ENDOPLASMIC RETICULUM-MEDIATED QUALITY-CONTROL OF TYPE-I COLLAGEN PRODUCTION BY CELLS FROM OSTEOGENESIS IMPERFECTA PATIENTS WITH MUTATIONS IN THE PRO-ALPHA-1(I) CHAIN CARBOXYL-TERMINAL PROPEPTIDE WHICH IMPAIR SUBUNIT ASSEMBLY

A heterozygous single base change in exon 49 of COL1A1, which converte d 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 ri,he 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 no rmal pro alpha 1(I) and pro alpha 2(I) to form pepsin-resistant triple -helical molecules, a proportion of which were secreted. Further evide nce 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, w as specifically bound to type I procollagen and was coimmunoprecipitat ed in the osteogenesis imperfecta cells but not control cells. Experim ents with brefeldin A, which inhibits protein export from the endoplas mic reticulum, demonstrated that unassembled mutant pro alpha 1(I) cha ins were selectively degraded within the endoplasmic reticulum resulti ng 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 wh en grown in the continuous presence of ascorbic acid to allow collagen matrix formation. Both these carboxyl-terminal propeptide mutants sho wed a marked reduction in collagen accumulation to 20% (or less) of co ntrol cultures, comparable to the reduced collagen content of tissues from OI26.