Abstract
Purpose. The sodium-dependent, purine-selective nucleoside transporter, SPNT, has a unique steady-state expression pattern in renal epithelial cells. In comparison with the concentrative nucleoside transporter, CNT1, which is confined to the apical membrane, SPNT is expressed predominantly on the apical membrane but with significant expression on the basolateral membrane as well. Alternate surface expression indicates that SPNT likely has different sorting and trafficking mechanisms from CNT1. Because glycosylation has been reported to be essential for apical targeting of other transporters, and SPNT contains three unique glycosylation sites, we examined the importance of glycosylation in sorting of SPNT. Preliminary studies suggested that glycosylation affects surface expression of SPNT but not CNT1.
Methods. All three unique glycosylation sites were mutated alone and in tandem. Wild-type and mutant SPNT, tagged with green fluorescence protein, were stably transfected into MDCK. Positive clones were assayed for polarized surface expression by immunofluorescence and functional analysis.
Results. Mutation at all three sites alone or in tandem resulted in functional proteins. Removal of sites N606 and N625 resulted in proteins of reduced molecular mass. None of the unglycosylated mutants localized differently than wild-type SPNT.
Conclusion. N-linked glycosylation is not essential for polarized sorting.
This is a preview of subscription content, log in via an institution to check access.
REFERENCES
M. Che, D. F. Ortiz, and I. M. Arias. Primary structure and functional expression of a cDNA encoding the bile canalicular, purine-specific Na(+)-nucleoside cotransporter. J. Biol. Chem. 270:13596-13599 (1995).
Q. Q. Huang, S. Y. Yao, M. W. Ritzel, A. R. Paterson, C. E. Cass, and J. D. Young. Cloning and functional expression of a complementary DNA encoding a mammalian nucleoside transport protein. J. Biol. Chem. 269:17757-17760 (1994).
M. W. Ritzel, A. M. Ng, S. Y. Yao, K. Graham, S. K. Loewen, K. M. Smith, R. G. Ritzel, D. A. Mowles, P. Carpenter, X. Z. Chen, E. Karpinski, R. J. Hyde, S. A. Baldwin, C. E. Cass, and J. D. Young. Molecular identification and characterization of novel human and mouse concentrative Na+-nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). J. Biol. Chem. 276:2914-2927 (2000).
M. Pennycooke, N. Chaudary, I. Shuralyova, Y. Zhang, and I. R. Coe. Differential expression of human nucleoside transporters in normal and tumor tissue. Biochem. Biophys. Res. Commun. 280:951-959 (2001).
L. M. Mangravite, J. H. Lipshutz, K. E. Mostov, and K. M. Giacomini. Localization of GFP-tagged concentrative nucleoside transporters in a renal polarized epithelial cell line. Am. J. Physiol. Renal Physiol. 280:F879-F885 (2001).
R. Martinez-Maza, I. Poyatos, B. Lopez-Corcuera, N. u. E, C. Gimenez, F. Zafra, and C. Aragon. The role of N-glycosylation in transport to the plasma membrane and sorting of the neuronal glycine transporter GLYT2. J. Biol. Chem. 276:2168-2173 (2001).
K. Petrecca, R. Atanasiu, A. Akhavan, and A. Shrier. N-linked glycosylation sites determine HERG channel surface membrane expression. J. Physiol. 515:41-48 (1999).
S. R. Hamilton, S. Y. Yao, J. C. Ingram, D. A. Hadden, M. W. Ritzel, M. P. Gallagher, P. J. Henderson, C. E. Cass, J. D. Young, and S. A. Baldwin. Subcellular distribution and membrane topology of the mammalian concentrative Na+-nucleoside cotransporter rCNT1. J. Biol. Chem. 276:27981-27988 (2001).
A. Felipe, R. Valdes, B. Santo, J. Lloberas, J. Casado, and M. Pastor-Anglada. Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells. Biochem. J. 330:997-1001 (1998).
C. Saunders, J. R. Keefer, A. P. Kennedy, J. N. Wells, and L. E. Limbird. Receptors coupled to pertussis toxin-sensitive G-proteins traffic to opposite surfaces in Madin-Darby canine kidney cells. A1 adenosine receptors achieve apical and alpha 2A adrenergic receptors achieve basolateral localization. J. Biol. Chem. 271:995-1002 (1996).
M. Pastor-Anglada, A. Felipe, F. J. Casado, B. del Santo, J. F. Mata, and R. Valdes. Nucleoside transporters and liver cell growth. Biochem. Cell. Biol. 76:771-777 (1998).
M. Pastor-Anglada, F. J. Casado, R. Valdes, J. Mata, J. Garcia-Manteiga, M. Molina, A. Felipe, B. del Santo, J. F. Mata, B. Santo, J. Lloberas, and J. Casado. Complex regulation of nucleoside transporter expression in epithelial and immune system cells. Mol. Membr. Biol. 18:81-85 (2001).
S. Lin, H. Y. Naim, A. C. Rodriguez, and M. G. Roth. Mutations in the middle of the transmembrane domain reverse the polarity of transport of the influenza virus hemagglutinin in MDCK epithelial cells. J. Cell. Biol. 142:51-57 (1998).
L. Olivares, C. Aragon, C. Gimenez, and F. Zafra. The role of N-glycosylation in the targeting and activity of the GLYT1 glycine transporter. J. Biol. Chem. 270:9437-9442 (1995).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mangravite, L.M., Giacomini, K.M. Sorting of Rat SPNT in Renal Epithelium Is Independent of N-Glycosylation. Pharm Res 20, 319–323 (2003). https://doi.org/10.1023/A:1022247826750
Issue Date:
DOI: https://doi.org/10.1023/A:1022247826750