期刊
FASEB JOURNAL
卷 29, 期 2, 页码 565-575出版社
FEDERATION AMER SOC EXP BIOL
DOI: 10.1096/fj.14-255414
关键词
EFEMP1; endoplasmic reticulum; malattia leventinese; unfolded protein response
资金
- NCRR NIH HHS [UL1 RR025774, RR025774] Funding Source: Medline
- NEI NIH HHS [EY020799, P30 EY020799] Funding Source: Medline
- NIA NIH HHS [R01 AG018917, AG018917] Funding Source: Medline
An R345W mutation in the N- glycoprotein, fibulin- 3 ( F3), results in inefficient F3 folding/ secretion and higher intracellular F3 levels. Inheritance of this mutation causes the retinal dystrophy malattia leventinese. N- Linked glycosylation is a common cotranslational protein modification that can regulate protein folding efficiency and energetics. Therefore, we explored how N- glycosylation alters the protein homeostasis or proteostasis of wild- type ( WT) and R345W F3 in ARPE- 19 cells. Enzymatic and lectin binding assays confirmed that WT and R345W F3 are both primarily N- glycosylated at Asn249. Tunicamycin treatment selectively reduced R345W F3 secretion by 87% ( vs. WTF3). Genetic elimination of F3 N- glycosylation ( via an N249Q mutation) caused R345W F3 to aggregate intracellularly and adopt an altered secreted conformation. The endoplasmic reticulum ( ER) chaperones GRP78 ( glucose- regulated protein 78) and GRP94 ( glucose- regulated protein 94), and the ER lectins calnexin and calreticulin were identified as F3 binding partners by immunoprecipitation. Significantly more N249Q and N249Q/ R345W F3 interacted with GRP94, while substantially lessN249Q andN249Q/ R345W interacted with the ER lectins than their N- glycosylated counterparts. Inhibition of GRP94 ATPase activity reduced only N249Q/ R345W F3 secretion ( by 62%), demonstrating this variant's unique reliance on GRP94 for secretion. These observations suggest that R345W F3, but not WT F3, requires N- glycosylation to acquire a stable, native- like structure.-
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