Abstract
Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.
Highlights
Protein misfolding is associated with human disorders, collectively known as “protein deposition diseases” [1] or “protein misfolding diseases” [2]
In this study we have investigated the stability of WT TGFBIp, three different mutants of the fasciclin 1 (FAS1)-1 domain (R124H, R124L, R124C) and three FAS1-4 mutants (A546T, R555Q, and R555W)
The TGFBIp variants were initially analyzed by Transverse Urea Gradient (TUG) gel electrophoresis, revealing that the overall stability of intact TGFBIp was affected only by amino acid substitutions within the FAS1-4 domain, whereas changes in the FAS1-1 failed to change the stability
Summary
Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. Amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation. More than 30 different mutations in the TGFBI gene have been reported to result in corneal protein deposits [21]. This study addresses the stability changes in TGFBIp brought about by amino acid substitutions associated with different types of corneal dystrophies.
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