Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity.
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.
Li
Hongyan
H
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0641, USA.
Pajor
Ana M
AM
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D027682:Cation Transport Proteins; D005260:Female; D006031:Glycosylation; D006801:Humans; D008297:Male; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D000077003:Sodium Sulfate Cotransporter; D013552:Swine; D027981:Symporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014982:Xenopus laevis
D027682:Cation Transport Proteins; D000077003:Sodium Sulfate Cotransporter; D027981:Symporters