hClC-6 is glycosylated upon overexpression.

<p>(A) Western blots showing the effect of tunicamycin and (a) PNGaseF and (b) EndoH on hClC-6. For tunicamycin COS-1 cells were incubated with tunicamycin (0.05 and 0.1 µg/ml) for 36 hours; for PNGaseF and EndoH membrane fractions of hClC-6 expressing COS-1 cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000474#s2" target="_blank">materials and methods</a>. ‘WT’ refers to untreated hClC-6. The small difference in molecular mass between PNGaseF and tunicamycin-treated hClC-6 might be due to the presence of oligosaccharides carrying fucose-linked α1–3 to the GlcNac attached directly to asparagines, which are PNGaseF resistant as described by Dwek <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000474#pone.0000474-Dwek1" target="_blank">[53]</a>. (B)(a), Model of the hClC multimeric structure of 2 homologous subunits with the possible glycosylation sites marked as spheres. (b), Partial sequence alignment (ClustalW) of all members of the CLC family reveals that Asn residues that possibly participate in glycosylation (marked in red) in hClC-6 are situated in a region that is poorly conserved among the other members of the CLC family and located between predicted helices K and M. (C) Western blots of membrane fractions of COS-1 cells expressing WT or mutant hClC-6. (a), Glycosylation pattern of the triple (AAA: N410A/N422A/N432A) and quadruple (AAAA: N137A/N410A/N422A/N432A) mutant compared to WT and WT with tunicamycin. (b), Glycosylation pattern of the double mutants (AAN: N410A/N422A; ANA: N10A/N432A; NAA: N422A/N432A) compared to glycosylated WT and the triple mutant (AAA). (c), Glycosylation pattern of the single mutants (ANN: N410A, NAN: N422A, NNA: N432A) compared to the glycosylated WT and deglycosylated WT treated with tunicamycin. (d), Effect of PNGaseF treatment on a single (ANN: N410A) and double (AAN: N410A/N422A) mutant compared to WT and triple (AAA) mutant. (e), Effect of EndoH treatment on a single (ANN: N410A) and double (AAN: N410A/N422A) mutant compared to WT and triple (AAA) mutant. Bands marked with an asterisk, occasionally observed in the WT protein and frequently observed in single mutants after sustained exposure represent possible intermediary biosynthetic products, which are PNGaseF-sensitive and EndoH-insensitive as shown in panels (d) and (e).