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Biosynthetic defect in platelet glycoprotein IX mutants associated with
Bernard-Soulier syndrome
G Sae-Tung, JF Dong and JA Lopez
Gladstone Institute of Cardiovascular Disease, Cardiovascular Research
Institute, San Francisco, CA, USA.
To evaluate the biosynthetic basis for decreased glycoprotein (GP) Ib- IX
expression resulting from GP IX mutations described in three siblings with
Bernard-Soulier syndrome, we introduced each mutation into the cDNA for GP
IX by site-directed mutagenesis (GP IX Asp21 --> Gly and GP IX Asn45
--> Ser) and examined the associations of the mutants with the two other
subunits of the GP Ib-IX complex in transfected cells. Unlike wild-type GP
IX, neither of the mutants was able to increase GP Ib expression on the
cell surface, either when transfected into Chinese hamster ovary (CHO)
alpha beta cells or when cotransfected with GP Ib alpha and GP Ib beta into
wild-type CHO cells. We also evaluated whether cotransfecting wild-type or
mutant GP IX with GP Ib beta would result in the appearance of GP IX on the
surface of the transfected cells; the wild-type protein was detected on the
surface of the cells, whereas neither mutant reached the cell surface in
appreciable quantities. Immunofluorescence microscopy of permeabilized
cells revealed that the failure to express mutant GP IX on the cell surface
did not result from failure to synthesize the polypeptide. Both mutants
were detected in intracellular compartments, albeit at lower levels than
the wild-type polypeptide (the fluorescence of cells expressing the GP IX
Asp21 --> Gly was consistently the lowest). Direct evidence that the
mutants associate poorly with Gp Ib beta was obtained of 35S-labeled cells
transiently expressing GP Ib beta and wild-type or mutant GP IX. The amount
of GP IX coprecipitated with GP Ib beta was greatly diminished in cells
expressing either mutant. These findings suggest an important role for the
conserved leucine-rich motif of GP IX in the association of this
polypeptide with GP Ib beta and provide further evidence for the importance
of GP IX in the stability of the GP-Ib-IX complex.
Volume 87,
Issue 4,
pp. 1361-1367,
02/15/1996
Copyright © 1996 by The American Society of Hematology
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