Linking Transforming Growth Factor Beta Induced Protein (TGFBIp) Turnover to the TGFBI-Associated Corneal Dystrophies

Abstract

Purpose: : Numerous point mutations in the TGFBI gene have been linked to corneal dystrophies, characterized by an extracellular deposition of TGFBIp. In the present study the turnover of wild-type and mutant TGFBIp was investigated in human corneal tissue and in vitro.

Methods: : Human corneal tissue was analyzed using 2D gel electrophoresis (2DGE) followed by Western blotting using an anti-TGFBIp antibody. Limited proteolysis of purified will type and mutant TGFBIp was performed by titrating a fixed concentration of TGFBIp with increasing amounts different proteases. The proteolytic fragments were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed by mass spectrometry (MS) and Edman degradation.

Results: : Visualization of TGFBIp derived from healthy human corneas following 2DGE revealed a distinct pattern of distribution indicating that TGFBIp is degraded in an orchestrated fashion. This pattern was observed in all fully developed corneas (from individuals of ages 2-80 years).Protein turnover was further investigated in vitro by digesting purified TGFBIp using MMPs. These results showed that particular physiologically relevant MMPs preferred to proteolyze wild-type TGFBIp while the cleavage of mutant TGFBIp was retarded. Differences in the fragmentation pattern of wild type and mutant TGFBIp indicated that the mutations altered the gross fold of TGFBIp. The limited proteolysis data also showed that particular proteases produce a fragment resistant to further proteolysis.

Conclusions: : The distribution pattern of TGFBIp following separation of normal human cornea proteins by 2DGE indicated that specific proteolytic events are taking place during normal TGFBIp turnover. Specifically a sequential trimming of the N-terminal was observed. In addition, corneal MMPs were found to preferentially cleave wild-type TGFBIp while the naturally occurring mutants resisted proteolysis suggesting that specific MMP-catalyzed proteolytic events are involved in the characteristic histopathology of the corneal dystrophies. Comparison of wild-type versus mutant TGFBIp by limited proteolysis indicated that differences in the overall structures might be the cause of this.

Keywords: cornea: basic science • protein structure/function • mutations