The glycan domain of thrombopoietin enhances its secretion.
Thrombopoietin (TPO) is the major regulator of megakaryocyte development and platelet production. The hormone is structurally characterized by an amino-terminal receptor binding domain (amino acid residues 1-152) predicted to encode a left-handed four-helix bundle structure, and a carboxyl-terminal domain (residues 153-335) that is remarkable for its abundant carbohydrate modification and a lack of homology to other proteins. To investigate the functional role of the carboxyl-terminal glycan domain, we generated truncated forms of murine TPO (TPO1-238, TPO1-174, and TPO1-152) and glycomuteins in which the predicted asparagine (N)-linked sites of glycosylation were sequentially mutated to glutamine (Q), and assayed their secretion and function by comparing them to the native sequence (TPO1-335). Following transient transfection of the corresponding cDNA expression vectors into mammalian cell lines, the secretory efficiencies of the proteins were compared with those of the native hormone. Transfection efficiencies were monitored by cotransfection and reporter gene assay, and TPO secretion was assessed by functional and immunologic assays. We found that full-length TPO was 5-29-fold more efficiently secreted than any of the truncated forms of the hormone in fibroblast and hepatocyte cell lines. Elimination of carboxyl-terminal sites of N-linked glycosylation had a minor impact on secretion of the protein. We conclude that the carboxyl-terminal domain of TPO serves the important role of enhancing secretion of the protein, and in this manner functions as a prosequence.
Linden
H M
HM
Division of Hematology, University of Washington, Box 357710, Seattle, Washington 98195, USA.
Kaushansky
K
K
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002474:Cell-Free System / Q000378:metabolism; D006224:Cricetinae; D006207:Half-Life; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D017384:Sequence Deletion; D013926:Thrombopoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D011134:Polysaccharides; D013926:Thrombopoietin