Disruption of N-linked glycosylation promotes proteasomal degradation of the human ATP-binding cassette transporter ABCA3

Abstract

The lipid transport protein, ABCA3, expressed in alveolar type 2 (AT2) cells, is critical for surfactant homeostasis. The first luminal loop of ABCA3 contains three putative N-linked glycosylation sites at residues 53, 124, and 140. A common cotranslational modification, N-linked glycosylation, is critical for the proper expression of glycoproteins by enhancing folding, trafficking, and stability through augmentation of the endoplasmic reticulum (ER) folding cycle. To understand its role in ABCA3 biosynthesis, we utilized EGFP-tagged fusion constructs with either wild-type or mutant ABCA3 cDNAs that contained glutamine for asparagine substitutions at the putative glycosylation motifs. In A549 cells, inhibition of glycosylation by tunicamycin increased the electrophoretic mobility (Mr) and reduced the expression level of wild-type ABCA3 in a dose-dependent manner. Fluorescence imaging of transiently transfected A549 or primary human AT2 cells showed that although single motif mutants exhibited a vesicular distribution pattern similar to wild-type ABCA3, mutation of N124 and N140 residues resulted in a shift toward an ER-predominant distribution. By immunoblotting, the N53 mutation exhibited no effect on either the Mr or ABCA3 expression level. In contrast, substitutions at N124 or N140, as well a N124/N140 double mutation, resulted in increased electrophoretic mobility indicative of a glycosylation deficiency accompanied by reduced overall expression levels. Diminished steady-state levels of glycan-deficient ABCA3 isoforms were rescued by treatment with the proteasome inhibitor MG132. These results suggest that cotranslational N-linked glycosylation at N124 and N140 is critical for ABCA3 stability, and its disruption results in protein destabilization and proteasomal degradation.

The biology of the ABCA3 lipid transporter in lung health and disease

“…N-linked glycosylation is one of the most common co-/posttranslational modifications that occurs during protein synthesis in the ER/Golgi and has been shown to have a pivotal role in the folding, stability, and cellular localization of proteins (Helenius and Aebi, 2001; Beers et al, 2011; Molinari, 2007). Cleavage profiles, generated using endoglycosidase H (Endo H) and N-gylycosidase F (PNGase F) that cleave N-linked glycans including complex carbohydrate chains, have confirmed the structural predictions that ABCA3 undergoes N-linked glycosylation (Cheong et al, 2006; Matsumura et al, 2006; Beers et al, 2013). Subsequent site directed mutagenesis studies have demonstrated that substitution mutations of asparagine to alanine at either or both of the 124 and 140 residues resulted in ER retention of glycan deficient ABCA3 isoforms and showed increased electrophoretic mobility of the expressed products by immunoblot (Beers et al, 2013).…”

“…Following synthesis of the primary translation product and translocation to the ER, ABCA3 is routed via the Golgi, sorting vesicles (SVs), and multivesicular bodies (MVBs) directly to the outer membrane of LBs in AT2 cells or to lysosomes and lysosomal-related organelles in A549 and HEK293 cell lines (Cheong et al, 2006; Mulugeta et al, 2002; Nagata et al, 2004; Beers et al, 2013). Functionally essential posttranslational modifications including glycosylation and proteolytic cleavage also occur during this trafficking (discussed below).…”

Mutations of rat surfactant protein A have distinct effects on its glycosylation, secretion, aggregation and degradation

“…Although the molecular mechanism of protein aggregation still needs to be elucidated, disruption of glycosylation is a potential factor that contributes to the degradation of proteins and their aggregates. For example, N-linked glycans on ABCG2 (ATP-binding cassette transporter) and ABCA3 (the lipid transport protein) have been shown to be important for protein stability, and disruption of N-linked glycosylation results in these proteins' destabilization, as well as enhanced degradation through the proteasome (Beers et al, 2013;Nakagawa et al, 2009). Moreover, proteins are also degraded through the autophagy-mediated lysosomal pathway (Ishida et al, 2009;Vembar and Brodsky, 2008).…”

“…O-linked glycosylation occurs at a serine (Ser) or threonine (Thr) residue by the attachment of N-acetylgalactosamine (Peter-Katalinic, 2005). Site-directed mutagenesis of glycan linked-amino acids (Asn, Ser or Thr) in a protein reduces its glycosylation and can disrupt its biological functions (Beers et al, 2013). However, mutations in non-glycan-linked amino acids could also lead to glycosylation defects.…”

The classification of ATP‐binding cassette subfamily A member 3 mutations using the cystic fibrosis transmembrane conductance regulator classification system

“…For example, missense mutations in the ABCA3 extracellular domain 1 (ECD1) location were classified as Type II mutations because specific ABCA3 missense mutations in ECD1 were experimentally shown to lead to the accumulation ABCA3 protein in the endoplasmic reticulum. 17,20 The same process was extended to mutations in extracellular domain 2 (ECD2). However, frameshift and nonsense mutations that were located in the nucleotide binding domain 1 (NBD1) were classified as Type I mutations as mutations of this type result in defective protein synthesis.…”