Identification of PDGF-BB binding to thymosin β₄ by chemical cross-linking

Abstract

Introduction: The purpose of our work was to identify unknown interaction partners of thymosin β₄ (Tβ₄). It was suggested that Tβ₄ could be an antifibrotic drug for treatment of liver fibrogenesis, because Tβ₄ prevents the platelet-derived growth factor-BB (PDGF-BB)-induced activation of hepatic stellate cells (HSCs). Very little information is available how Tβ₄ counteracts the PDGF-BB-induced activation of HSCs. We propose the hypothesis that Tβ₄ could bind directly to PDGF-BB and thereby reduce the concentration of free PDGF-BB available for binding to the PDGF-β receptor.

Methods: To prove our suggestion of a direct interaction between Tβ₄ and PDGF-BB, we carried out chemical as well as photochemical cross-linking experiments between the two pure proteins in vitro.

Results: We identified an interaction between Tβ₄ and PDGF-BB by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-linking as well as through biotin label transfer using a bifunctional photoactivatable derivative of Tβ₄. In an in vitro system, PDGF-BB was identified as the first extracellular partner interacting with Tβ₄. This interaction could influence PDGF-BB binding to its receptor and abolish PDGF-BB-related effects.

Conclusion: Direct interaction of Tβ₄ with extracellular factors should be considered as a potential mechanism to explain the pleiotropic effects of β-thymosins.

Keywords:

• biotin label transfer
• cross-linking
• platelet-derived growth factor
• β-thymosin

Acknowledgments

We want to give special thanks to the deceased Dr. Marcus Rojkind and to the members of his laboratory (Reyes-Gordillo, Sha, and Arellanes-Robledo) at The George Washington University for the opportunity to carry out all experiments with HSCs in their facility. We express our appreciation to Doris Jaegers for excellent technical assistance and support.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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