Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein\u2013protein interactions of the fused protein

Hyo-Jun Kim,Soojin Lee,Jin-Haeng Lee,Sung-Yup Cho,Ki Baek Lee,Mee-Ae Kwon,Ji-Woong Shin,In-Gyu Kim,Eui Man Jeong

doi:10.1038/s12276-020-00549-9

Hyo-Jun Kim, Soojin Lee + Show 7 more

Open Access

https://doi.org/10.1038/s12276-020-00549-9

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Abstract

Glutathione S-transferase (GST) from Schistosoma japonicum has been widely used as a tag for affinity purification and pulldown of fusion proteins to detect protein–protein interactions. However, the reliability of this technique is undermined by the formation of GST-fused protein aggregates after incubation with cell lysates. It remains unknown why this aggregation occurs. Here, we demonstrate that the GST tag is a substrate of transglutaminase 2 (TG2), which is a calcium-dependent enzyme that polyaminates or crosslinks substrate proteins. Mutation analysis identified four glutamine residues in the GST tag as polyamination sites. TG2-mediated modification of the GST tag caused aggregate formation but did not affect its glutathione binding affinity. When incubated with cell lysates, GST tag aggregation was dependent on cellular TG2 expression levels. A GST mutant in which four glutamine residues were replaced with asparagine (GST4QN) exhibited a glutathione binding affinity similar to that of wild-type GST and could be purified by glutathione affinity chromatography. Moreover, the use of GST4QN as a tag reduced fused p53 aggregation and enhanced the induction of p21 transcription and apoptosis in cells treated with 5-fluorouracil (5-FU). These results indicated that TG2 interferes with the protein–protein interactions of GST-fused proteins by crosslinking the GST tag; therefore, a GST4QN tag could improve the reproducibility and reliability of GST pulldown experiments.

Highlights

Introduction GlutathioneS-transferase (GST) from Schistosoma japonicum is often included as a protein tag in a commonly used molecular biology technique
The Glutathione S-transferase (GST) tag is a Transglutaminase 2 (TG2) substrate To determine whether TG2 is involved in the aggregate formation of the GST fusion protein, we investigated whether the GST tag was a TG2 substrate using an incorporation assay that utilized the synthetic polyamine biotinylated pentylamine (BP)
To determine whether the polyamine levels found in cell lysates are sufficient to incorporate into the GST tag, we determined the Km and catalytic efficiency of TG2

Summary

Introduction

S-transferase (GST) from Schistosoma japonicum is often included as a protein tag in a commonly used molecular biology technique. GST fusion proteins purified by GSH affinity chromatography have been used to identify new protein–protein interactions. In this case, cell lysates are incubated with a GST fusion protein, and Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that catalyzes the calcium-dependent transamidation reaction between glutamine and lysine residues of proteins to form crosslinked proteins. During pulldown experiments to identify TG2-interacting proteins and to analyze domain–domain interactions, lysates from cells that express a high level of TG2 exhibited an increase in aggregate formation. We investigated the role of TG2 in GST fusion protein aggregation

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