Functional characterization of wild-type and mutant human sialin

Abstract

The modification of cell surface lipids or proteins with sialic acid is essential for many biological processes and several diseases are caused by defective sialic acid metabolism. Sialic acids cleaved off from degraded sialoglycoconjugates are exported from lysosomes by a membrane transporter, named sialin, which is defective in two allelic inherited diseases: infantile sialic acid storage disease (ISSD) and Salla disease. To develop a functional assay of human sialin, we redirected the protein to the plasma membrane by mutating a dileucine‐based internalization motif. Cells expressing the plasmalemmal construct accumulated neuraminic acid at acidic pH by a process equivalent to lysosomal efflux. The assay was used to determine how pathogenic mutations affect transport. Interestingly, while two missense mutations and one small, in‐frame deletion associated with ISSD abolished transport, the mutation causing Salla disease (R39C) slowed down, but did not stop, the transport cycle, thus explaining why the latter disorder is less severe. Since neurological symptoms predominate in Salla disease, our results suggest that sialin is rate‐limiting to specific sialic acid‐dependent processes of the nervous system.

Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy

“…Deconvolution microscopy was performed as described (66). HEK-293 cells were transfected by lipofection and assayed for transport as described (38).…”

“…The LL/AA sorting mutant provided favorable conditions for testing this hypothesis because it allows replacing the poorly tractable lysosomal activity by a classic, whole-cell influx equivalent to lysosomal efflux. Several lysosomal transporters have been successfully characterized using this whole-cell approach (6,12,18,37,38). In preliminary experiments, we expressed PQLC2-LL/AA-EGFP in HEK-293 cells and examined their ability to take up…”

Sialin ( SLC17A5 ) functions as a nitrate transporter in the plasma membrane

“…3 E-G). Importantly, expression of the sialin mutant L22A-L23A, which reportedly is targeted to the plasma membrane (24,25), increased the currents by approximately twofold with either NO 3 − or SA in the external solution ( Fig. 3 H and I).…”

“…To establish the link between sialin and nitrate transport, we examined the effect of two nonfunctional sialin mutants that have been associated with Salla disease (R39C) and ISSD (H183R) (23)(24)(25). Expression of each of these mutants in HSG cells induced dominant suppression of both NO 3 − and SA − currents without altering the current-voltage (I-V) characteristics (Fig.…”

Infantile Sialic Acid Storage Disease: Two Unrelated Inuit Cases Homozygous for a Common Novel SLC17A5 Mutation

“…General genotype-phenotype correspondences have been established, and (with exceptions) most Salla disease patients have at least one copy of the common Finnish founder allele, p.Arg39Cys (NM_012434.4:c.115C>T) bearing some residual transporter activity in vitro (Aula et al 2000;Morin et al 2004;Verheijen et al 1999;Wreden et al 2005;Mochel et al 2009). In contrast, a wide range of mutation types are seen in ISSD; those studied in vitro appear to be functionally null (Aula et al 2000;Morin et al 2004;Myall et al 2007;Ruivo et al 2008;Wreden et al 2005). …”