Abstract
Distinct mutations in the secreted extracellular matrix protein, fibulin-3 (F3), have been associated with a number of ocular diseases ranging from primary open angle glaucoma to cuticular age-related macular degeneration to a rare macular dystrophy, Malattia Leventinese (ML). The R345W F3 mutation that causes ML leads to F3 misfolding, inefficient secretion and accumulation at higher intracellular steady state levels in cultured cells. Herein, we determined whether fifteen other clinically-identified F3 mutations also led to similar levels of misfolding and secretion defects, which might provide insight into their potential pathogenicity. Surprisingly, we found that only a single F3 variant, L451F, presented with a significant secretion defect (69.5 ± 2.4% of wild-type (WT) F3 levels) and a corresponding increase in intracellular levels (226.8 ± 25.4% of WT F3 levels). Upon follow-up studies, when this conserved residue (L451) was mutated to a charged (Asp or Arg) or bulky (Pro, Trp, Tyr) residue, F3 secretion was also compromised, indicating the importance of small side chains (Leu, Ala, or Gly) at this residue. To uncover potential inherent F3 instability not easily observed under typical culture conditions, we genetically eliminated the sole stabilizing N-linked glycosylation site (N249) from select clinically-identified F3 mutants. This removal exacerbated R345W and L451F secretion defects (19.8 ± 3.0% and 12.4 ± 1.2% of WT F3 levels, respectively), but also revealed a previously undiscovered secretion defect in another C-terminal variant, Y397H (42.0 ± 10.1% of WT F3 levels). Yet, glycan removal did not change the relative secretion of the N-terminal mutants tested (D49A, R140W, I220F). These results highlight the uniqueness and molecular similarities between the R345W and L451F variants and also suggest that previously identified disease-associated mutations (e.g., R140W) are indistinguishable from WT with respect to secretion, hinting that they may lead to disease by an alternative mechanism.
Highlights
Distinct mutations in the secreted extracellular matrix protein, fibulin-3 (F3), have been associated with a number of ocular diseases ranging from primary open angle glaucoma to cuticular agerelated macular degeneration to a rare macular dystrophy, Malattia Leventinese (ML)
Age-related macular degeneration (AMD) is an etiologically complex disease due to a variety of genetic and environmental risk factors[15,16,17], insight into its pathogenesis can be gained by studying phenotypically similar, early-onset monogenic macular diseases. One such disease is Malattia Leventinese (ML), a rare macular dystrophy caused by an autosomal dominant Arg345Trp (R345W) mutation in the fibulin-3 (F3) protein, wherein patients develop AMD-like symptoms including drusen formation as early as 20 years of a ge[18,19]
We found that only R345W and L451F F3 displayed a significant secretion propensity defect compared to WT F3
Summary
Distinct mutations in the secreted extracellular matrix protein, fibulin-3 (F3), have been associated with a number of ocular diseases ranging from primary open angle glaucoma to cuticular agerelated macular degeneration to a rare macular dystrophy, Malattia Leventinese (ML). AMD is an etiologically complex disease due to a variety of genetic and environmental risk factors[15,16,17], insight into its pathogenesis can be gained by studying phenotypically similar, early-onset monogenic macular diseases One such disease is Malattia Leventinese (ML), a rare macular dystrophy caused by an autosomal dominant Arg345Trp (R345W) mutation in the fibulin-3 (F3) protein, wherein patients develop AMD-like symptoms including drusen formation as early as 20 years of a ge[18,19]. C-terminus fibulin domain the EFEMP1 promoter (the gene that encodes for F3) were linked to increased risk for A MD21, and (3) a sequence variant, Asp49Ala (D49A), was discovered in a patient with cuticular drusen, a clinical subtype of A MD22 The combination of these findings strongly suggests that both mutant and WT F3 may play an important role in the development of AMD or AMD-like retinal d ystrophies[23]. Additional mutations have been identified in the EFEMP1 gene in patients with ocular disorders (information obtained through C linVar35), but the clinical significance of these genetic alterations is unclear
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