Am J Physiol Cell Physiol 2003 Nov 01;2855:C1188-96. doi: 10.1152/ajpcell.00162.2003.

Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity.

???displayArticle.abstract???
The human Na+-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na+-sulfate cotransporter (hSUT-1) and the Na+-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide-N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn591 is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced Vmax, with no change in Km. These results suggest that Asn591 and/or N-glycosylation is critical for transport activity in NaSi-1.

???displayArticle.pubmedLink??? 12867358
???displayArticle.link??? Am J Physiol Cell Physiol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: slc13a1