TPO is synthesized in the liver as a 353-amino acid precursor protein with a molecular weight of 36 kDa (5,6,12). After the removal of the 21-amino acid signal peptide, the remaining 332 amino acids undergo glycosylation to produce a glycoprotein with a molecular weight of 80-90 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 57,539 Daltons by mass spectrometry (13). The glycoprotein is then released into the circulation with no apparent intracellular storage in the liver.

TPO is a member of the four-helix-bundle cytokine superfamily and has a number of unusual properties (Fig. 1). First, it is much larger than most other regulators of blood cell production such as granulocyte colony-stimulating factor (G-CSF) and EPO. Second, it has an unusual structure in that it has two domains, an EPO-like domain (residues 1-153) and a carbohydrate-rich domain (residues 154-332) separated by a dibasic site of potential proteolytic cleavage (Arg153-Arg154). It is unclear whether this cleavage site is biologically relevant (14).

The first 153 amino acids of the mature protein are 23% identical with human EPO (15,16) and 50% similar if conservative amino acid substitutions are considered. This

Fig. 1. The primary structure of human thrombopoietin. (Courtesy of Dr. T. Kato, Pharmaceutical Research Laboratory, Kirin Brewery, Takasaki, Gunma, Japan.)

region also contains four Cys residues, Cys7-Cys151 and Cys29-Cys85 (17), comparable to those in EPO, which are highly conserved among different species. Replacement of Cys7 or Cys151 abrogates all activity, whereas disruption of Cys29 or Cys85 reduces activity (18). This region also contains four a-helices as in EPO. All the TPO receptor binding activity is located in this region.

Amino acids 154-332 comprise a unique sequence that contains six ^-linked glyco-sylation sites and is less well conserved among different species. It is rich in Ser, Thr, and Pro residues. Structure-function studies have shown that whereas the first 153 amino acids of the c-mpl ligand are all that are required for its thrombopoietic effect in vitro (5,6), this truncated molecule has a markedly decreased circulatory half-life compared with the 20-40-h half-life of the native protein (19). Paradoxically, the truncated molecule has a specific activity in vitro 20-fold greater than the full-length molecule (20).

Glycosylation is an important post-translational modification of TPO. Other post-translational modifications, such as hydroxylation, methylation, or deamidation of TPO, are not evident. Presumably, the glycosylated second half of the molecule confers stability and prolongs the circulatory half-life. Similar carbohydrate sequences regulate the stability of EPO (21). In addition, this part of the molecule assists in the secretion of the intact molecule from the hepatocytes by serving as a molecular chaperone or guide in protein folding; truncated muteins of this portion of the molecule have diminished secretion (22). All six sites of N-glycosylation are found in residues 154-332. The N-glycans are of the complex type, with the core-fucosylated disialylated biantennary and trisialylated triantennary structures predominating. N185FT and N234GT appear to be 100% occupied, whereas N176RT and N213QT are partially occupied, and the status of N319TS and N327LS is uncertain (13). In addition, there are 3 O-glycosylation sites (S1, T37, T110) in the EPO-like domain and at least 5 (T158, T159, S163, T192, T244) and possibly 16 others in the carbohydrate-rich domain. The O-glycans are of the mucin type with the monosialylated and disialylated GalGalNAc-S/T structures predominating. This formation has led to the proposal that the carbohydrate-rich domain of TPO can be further divided into two subdomains on the basis of sequence homology among the cloned sequences and glycosylation: an N-glycan domain (amino acids 154-246) and an O-glycan domain (amino acids 247-332) (13).

Although the crystal-structure analysis of TPO has been completed, it has not been published. The areas of TPO that bind the TPO receptor are probably analogous to those of human growth hormone and EPO. Based on the similarity of these three cytokines, it has been proposed that Lys138 of helix D and Pro42 and Glu50 of loop AB may constitute one binding region, and Arg10 and Lys14 of helix A may constitute a second binding region that participates in the dimerization of TPO receptors (23). This suggestion has been partly confirmed by monoclonal antibody (MAb) studies. Two MAbs that block TPO binding to its receptor have determinants in helix D (24). These results again suggest that TPO has two receptor binding sites that help dimerize its receptor.

0 0

Post a comment