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Am J Physiol Cell Physiol (July 16, 2003). doi:10.1152/ajpcell.00162.2003
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Submitted on April 24, 2003
Accepted on July 10, 2003

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

Hongyan Li1 and Ana M Pajor1*

1 Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX, USA

* To whom correspondence should be addressed. E-mail: ampajor{at}utmb.edu.

The human Na+/sulfate cotransporter, hNaSi-1, belongs to the SLC 13 gene family that 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 (called 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. The wild-type and two N-glycosylation site mutants, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by PNGase F treatment in contrast to the mass of the wild-type hNaSi-1 which was reduced by up to 15 kDa, indicating that Asn-591 is the N-glycosylation site. Although the cell-surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of the wild-type, both mutants had greatly reduced Vmax with no change in Km. These results suggest that Asn-591 and/or N-glycosylation are critical for transport activity in NaSi-1.




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