Trafficking defects of the Southeast Asian ovalocytosis deletion mutant of anion exchanger 1 membrane proteins

Abstract

Human AE1 (anion exchanger 1) is a membrane glycoprotein found in erythrocytes and as a truncated form (kAE1) in the BLM (basolateral membrane) of a-intercalated cells of the distal nephron, where they carry out electroneutral chloride/bicarbonate exchange. SAO (Southeast Asian ovalocytosis) is a dominant inherited haematological condition arising from deletion of Ala400-Ala408 in AE1, resulting in a misfolded and transport-inactive protein present in the ovalocyte membrane. Heterozygotes with SAO are able to acidify their urine, without symptoms of dRTA (distal renal tubular acidosis) that can be associated with mutations in kAE1. We examined the effect of the SAO deletion on stability and trafficking of AE1 and kAE1 in transfected HEK-293 (human embryonic kidney) cells and kAE1 in MDCK (Madin-Darby canine kidney) epithelial cells. In HEK-293 cells, expression levels and stabilities of SAO proteins were significantly reduced, and no mutant protein was detected at the cell surface. The intracellular retention of AE1 SAO in transfected HEK-293 cells suggests that erythroid-specific factors lacking in HEK-293 cells may be required for cell-surface expression. Although misfolded, SAO proteins could form heterodimers with the normal proteins, as well as homodimers. In MDCK cells, kAE1 was localized to the cell surface or the BLM after polarization, while kAE1 SAO was retained intracellularly. When kAE1 SAO was co-expressed with kAE1 in MDCK cells, kAE1 SAO was largely retained intracellularly; however, it also co-localized with kAE1 at the cell surface. We propose that, in the kidney of heterozygous SAO patients, dimers of kAE1 and heterodimers of kAE1 SAO and kAE1 traffic to the BLM of a-intercalated cells, while homodimers of kAE1 SAO are retained in the endoplasmic reticulum and are rapidly degraded. This results in sufficient cell-surface expression of kAE1 to maintain adequate bicarbonate reabsorption and proton secretion without dRTA.

Acid-Base Transport by the Renal Proximal Tubule

“…Various approaches (e.g., see references [89,90]) generally have yielded similar conclusions about the amino acid residues that contribute to transmembrane segments 1 through 5 (TM1 through TM5). Therefore, investigators feel reasonably confident about these assignments.…”

Cell Surface Rescue of Kidney Anion Exchanger 1 Mutants by Disruption of Chaperone Interactions

“…Disruption of chaperone interactions may allow for the release of ER-retained membrane glycoproteins and permit their trafficking to the cell surface. We have demonstrated previously that dominant dRTA kAE1 mutants are retained in the ER when expressed in HEK-293 and MDCK cells (14,15). We also have shown that erythroid AE1 is able to interact with calnexin in a glycosylation-dependent manner both in vitro and in HEK and K562 cells (22,23).…”

“…Immunofluorescence and Microscopy-Immunofluorescence staining and confocal microscopy of MDCK cells stably expressing kAE1 was performed as described previously (15). Imaging equipment includes a laser scanning confocal Zeiss LSM 510 microscope, Zeiss AxioCam, AxioVision, and LSM image browser.…”

“…The presence of this misfolded protein at the cell surface of red blood cells result in an ovalocytic and rigid shape. However, the presence of sufficient functional wild-type AE1 in erythrocytes, or kWT in kidney cells in the heterozygous state, compensates for the lack of kSAO transport activity resulting in asymptomatic anemia or dRTA (15,19). Co-expression of kSAO and dRTA mutants in the compound heterozygote state, however, results in severe dRTA due to the enhanced intracellular retention of the dRTA mutant by heterodimer formation with kSAO and the complete lack of transport activity of kSAO (16,19).…”

Loss of Specific Chaperones Involved in Membrane Glycoprotein Biosynthesis during the Maturation of Human Erythroid Progenitor Cells

“…The oligosaccharide on AE1 in HEK-293 cells remains in the high mannose form and is sensitive to digestion with endoglycosidase H. At steady state, about 30% of wild type AE1 in the high mannose form is present at the cell surface with the remainder in the ER (25). The expression of SAO and HS R760Q in HEK-293 cells results in their intracellular retention in the ER (23,26); however, no enhanced interaction of chaperones with the mutants relative to the wild type protein was detected by co-immunoprecipitation (supplemental Fig. S2).…”