PMID: 22159084

 

    Legend: Sugar

Title : Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3

Abstract :

1. SLC26A3 is a Cl(-)/HCO(3)(-) exchanger that plays a major role in Cl(-) absorption from the intestine
2. Its mutation causes congenital chloride-losing diarrhea
3. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function
4. However, the role of glycosylation has yet to be clearly determined
5. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation
6. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates.
7. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity
8. To localize glycosylation sites , we mutated the five consensus sites by replacing asparagine (N) with glutamine
9. Immnoblotting suggests that SLC26A3 is glycosylated at N153 , N161, and N165
10. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression
11. We also assessed whether SLC26A3 is protected from tryptic digestion
12. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin , deglycosylated SLC26A3 exhibited increased susceptibility to trypsin , suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion
13. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation