Identification of three distinct glycosylation sites in the ATP binding cassette class A3 (ABCA3) transporter

Abstract

Glycosylation plays a critical role in the proper maintenance of protein structure to promote stability and/or to mediate trafficking and function of various proteins. ABCA3 is a polytopic transmembrane transporter that functions as a lipid and phospholipid pump. Within the first luminal loop of ABCA3, three putative glycosylation sites exist containing NATI (a), NCSS (b), and NHSK (c) motifs. To determine if these sites undergo glycosylation, we used alanine mutagenesis to generate fusion constructs encoding EGFP‐tagged to either wild type ABCA3 (ABCA3WT), single motif mutants (ABCA3a, ABCA3b, ABCA3c), double mutants (ABCA3a+b, ABCA3b+c), or a triple mutant (ABCA3a+b+c). Fluorescence examination of transiently transfected A549 cells showed cytosolic vesicle distribution of the single motif mutants similar to ABCA3WT. By Western blot, removal of N‐linked glycosylation by PNGaseF resulted in the migration of the ABCA3WT primary translation product band at a faster rate. Moreover, all three single motif mutant bands also migrated in a similar manner. However, double and triple mutants resulted in ER retained, unprocessed forms of ABCA3. Although the exact role of glycosylation in ABCA3 is yet to be fully explained, these results suggest that ABCA3 transporter is glycosylated on at least three specific sites and amino acid alteration of these motifs leads to abnormal trafficking of the transporter.