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11:25
Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
Published on: October 4, 2017
N-Glycosylation is required for Na+-dependent vitamin C transporter functionality
Veedamali S Subramanian1, Jonathan S Marchant, Jack C Reidling
1Departments of Medicine, Physiology and Biophysics, University of California, Irvine, CA 92697, USA. vsubrama@uci.edu
Biochemical and Biophysical Research Communications
|July 16, 2008
Summary
N-glycosylation is crucial for the function of human sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2). Removing N-glycosylation sites impairs their ability to transport ascorbic acid.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Human sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2) are essential for cellular ascorbic acid uptake.
- Potential N-glycosylation sites exist in the extracellular domains of hSVCT1 and hSVCT2, but their functional significance remains unknown.
Purpose of the Study:
- To investigate the role of N-glycosylation in the function, expression, and cellular targeting of hSVCT1 and hSVCT2.
- To determine if N-glycosylation is essential for ascorbic acid transport mediated by these transporters.
Main Methods:
- Systematic ablation of consensus N-glycosylation sites in hSVCT1 and hSVCT2 using site-directed mutagenesis.
- Assessment of ascorbic acid uptake using (14)C-ascorbic acid.
- Analysis of transporter expression and cellular localization via Western blotting and cellular fractionation.
Main Results:
- Tunicamycin treatment reduced (14)C-ascorbic acid uptake in HepG2 cells, suggesting a role for N-glycosylation.
- Mutagenesis of individual N-glycosylation sites significantly impaired protein expression and ascorbic acid uptake for both hSVCT1 and hSVCT2.
- One hSVCT1 mutant (N138Q) was retained intracellularly, while all hSVCT2 mutants reached the cell surface, indicating differential effects on targeting.
Conclusions:
- N-glycosylation is essential for the proper expression and functionality of human vitamin C transporters.
- Disruption of N-glycosylation significantly impacts ascorbic acid uptake, highlighting its critical role in transporter activity.
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