Title : N-Linked glycosylation of the human ABC transporter
ABCG2 on
asparagine 596 is not essential for expression, transport activity, or trafficking to the plasma membrane
Abstract :
- The human ATP-binding cassette half-transporter ABCG2 is a 72 kDa plasma membrane protein that can confer multidrug resistance to cells in culture when overexpressed
- Both transiently and stably expressed ABCG2 are glycosylated, and treatment with peptide N-glycosidase F reduces the apparent molecular mass on SDS-PAGE gels to approximately 60 kDa
- Sequence analysis revealed three potential N-linked glycosylation sites in human ABCG2 at amino acids 418, 557, and 596
- Site-directed mutagenesis experiments, in which each Asn was changed to Gln independently, revealed that only asparagine 596 is N-linked glycosylated
- These data provide the first direct identification of the modified residue in ABCG2 and evidence for the localization of loop 5 to the extracellular space, previously only predicted from hydropathy analysis
- Immunoblot and pulse-chase analyses revealed that the glycosylation-deficient ABCG2 ( N596Q ) variant and the glycosylated parent transporter are expressed equivalently at steady state and have similar half-lives
- Cell surface analysis of ABCG2 expression showed comparable amounts of the N596Q variant present at the plasma membrane compared to the glycosylated ABCG2 protein
- The ABCG2 ( N596Q ) variant is also functional, demonstrating rhodamine 123 transport in intact cells comparable to that in cells expressing glycosylated ABCG2
- Furthermore, in crude membrane preparations, neither the basal nor the prazosin-stimulated ( approximately 2-fold) ATPase activities of ABCG2 ( N596Q ) were affected compared to glycosylated ABCG2
- Although subtle defects in transporter trafficking and function may exist, these data taken together suggest that N-glycosylation at arginine 596 is not essential for the expression, trafficking to the plasma membrane, or the overall function of ABCG2