Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization

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.

A loss-of-function cysteine mutant in fibulin-3 (EFEMP1) forms aberrant extracellular disulfide-linked homodimers and alters extracellular matrix composition

“…After electroporation, cells were expanded and submitted for cell sorting (UT Southwestern Flow Cytometry Core). Single colony clones were isolated, grown, and their mRNA was extracted (Aurum Total RNA Mini kit, BioRad) and analyzed for F3 expression (described previously(Woodard, Nakahara, et al, 2021)), identifying KO cells in the process. These cells were then used as the parental cells to generate the 3xFT WT F3 and 3xFT C55R F3 overexpression lines.…”

“…Human embryonic kidney cells (HEK-293A, Life Technologies, Carlsbad, CA) were cultured at 37°C with 5% CO 2 in Dulbecco’s minimal essential medium (DMEM) supplemented with high glucose, (4.5 g/L, Corning, Corning, NY, USA), 10% fetal bovine serum (FBS, Omega Scientific, Tarzana, CA, USA), and 1% penicillin-streptomycin-glutamine (Gibco, Waltham, MA, USA). Cells were plated at a density of ∼100,000 cells/well in a 24 well plate and transfected the following day with 500 ng of midi-prepped endotoxin-free plasmid DNA (Qiagen, Germantown, MD, USA) using Lipofectamine 3000 (Life Technologies) as described previously(Woodard, Nakahara, et al, 2021; Woodard, Xing, et al, 2021). Forty-eight hours after transfection, fresh serum-free media was added.…”

“…Fibulin-3 (F3, encoded by the EFEMP1 gene) is a secreted, disulfide rich, extracellular matrix (ECM) glycoprotein of unclear function that is broadly expressed throughout the body(Daniel et al, 2020; Zhang & Marmorstein, 2010). Increasing evidence suggests the involvement of F3 in a variety of diseases ranging from gliomas(Hu et al, 2011), to marfanoid syndrome(Bizzari et al, 2020; Driver et al, 2020), to primary open angle glaucoma(Mackay, Bennett, & Shiels, 2015), to age-related macular degeneration(Meyer et al, 2011), macular dystrophies(Hulleman, 2016; Stone et al, 1999), and general retinal dysfunction(Woodard, Nakahara, & Hulleman, 2021). Yet knowledge of the factors that influence F3 protein homeostasis is limited.…”

Development of a new DHFR-based destabilizing domain with enhanced basal turnover and applicability in mammalian systems

“…Individual colonies were collected and combined, and then shuttled as a mixture of DNAs into the pLenti CMV Puro DEST vector (gift from Eric Campeau and Paul Kaufman, Addgene plasmid # 17452) via an LR clonase II reaction (Invitrogen, Waltham, MA). VSV-G-pseudotyped lentivirus was generated by cotransfection of HEK-293T cells (CRL-3216, ATCC, Manassas, VA) with psPAX2 (gift from Didier Trono, Addgene plasmid #12260), pMD2.G (gift from Didier Trono, Addgene plasmid #12259), and the pLenti library as described previously 26 . To generate the ecDHFR-expressing cell library, Chinese hamster ovary cells (CHO/dhFr-, CRL-9096, ATCC) were seeded into a 10 cm dish (Costar, Corning, Corning, NY) at a density of 3 million cells/dish and allowed to attached overnight.…”

Important anatomical, age-related, and species considerations regarding ocular fibulin-3 (EFEMP1) analysis

“…Conversely, and somewhat paradoxically, increases in F3 copy number/expression have been associated with a higher chance of development of ocular diseases such as age-related macular degeneration (AMD) 11,12 , the leading cause of blindness in the elderly in industrialized nations 13 . Moreover, select autosomal dominant mutations in F3 appear to cause a range of diverse and devastating vision disorders including juvenile or primary open angle glaucoma [14][15][16] , generalized retinal degeneration 17 , and the rare AMD-like disease, Doyne honeycomb retinal dystrophy/Malattia Leventinese (DHRD/ML) 18 .…”