TY - JOUR
T1 - Posttranslational processing of the thrombopoietin receptor is impaired in polycythemia vera
AU - Moliterno, Alison R.
AU - Spivak, Jerry L.
PY - 1999/10/15
Y1 - 1999/10/15
N2 - Recently, we demonstrated a marked reduction in the expression of the thrombopoietin receptor, Mpl, in polycythemia vera (PV) platelets and megakaryocytes using an antiserum against the Mpl extracellular domain. To further examine this abnormality, we raised an antibody to the Mpl C- terminus. Immunologic analysis of PV platelets with this antiserum confirmed the reduction in Mpl expression. However, the C-terminal antiserum detected 2 forms of Mpl in PV platelets in contrast to normal platelets, in which a single form of Mpl was detected by both the extracellular domain and C- terminal antisera. Two-dimensional gel electrophoresis studies with isoelectric focusing in the first dimension identified normal platelet Mpl as an 85 to 92 kD protein with an isoelectric point (pl) of 5.5. PV platelets contained an additional 80 to 82 kD Mpl Mpl isoform with a pl of 6.5. Analysis of Mpl expressed by the human megakaryocytic cell line, Dami, showed 2 isoforms similar to those found in PV platelets suggesting a precursor- product relationship. Digestion of Dami cell and normal platelet lysates with neuraminidase converted the more acidic Mpl isoform to the more basic one, indicating that the 2 isoforms differed with respect to posttranslational glycosylation. Furthermore, in contrast to normal platelet Mpl, PV platelet Mpl was susceptible to endoglycosidase H digestion, indicating defective Mpl processing by PV megakaryocytes. The glycosylation defect was specific for Mpl, as 2 other platelet membrane glycoproteins, glycoprotein IIb and multimerin, were processed normally. Importantly, the extent of the PV platelet Mpl glycosylation defect correlated with disease duration and extramedullary hematopoiesis.
AB - Recently, we demonstrated a marked reduction in the expression of the thrombopoietin receptor, Mpl, in polycythemia vera (PV) platelets and megakaryocytes using an antiserum against the Mpl extracellular domain. To further examine this abnormality, we raised an antibody to the Mpl C- terminus. Immunologic analysis of PV platelets with this antiserum confirmed the reduction in Mpl expression. However, the C-terminal antiserum detected 2 forms of Mpl in PV platelets in contrast to normal platelets, in which a single form of Mpl was detected by both the extracellular domain and C- terminal antisera. Two-dimensional gel electrophoresis studies with isoelectric focusing in the first dimension identified normal platelet Mpl as an 85 to 92 kD protein with an isoelectric point (pl) of 5.5. PV platelets contained an additional 80 to 82 kD Mpl Mpl isoform with a pl of 6.5. Analysis of Mpl expressed by the human megakaryocytic cell line, Dami, showed 2 isoforms similar to those found in PV platelets suggesting a precursor- product relationship. Digestion of Dami cell and normal platelet lysates with neuraminidase converted the more acidic Mpl isoform to the more basic one, indicating that the 2 isoforms differed with respect to posttranslational glycosylation. Furthermore, in contrast to normal platelet Mpl, PV platelet Mpl was susceptible to endoglycosidase H digestion, indicating defective Mpl processing by PV megakaryocytes. The glycosylation defect was specific for Mpl, as 2 other platelet membrane glycoproteins, glycoprotein IIb and multimerin, were processed normally. Importantly, the extent of the PV platelet Mpl glycosylation defect correlated with disease duration and extramedullary hematopoiesis.
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U2 - 10.1182/blood.v94.8.2555.420k14_2555_2561
DO - 10.1182/blood.v94.8.2555.420k14_2555_2561
M3 - Article
C2 - 10515857
AN - SCOPUS:0032830543
SN - 0006-4971
VL - 94
SP - 2555
EP - 2561
JO - Blood
JF - Blood
IS - 8
ER -