G328E and G409E sialin missense mutations similarly impair transport activity, but differentially affect trafficking

Abstract

Two disease-associated missense mutations in the sialin gene (G328E and G409E) have recently been identified in patients with lysosomal free sialic acid storage disease. We have assessed the effect of these mutations and find complete loss of measurable transport activity with both and impaired trafficking of the G409E protein. These results suggest that the two residues are important for proper function of sialin and confirm the association of loss of transport with disease causative mutations.

Figure 1

A) Comparison of the sequences corresponding to G328 and G409 and the surrounding residues in human, rat and drosophila sialin and human isoforms of two related proteins, VGLUT1 and NaPi1 demonstrate that these specific residues are completely conserved. B) Immunofluorescence colocalization of HA-tagged G328E and G409E (left panels, red) and myc-tagged wild type sialin (middle panels, green) demonstrate strong colocalization of wild-type and G328E sialin, but less overlap of the wild-type and G409E patterns. In higher magnification view of indicated region (bottom panel right column) arrows indicate G409E puncta that do not colocalize with wild-type sialin. C) Sialic acid transport activity of cells expressing plasma membrane targeted isoforms of G328E and G409E demonstrates less than 1% of the uptake measured in the cells transfected with the wild-type plasma membrane targeted sialin. Uptake by cells transfected with the G328E and G409E isoforms were not statistically different from that measured for LYAAT transfected cells (p=0.48 and p=0.60 respectively, two-tailed unpaired t-test). Duplicate samples were assayed in three separate experiments. The mean values are plotted with the standard deviation.