Abstract
Purpose :
Distinct mutations in fibulin-3 (F3), a secreted extracellular matrix glycoprotein, have been associated with various ocular diseases including Malattia Leventinese (ML, p.R345W), primary open angle glaucoma (POAG, p.R140W), and age-related macular degeneration (AMD, p.D49A). Previous research has demonstrated that the R345W mutation, which causes ML, appears to lead to protein misfolding and inefficient secretion. However, it is unclear whether other potentially pathogenic or clinically-identified F3 variants reported in the human population also share these features, which we have tested herein.
Methods :
Fifteen synonymous and missense mutations located throughout the F3 gene (EFEMP1) were identified using clinical databases. Site-directed mutagenesis was performed to generate the variants. F3 constructs were expressed in HEK293A and ARPE-19 cells and secretion/intracellular F3 levels were monitored by western blotting. Equal expression was confirmed by quantitative PCR.
Results :
Of the fifteen new variants, only a single leucine (Leu) to phenylalanine (Phe) mutation at 451 (L451F) in the C-terminal fibulin type domain caused a significant (p<0.01) secretion defect relative to WT F3 (n ≥ 6). This mutation was found in two unrelated individuals, one with retinal dystrophy and the other with nystagmus. Surprisingly, charged (Arg, Asp), aromatic (Phe, Trp, Tyr), and restrictive (Pro) residues appear to be disfavored at this 451 position, also causing secretion defects (p<0.05). Furthermore, the L451F variant relies heavily on N-linked glycosylation at Asn249 for secretion, consistent with observations made with R345W F3, suggesting that this new variant is also intrinsically unstable.
Conclusions :
Secretion defects are not a shared feature of all pathogenic or clinically-identified F3 variants. The previously identified disease-associated D49A (cuticular AMD) and R140W (POAG) F3 variants displayed no signs of misfolding. The novel L451F variant has molecular properties very similar to that of R345W, suggesting that it is likely misfolded, intrinsically unstable, and potentially contributes to ocular disease.
This is a 2021 ARVO Annual Meeting abstract.