DEPOSITION AND SELECTIVE DEGRADATION OF STRUCTURALLY-ABNORMAL TYPE-1 COLLAGEN IN A COLLAGEN MATRIX PRODUCED BY OSTEOGENESIS IMPERFECTA FIBROBLASTS IN-VITRO

Collagen matrix deposition and turnover were studied in skin fibroblas ts from a control and from a patient with lethal perinatal osteogenesi s imperfecta (OI) identified as a Gly(667) to Arg substitution in the alpha 1(I) chain. A culture system where ascorbic acid was included to stimulate collagen matrix formation over extended culture periods was used. Serial extraction of the control cell collagen matrix confirmed that a substantial mature crosslinked collagen matrix was formed in t he control fibroblast cell layer. In contrast, total collagen depositi on by the OI fibroblasts was poor, with the quantity of collagen depos ited only about a quarter of that of the control cells. Detailed analy sis of the OI fibroblast matrix revealed that the mutant collagen chai ns were incorporated into the collagenous matrix. These data indicate that, when grown with ascorbate in long-term culture, OI fibroblasts r eproduced the abnormal matrix deposition pattern of OI tissues in vivo . The overall dramatic reduction in collagen matrix formation was not accounted for by reduced collagen production, since during the period of matrix deposition (days 8-12) the rate of production by the OI cell s was only slightly less than that of the control cells. The incorpora tion of the newly-synthesized OI collagen into the matrix was less eff icient than in control cells, reflecting the cooperative nature of mat rix deposition. The fate of this mutant collagen containing the Gly to Arg charge-change was followed in the matrix by a pulse-chase experim ent and two-dimensional electrophoresis. These data demonstrated that the mutant incorporated into the matrix was unstable, with the proport ion of mutant declining during the chase. The deposition of the mutant monomers into a pool more accessible to proteolytic degradation indic ated that the mutant and normal collagens did not copolymerize to form collagen fibers of even collagen distribution, but rather the mutant collagen was either enriched on the exposed surfaces of mixed-composit ion fibers, or was unable to form copolymers efficiently and polymeriz ed into mutant-only fibrillar assemblies more prone to proteolytic att ack.