Molecular characterization of equine thymidine kinase 1 and preliminary evaluation of its suitability as a serum biomarker for equine lymphoma

Abstract

Background: Thymidine kinase 1 (TK1) plays a key role in the synthesis of deoxythymidine triphosphate (dTTP) and is thus important for DNA replication and cell proliferation. The expression of TK1 is highest during S-phase, and it is rapidly degraded after mitosis. In cancer cells, TK1 is upregulated, resulting in leakage of excess TK1 into the blood. Consequently, serum TK1 has been used as a diagnostic and prognostic cancer biomarker, mainly in human medicine. The aims of this work were to characterize equine TK1 and to evaluate its suitability as a serum biomarker for equine lymphoma.

Results: Equine TK1 was cloned, expressed in E. coli and affinity purified. The purified recombinant horse TK1 showed broad substrate specificity, phosphorylating pyrimidine deoxyribo- and ribonucleosides and, to some extent, purine deoxynucleosides, including anticancer and antiviral nucleoside analogues. ATP was the preferred phosphate donor. Serum TK1 activity was measured in samples collected from horses with confirmed or suspected lymphoma and control horses with and without concurrent diseases. Serum TK1 activity levels were significantly higher in horses with lymphoma (p < 0.0005) and suspected lymphoma (p < 0.02) and in tumour-free groups with diverse diseases (p < 0.03) than in controls without concurrent diseases. There was a significant difference between the lymphoma group and the tumour-free group with diverse diseases (p < 0.0006). Furthermore, receiver operating characteristic analysis revealed a sensitivity of 0.86, a specificity of 0.95 and an AUC (area under the curve) of 0.92 compared to the controls without concurrent diseases, with a sensitivity of 0.97, a specificity of 0.71 and an AUC of 0.88 when compared with the tumour-free group with diverse diseases.

Conclusion: Equine TK1 showed high specific activity and broader substrate specificity than human TK1. Anticancer and antiviral thymidine analogues were efficiently phosphorylated by horse TK1, suggesting that these analogues might be good candidates for chemotherapy in horses. Serum TK1 activity was significantly higher in horses with lymphoma than in controls. ROC analysis indicated that serum TK1 could serve as a promising cancer biomarker in horses.

Keywords: Enzyme kinetics; Equine lymphoma; Equine thymidine kinase 1; Nucleoside analogues; Serum biomarker; cancer.

Fig. 1

A Amino acid sequence alignment of equine TK1 isoforms with human TK1 using the Clustal Omega algorithm (https://www.ebi.ac.uk/Tools/msa/clustalo/) and structural alignment using ENDscript 2.0 software (http://endscript.ibcp.fr/ESPript/cgi-bin/ENDscript.cgi) with the human TK1 structure as the template. B Predicted 3D structure for isoform 1. C Predicted 3D structure for isoform 3. D Superimposed structures of isoforms 1 and 3. Structure prediction was performed with fully automated structural modelling software (https://sissmodel.expasy.org) using the human TK1 structure in complex with dTTP as a template (PDB code: 1W4R)

Fig. 2

Characterization of recombinant horse TK1. SDS-PAGE analysis A and western blot analyses of recombinant horse TK1 with antibodies against 6xHistidine-tag B and TK1 C. Lane 1, E. coli extracts of uninduced culture; lane 2, E. coli extracts of induced culture; lane 3, purified recombinant horse TK1

Fig. 3

Steady-state kinetic analysis of horse TK1. Plots of initial velocity versus substrate concentration, dThd A, dUrd B, Urd C and dGuo D. The ATP concentration was kept at 1 mM

Fig. 4

Steady-state kinetic analysis of horse TK1. ATP was the variable substrate, and the dThd concentration was kept at 0.1 mM

Fig. 5

Analysis of serum TK1 levels. A. Comparison of serum TK1 levels in the control group without concurrent disease (Controls, n = 40), the lymphoma (n = 7) and suspected lymphoma (n = 5) groups and the tumour-free group with concurrent diseases (nonneoplastic diseases, n = 107). Bars represent the median. Receiver operating characteristic (ROC) analysis of serum TK1 activity to distinguish horses with lymphoma B and nonneoplastic diseases (nontumour diseases) C from the control group without concurrent diseases. The sensitivity and specificity were determined based on the chosen cut-off of 0.65 pmol/min/ml. To distinguish lymphoma from nonneoplastic diseases (nontumour disease group) D, the sensitivity and specificity were determined at a chosen cut-off value of 1.0 pmol/min/ml (mean + 2 SD)