP-D also proceeds through a dimeric intermediate, the three chains may not be within an equivalent environment as compared with the context of the intact neck ϩ CRD domain.…”

“…Increasing amounts of BS 3 (0, 0.1, 0.5, 1, and 2 mM final concentration) prepared in 5 mM sodium citrate, pH 5, were added to each recombinant protein for 1 h at room temperature. Addition of SDS-PAGE loading buffer containing Tris-HCl (0.5 M) inhibited the reaction.…”

“…Precipitated proteins were washed three times in saturated ammonium sulfate, resuspended in PBS, and dialyzed overnight in cold PBS. The collagen deletion mutant protein (mIIA-coll-218) was crosslinked using BS 3 and detected by SDS-PAGE and Western blotting using the anti-IIA polyclonal antibody.…”

“…Trimerization of most fibrillar collagens is dependent on the globular COOH-propeptide for the recognition and association of the three polypeptide chains resulting in registered nucleation of triple helix formation in a zipper-like fashion from the C to N terminus (3)(4)(5)(6)(7). Functions proposed for procollagen NH 2 -propeptides include the regulation of collagen fibrillogenesis (8) and a feedback control of net collagen biosynthesis (9 -11).…”

Folding defects in fibrillar collagens

“…These mutations lead to activation of the unfolded protein response pathway. In cells that carry many of these mutations the stress^response proteins BiP (GRP78) and GRP94 are synthesized at high levels and appear to interact with the abnormal proteins, perhaps directing them to sites of degradation as they are often short lived (Chessler & Byers 1993;Lamande et al 1995). These abnormal molecules may also be subject to degradation through the proteosome (Fitzgerald et al 1999).…”