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

Abstract

The production of erythrocytes requires the massive synthesis of red cell-specific proteins including hemoglobin, cytoskeletal proteins, as well as membrane glycoproteins glycophorin A (GPA) and anion exchanger 1 (AE1). We found that during the terminal differentiation of human CD34؉ erythroid progenitor cells in culture, key components of the endoplasmic reticulum (ER) protein translocation (Sec61␣), glycosylation (OST48), and protein folding machinery, chaperones BiP, calreticulin (CRT), and Hsp90 were maintained to allow efficient red cell glycoprotein biosynthesis. Unexpected was the loss of calnexin (CNX), an ER glycoprotein chaperone, and ERp57, a protein-disulfide isomerase, as well as a major decrease of the cytosolic chaperones, Hsc70 and Hsp70, components normally involved in membrane glycoprotein folding and quality control. AE1 can traffic to the cell surface in mouse embryonic fibroblasts completely deficient in CNX or CRT, whereas disruption of the CNX/CRT-glycoprotein interactions in human K562 cells using castanospermine did not affect the cell-surface levels of endogenous GPA or expressed AE1. These results demonstrate that CNX and ERp57 are not required for major glycoprotein biosynthesis during red cell development, in contrast to their role in glycoprotein folding and quality control in other cells.

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

“…Previous immunofluorescence studies of the kAE1 mutants kSAO, R589H, and R901stop co-localize these mutants to the ER, suggesting that the ER chaperone calnexin may play a role in folding of kAE1, a membrane glycoprotein (14,15). Indeed, we have shown that erythroid AE1 is able to interact with calnexin in a glycosylation-dependent manner both in vitro and in HEK-293 cells (22,23).…”

“…Thus, the cellular context and nature of interacting proteins is very important in producing a phenotype of mutations with the same genotype. Indeed, human red cell precursors lose specific chaperones like calnexin during differentiation (22). This loss of protein quality control may allow misfolded forms of AE1, such as the SAO mutant, to traffic to the cell surface during erythropoiesis and be expressed in the mature red cell.…”

“…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). Chaperones therefore may play a role in the intracellular retention of dRTA mutants and may be therapeutic targets to promote ER exit and trafficking to the cell surface.…”

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

“…Glycophorin A can rescue the trafficking of some AE1 mutants to the cell surface (see below), including AE1 Southeast Asian ovalocytosis (SAO) (Patterson et al, 2009), certain hereditary spherocytosis (HS) mutants , and in kAE1, dominant mutations causing distal renal tubular acidosis (dRTA) Young et al, 2000). AE1 HS mutants that are not rescued by Glycophorin A may have lost the ability to interact with this protein partner due to misfolding.…”

Enhanced calreticulin expression in red cells of polycythemia vera patients harboring the JAK2V617F mutation

“…12 CANX was shown to be lost during late erythroid differentiation, 13 so its presence in the membrane of circulating PV red blood cells is potentially interesting as it might affect physiological functions. Indeed, little is known about the role of CALR and CANX as calcium-binding chaperones …”