Trafficking and Folding Defects in Hereditary Spherocytosis Mutants of the Human Red Cell Anion Exchanger

Abstract

Hereditary spherocytosis (HS) is a common inherited hemolytic anemia caused by mutations in erythrocyte proteins including the anion exchanger, AE1 (band 3). This study examined seven missense mutations (L707P, R760Q, R760W, R808C, H834P, T837M, and R870W) located in the membrane domain of the human AE1 that are associated with this disease. The HS mutants, constructed in full-length AE1 cDNA, could be transiently expressed to similar levels in HEK 293 cells. Immunofluorescence, cell surface biotinylation, and pulse chase labeling showed that the HS mutants all exhibited defective cellular trafficking from the endoplasmic reticulum to the plasma membrane. Impaired binding to an inhibitor affinity matrix indicated that the mutant proteins had non-native structures and may be misfolded. Further characterization of the HS R760Q mutant showed no change in its oligomeric structure or turnover (half-life = 15 h) compared to wild-type AE1, suggesting the mutant was not aggregated or targeted for rapid degradation via the proteasome. Intracellular retention of HS mutant AE1 would lead to destruction of the protein during erythroid development and would account for the lack of HS mutant AE1 in the plasma membrane of the mature red cell.

Novel Topology in C-terminal Region of the Human Plasma Membrane Anion Exchanger, AE1

“…The NMR structure of a synthetic peptide corresponding to Gly 796 -Ile 841 revealed Ile 803 -Leu 810 in ␣-helical conformation (40). The R808C mutation, found in this region, causes hereditary erythroid spherocytosis because of a failure to process AE1 to the cell surface (36). Consistent with that finding, R808C AE1 was not functional and was not biotinylated in the present study.…”

“…7 and Table I). Human mutations H834P and T837M both cause erythrocyte spherocytosis because of a failure of AE1 to be processed to the cell surface (36). In the present study, mutation to cysteine at each of these sites resulted in inactive protein, which was not labeled by biotin maleimide.…”

“…Thus, it is important to determine if any intracellular AE1 is labeled by biotin maleimide, as this protein may not have native structure. The R808C mutation has previously been shown to cause retention of AE1 in intracellular membranes (36). To determine whether intracellular AE1 is accessible to labeling by biotin maleimide we constructed the R808C/C479S double mutant in a wild type AE1 background.…”

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).…”

“…The non-functional SAO AE1 protein that arises from a nine-amino acid deletion (⌬400 -408), however, is present in nearly equal amounts to the normal protein in the plasma membrane of red blood cells of heterozygotes (22). HS point mutations in the membrane domain of AE1 lead to a protein that is retained in the ER of transfected HEK-293 cells (23). The HS mutant, AE1 R760Q (Prague II), is completely absent in mature red blood cells (24), indicating defective trafficking to the plasma membrane during erythropoiesis.…”