Loss of Specific Chaperones Involved in Membrane Glycoprotein
Biosynthesis during the Maturation of Human Erythroid Progenitor
Cells |
| |
Authors: | Sian T Patterson Jing Li Jeong-Ah Kang Amittha Wickrema David B Williams and Reinhart A F Reithmeier |
| |
Institution: | ‡Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada and the §Section of Hematology/Oncology, Department of Medicine, University of Chicago, Chicago, Illinois 60637 |
| |
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 (Sec61α), 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.The production of red blood cells involves the terminal differentiation of
hematopoietic stem cells in the bone marrow followed by release into the
peripheral blood (1,
2). Red blood cells remain in
circulation for ~120 days and require the prior production of abundant red
cell-specific proteins including hemoglobin, cytoskeletal proteins, and
membrane glycoproteins such as anion exchanger 1
(AE1)3 and glycophorin
A (GPA). During differentiation, erythroid progenitor cells undergo extensive
remodeling of their cytoskeleton and loss of nuclei and other organelles like
the endoplasmic reticulum (ER). AE1 and GPA are known to be synthesized late
in differentiation when these key cellular components are lost
(3). The efficient biosynthesis
of these red cell membrane glycoproteins, however, is expected to require
robust ER assembly machinery involving protein translocation,
N-glycosylation, and protein folding chaperones.The proper folding of membrane glycoproteins engages the quality control
function of cytosolic and ER chaperone proteins
(4,
5). Newly synthesized proteins
undergo cycles of binding and release with chaperones, minimizing aggregation
and facilitating folding. Chaperones also play a role in the retention and
degradation of misfolded proteins and in apoptosis
(6-8).
The membrane-bound ER chaperone calnexin (CNX) and its luminal paralog
calreticulin (CRT) interact with folding intermediates via their lectin and
protein binding domains, thereby preventing aggregation
(9). A wide variety of
glycoprotein substrates have been identified, with some binding to one or both
chaperones, and both have been shown to be vital in the prevention of
aggregation and proper maturation of membrane glycoproteins
(9,
10). Disruption of
interactions with CNX and CRT can allow misfolded membrane glycoproteins to
escape the ER and traffic to the plasma membrane
(9).In the present study, we examined the integrity of the ER protein
translocation, N-glycosylation, and quality control machinery during
the differentiation of human CD34+ erythroid cells in culture. We
found that specific components of the protein quality control system were
completely lost (CNX and ERp57) or diminished (Hsc70 and Hsp70) before the
production of the major glycoproteins, AE1 and GPA, was completed. Components
of the protein translocation (Sec61α) and N-glycosylation
machinery (OST48) were, however, maintained. Chaperones that play other roles
in erythrocyte maturation and survival (CRT, BiP, and Hsp90) were also
retained (11). AE1 was found
to traffic efficiently to the plasma membrane in mouse embryonic fibroblasts
completely lacking the ER chaperone CNX or CRT. Furthermore, disruption of
CNX/CRT-glycoprotein interactions in human K562 cells did not affect the
cell-surface expression of GPA or AE1. These results demonstrate that CNX and
ERp57 are not required for the efficient synthesis and folding of red cell
membrane glycoproteins during terminal erythropoiesis. The lack of engagement
with the quality control and disulfide folding machinery may allow the more
rapid production of red cell glycoproteins late in differentiation,
sacrificing quality for quantity. |
| |
Keywords: | |
|
|