Structural and functional consequences of an N-glycosylation mutation (HEMPAS) affecting human erythrocyte membrane glycoproteins

Abstract

Band 3, the human erythrocyte anion exchanger (AE1), and the glucose transporter (GLUT1) proteins each contain a single site of N-glycosylation that is heterogeneously glycosylated. Lectin binding and enzymatic deglycosylation assays showed that the polylactosaminyl oligosaccharide structure of these glycoproteins was altered to a high mannose or hybrid glycan form in three patients with hereditary erythroblastic multinuclearity, with a positive acidified-serum lysis test (HEMPAS). Offspring from one of the HEMPAS patients had intermediate levels of polylactosaminyl oligosaccharide associated with AE1 and GLUT1, suggesting they may have been heterozygous for the genetic defect. The array of polylactosaminyl-containing glycoproteins present in EBV-transformed lymphoblasts derived from fresh blood of HEMPAS patients was similar to control lymphoblasts. HEMPAS lymphoblasts do not therefore express the defect in polylactosamine synthesis found in erythroid cells, indicating that lymphoid cells are not deficient in the processing enzymes or contain an alternative oligosaccharide processing pathway. Purified HEMPAS band 3 had an unaltered oligomeric structure but dimers aggregated more rapidly in detergent solution than normal band 3. The altered oligosaccharide structure did not affect the sensitivity of band 3 to proteolytic digestion in intact red cells but a greater amount of HEMPAS band 3 was associated with the cytoskeleton. The transport activities of AE1 and GLUT1 in HEMPAS erythrocytes were similar to those in normal controls. This shows that the HEMPAS glycosylation defect does not impair the functional accumulation of these two important erythrocyte membrane transporters even though it produces subtle structural changes in band 3 that result in its increased cytoskeletal interaction and self association in detergent solution.

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

“…Immunoblots of MDCK cells expressing dRTA mutants showed no change in glycosylation pattern in the presence of MAL3 or the small molecule correctors (data not shown). As in previous studies with erythroid AE1 (47), and as we have now shown with the CST results, formation of complex oligosaccharide is not required for the trafficking of kAE1 to the cell surface. It is therefore the interaction with the quality control mechanisms in the cell that determine the intracellular localization of this membrane glycoprotein.…”

Characterization of the N-glycosylation phenotype of erythrocyte membrane proteins in congenital dyserythropoietic anemia type II (CDA II/HEMPAS)

“…The truncation of erythrocyte N-glycans was the predominant finding in CDA II. α1,6 linkage of fucose to the reducing GlcNAc of the Nglycan (core fucosylation) is a frequent but optional modification of numerous N-glycans [27]. α1,6 fucosyltransferase residing in the medial Golgi (Fut8) exclusively catalyzes this reaction.…”

Structure, Function, and Trafficking of SLC4 and SLC26 Anion Transporters

“…AE1 in these red cells contains a high-mannose oligosaccharide rather than the normal complex structure, suggesting a defect in trafficking and oligosaccharide processing (Kameh, Landolt-Marticorena, Charuk, Schachter, & Reithmeier, 1998). HEMPAS is due to a defect in the SEC23B component of the COPII complex (Dancourt & Barlowe, 2010) that is responsible for the budding of vesicles from the ER that carries cargo to the Golgi (Schwarz et al, 2009).…”