PURPOSE: Specific components of transforming growth factor-beta-induced protein (TGFBIp) responsible for amyloid deposits in lattice corneal dystrophy (LCD) have not been delineated. LCD has been associated with various TGFBIp mutations such as R124C, L518P, and L527R. Using recombinant TGFBIp, this study was undertaken to identify TGFBIp components potentially contributing to the protein deposits in LCD. METHODS: Recombinant wild-type (WT) TGFBIp and four mutants (R124C, R124H, L518P, and L527R) were generated in HEK293FT cells. WT and mutant TGFBIp were collected from crude cell lysates or purified from culture media. Immunoblot analyses were performed with four different anti-TGFBIp antibodies raised against various regions of TGFBIp. RESULTS: Consistent with the authors' previous findings, purified recombinant proteins are more prone to polymerize than crude cell lysates. As expected, all monomers and polymers of TGFBIp WT and mutants were detected by these antibodies. However, the authors noted WT and TGFBIp mutants showed differential reactivities with these antibodies. A 47-kDa band was detected in purified 2-tag proteins of L518P by all four antibodies. A unique 43-kDa band was detected in both 1-tag cell lysates and purified proteins of R124C by the authors' custom-made antibody (KE50) and a commercial anti-TGFBIp. CONCLUSIONS: Based on its universal reactivity with various antibodies, the authors surmise that the 47-kDa protein is a ubiquitous TGFBIp fragment derived from the N-terminus of the L518P mutant. The fact that the 43-kDa protein fragment was present primarily in R124C and R124H but not in WT implicates its potential role in the protein deposits of LCD.
PURPOSE: Specific components of transforming growth factor-beta-induced protein (TGFBIp) responsible for amyloid deposits in lattice corneal dystrophy (LCD) have not been delineated. LCD has been associated with various TGFBIp mutations such as R124C, L518P, and L527R. Using recombinant TGFBIp, this study was undertaken to identify TGFBIp components potentially contributing to the protein deposits in LCD. METHODS: Recombinant wild-type (WT) TGFBIp and four mutants (R124C, R124H, L518P, and L527R) were generated in HEK293FT cells. WT and mutant TGFBIp were collected from crude cell lysates or purified from culture media. Immunoblot analyses were performed with four different anti-TGFBIp antibodies raised against various regions of TGFBIp. RESULTS: Consistent with the authors' previous findings, purified recombinant proteins are more prone to polymerize than crude cell lysates. As expected, all monomers and polymers of TGFBIp WT and mutants were detected by these antibodies. However, the authors noted WT and TGFBIp mutants showed differential reactivities with these antibodies. A 47-kDa band was detected in purified 2-tag proteins of L518P by all four antibodies. A unique 43-kDa band was detected in both 1-tag cell lysates and purified proteins of R124C by the authors' custom-made antibody (KE50) and a commercial anti-TGFBIp. CONCLUSIONS: Based on its universal reactivity with various antibodies, the authors surmise that the 47-kDa protein is a ubiquitous TGFBIp fragment derived from the N-terminus of the L518P mutant. The fact that the 43-kDa protein fragment was present primarily in R124C and R124H but not in WT implicates its potential role in the protein deposits of LCD.
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