The Glycan Domain of Thrombopoietin (TPO) Acts in
trans to Enhance Secretion of the Hormone and Other
Cytokines*
Hannah M.
Linden and
Kenneth
Kaushansky
From the Division of Hematology, University of Washington School of
Medicine, Seattle, Washington 98195
Thrombopoietin (TPO), the primary regulator of
platelet production, is composed of an amino-terminal 152 amino acids,
sufficient for activity, and a carboxyl-terminal region rich in
carbohydrates (183 residues) that enhances secretion of the molecule.
Full-length TPO is secreted at levels 10-20-fold greater than
truncated TPO. By introducing into mammalian cells a novel cDNA
encoding the TPO secretory leader linked to its carboxyl-terminal
domain (TPO glycan domain (TGD)), we tested whether TGD could function
in trans to enhance secretion of TPO. The artificial TGD
was secreted, inactive in proliferation assays, and did not inhibit TPO
activity. However, when co-transfected with a cDNA encoding
truncated TPO, TGD enhanced secretion 4-fold, measured by specific
bioassay and immunoassay. TGD also enhanced secretion of granulocyte
monocyte colony-stimulating factor and stem cell factor but did not
affect the production of erythropoietin, interleukin-3, growth hormone, or of full-length TPO. To localize TGD function, we added an
endoplasmic reticulum (ER) retention signal to TGD and, separately,
deleted the secretory leader. Deletion of the secretory leader
attenuated the secretory function of TGD, whereas addition of the ER
retention signal did not alter its function. To investigate the
physiologic role of TGD in folding and proteasomal protection, we
tested full-length and truncated TPO in assays of protein refolding,
and we examined protein stability in the presence of proteasome
inhibitors. We found that truncated TGD re-folds readily and that
proteasome-mediated degradation contributes to the poor secretion of
truncated TPO. We conclude that TGD enhances secretion of TPO and can
additionally function as an inter-molecular chaperone, in part because
of its ability to prevent degradation of the hormone. The cellular
location of TGD action is likely to be within the ER or earlier in the secretory pathway.
*
This work was supported by National Institutes of Health
Grants K08DK02665-02 (to H. L.), R01DK49855, and R01CA31615 (to
K. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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