Purpose: Types 1 and 2 granular corneal dystrophies (GCD) are primarily associated with accumulation of the R555W and R124H mutant transforming growth factor β-inducible proteins (TGFBIp) in corneal stroma, respectively. However, specific components of TGFBIp responsible for granular deposits have not been delineated. This study was undertaken to identify the mutant TGFBIp components potentially responsible for GCD.
Methods: Recombinant TGFBIp of wild-type (WT) and three mutants, R124C, R124H, and R555W, were generated in HEK293FT cells. WT and TGFBIp mutants were collected from cell lysates. Immunoblot analyses were performed with five different antibodies directed against various regions of WT TGFBIp.
Results: WT and TGFBIp mutants showed differential reactivities with these antibodies. In contrast to our prior observation in purified WT and TGFBIp mutants, TGFBIp from cell lysates were less prone to polymerize. A unique 35 kD fragment was detected in cell lysates of R555W and R124H, but not in those of WT or R124C, by a commercial antibody raised against amino acids (a.a.) 199-406 of TGFBIp.
Conclusions: Monomeric and polymeric WT and TGFBIp mutants were observed in vitro. The 35 kD fragment found only in R555W and R124H, but not in WT and R124C cell lysates, is likely a degraded TGFBIp derived from the central domain of these mutants and this fragment may be contributory to the nonamyloid granular deposits observed in GCD 1 and 2.