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...

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Autores principales:	DaNae R. Woodard, Emi Nakahara, John D. Hulleman
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f4d1cbf2ae3f44988272f1e62004c969
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spelling	oai:doaj.org-article:f4d1cbf2ae3f44988272f1e62004c9692021-12-02T14:06:51ZClinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization10.1038/s41598-020-79570-x2045-2322https://doaj.org/article/f4d1cbf2ae3f44988272f1e62004c9692021-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-79570-xhttps://doaj.org/toc/2045-2322Abstract 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.DaNae R. WoodardEmi NakaharaJohn D. HullemanNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q DaNae R. Woodard Emi Nakahara John D. Hulleman Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization
description	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.
format	article
author	DaNae R. Woodard Emi Nakahara John D. Hulleman
author_facet	DaNae R. Woodard Emi Nakahara John D. Hulleman
author_sort	DaNae R. Woodard
title	Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization
title_short	Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization
title_full	Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization
title_fullStr	Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization
title_full_unstemmed	Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization
title_sort	clinically-identified c-terminal mutations in fibulin-3 are prone to misfolding and destabilization
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/f4d1cbf2ae3f44988272f1e62004c969
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Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization

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