The role of N-linked glycosylation in protein folding, membrane targeting, and substrate binding of human organic anion transporter hOAT4.

We used a novel approach to evaluate how the addition/acquisition and processing/modification of N-linked oligosaccharides play a role in the functional maturation of human organic anion transporter hOAT4. Inhibition of acquisition of oligosaccharides in hOAT4 by mutating asparagine to glutamine and by tunicamycin treatment was combined with the expression of wild-type hOAT4 in a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing. We showed that both the disruption of the glycosylation sites by mutagenesis and the inhibition of glycosylation by tunicamycin treatment resulted in a nonglycosylated hOAT4, which was unable to target to the cell surface. In contrast, hOAT4 synthesized in mutant CHO-Lec cells, carrying different structural forms of sugar moieties (mannose-rich in Lec1 cells, sialic acid-deficient in Lec2 cells, and sialic acid/galactose-deficient in Lec8 cells) were able to traffic to the cell surface. However, hOAT4 expressed in CHO-Lec1 cells had significantly lower binding affinity for its substrates compared with that expressed in parental CHO cells. This study provided novel information that addition/acquisition of oligosaccharides but not the processing of the added oligosaccharides participates in the membrane insertion of hOAT4. Processing of added oligosaccharides from mannose-rich type to complex type is important for enhancing the binding affinity of hOAT4 for its substrates. Glycosylation could therefore serve as a means to specifically regulate hOAT4 function in vivo.