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Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
Published on: October 4, 2017
Glycosylation regulates prestin cellular activity.
Lavanya Rajagopalan1, Louise E Organ-Darling, Haiying Liu
1Bobby R. Alford Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. lrajagop@bcm.edu
Journal of the Association for Research in Otolaryngology : JARO
|November 10, 2009
Summary
Glycosylation regulates prestin oligomerization and membrane trafficking, impacting auditory function. This study reveals how N-glycosylation affects prestin
Area of Science:
- Biochemistry
- Cell Biology
- Auditory Neuroscience
Background:
- Glycosylation is a key post-translational modification influencing protein function.
- Prestin, crucial for auditory transduction in outer hair cells, has known N-glycosylation sites.
- Previous studies showed minor effects of N-glycosylation site mutations on prestin function.
Purpose of the Study:
- To investigate the role of prestin N-glycosylation in protein oligomerization, membrane trafficking, and nonlinear capacitance (NLC) function.
- To determine if glycosylation is essential for NLC generation.
- To elucidate the impact of cholesterol on glycosylation-deficient prestin function.
Main Methods:
- Site-directed mutagenesis to create a double N-glycosylation mutant prestin (prestin(NN163/166AA)).
- HEK 293 cell culture and transfection with WT or mutant prestin.
- Nonlinear capacitance (NLC) measurements under varying cholesterol conditions.
- Analysis of prestin oligomerization, membrane mobility, and cell-surface expression.
Main Results:
- The prestin(NN163/166AA) mutant is not glycosylated and exhibits normal NLC in untreated/cholesterol-depleted cells.
- Unlike WT prestin, the mutant prestin is enriched as monomers and shows increased plasma membrane mobility.
- In cholesterol-rich conditions, prestin(NN163/166AA) loses NLC function, showing decreased cell-surface expression, unlike WT prestin.
Conclusions:
- Prestin glycosylation regulates its self-association (oligomerization) and cellular trafficking.
- Glycosylation is not essential for NLC generation but is critical for proper function under cholesterol-rich conditions.
- Cholesterol-dependent trafficking, potentially via clathrin- and caveolin-mediated pathways, influences prestin localization and function.
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