Thymidine phosphorylase (TP) first identified as platelet derived endothelial cell growth factor (PD-ECGF) plays a key role in nucleoside metabolism. Human TP (hTP) is implicated in angiogenesis and is overexpressed in several solid tumors. Here, we report the crystal structures of recombinant hTP and its complex with a substrate 5-iodouracil (5IUR) at 3.0 and 2.5 Å, respectively. In addition, we provide information on the role of specific residues in the enzymatic activity of hTP through mutagenesis and kinetic studies.

Recombinant human platelet-derived endothelial cell growth factor, expressed in the yeast Saccharomyces cerevisiae was purified to greater than 98 % purity by anion-exchange and hydroxyapatite chromatography. It was shown to possess thymidine phosphorolytic activity in vitro (pH optimum, pH 5.3; K,, 0.11 mM; V, , , 12.5 mmol min-' mg-'; turnover number, 9.4 s -I ) . Covalent modification simultaneously inhibited the enzymatic and mitogenic properties of the protein, while interaction with a cell-surface receptor was not required to stimulate mitogenesis. Purified Escherichia coli thymidine phosphorylase was also mitogenic toward endothelial cells. It is proposed that platelet-derived endothelial cell growth factor is human thymidine phosphorylase which promotes endothelial cell proliferation by reducing thymidine levels that would otherwise be inhibitory to endothelial cell growth.

Monomeric human platelet-derived endothelial cell growth factor (PD-ECGF) is a single-chain protein of relative molecular mass (M,) =45 kDa, which stimulates the growth and chemotaxis of endothelial cells in vitro and possesses angiogenic activity in vivo (Miyazono et al., 1987;Ishikawa et al., 1989). As angiogenesis is central to the pathological conditions of tumor growth, rheumatoid arthritis, diabetic retinopathy, psoriasis, and hemangiomas, a detailed understanding of the molecular action of PD-ECGF would provide clues to therapeutic strategies for these disease states.Here we report the striking similarity between the primary sequences of PD-ECGF and thymidine phosphorylase (TP) from Escherichia cofi (Shwartz, 1978). Human TP catalyzes the reversible phosphorolysis of thymidine and other pyrimidine 2'-deoxyribosides, with the exception of 4-amino-substituted compounds and has nucleoside deoxyribosyl transferase activity. TP is one of two pyrimidine phosphorylases in the base and nucleoside salvage pathway. Under near-physiological conditions TP is a homodimer with a molecular mass of 110 kDa in mammals (Desgranges, 1981) and 90 kDa in E. cofi (Shwartz, 1978). Specific inhibitors of TP are considered as potential chemotherapeutics either to reduce clearance of thymidine and other deoxyuridine analogues presently in use as antineoplastic and antiviral agents or by interfering with the salvage process (Desgranges et al., 1983). The design of such inhibitors may be aided by the 2.8-A-resolution crystal structure of E. cofi TP, which shows TP to consist of a small helical domain and a larger a//3 domain both of which comprise two noncontinuous segments of polypeptide (residues 1-65, 163-193 and 80-154 phosphate moieties appear to be bound in a cleft between these two domains. Figure 1 highlights the sequence similarity between the human PD-ECGF and E. cofiTP sequences. The similarity extends over all but the N-terminal32 and the C-terminal 4 residues of PD-ECGF. Human PD-ECGF apparently undergoes posttranslational maturation, whereby 10 and 4 amino acids are removed from the amino and carboxyl termini, respectively . Mature PD-ECGF has a 22-amino acid N-terminal extension with respect to the E. coli TP sequence. The sequences show 40% identity calculated over the 438 common amino acid positions (see legend to Fig. 1) and are therefore likely to have diverged from a common genetic ancestor and share the same overall tertiary fold (Barton & Sternberg, 1987). Thus, the crystal structure of E. cofi TP may be used as a scaffold on which to model the structure of human PD-ECGF. The amino acids of the putative phosphate binding sites of TP are conserved in PD-ECGF (indicated by P in Fig. l), as are the thymidine-binding residues (Arg 171, Ser 186, and Lys 190, indicated by T in Fig. 1). The small helical domain of TP has 46% identity with PD-ECGF, whereas the larger domain has 37% identity. Further subdivision of these domains shows the segments containing binding residues for thymidine (163-193, helical domain) and ...

A platelet-derived endothelial cell growth factor cDNA has been cloned, sequenced and expressed using the Saccharomyces cerevisiae PRB1 promoter. Soluble recombinant platelet-derived endothelial cell growth factor constituted 0.5-1.0% of total soluble protein. Yeast soluble protein extracts containing recombinant platelet-derived endothelial cell growth factor stimulate the growth of calf pulmonary artery endothelial cells in vitro.

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