Glutathione S-transferase

Enzymes of the glutathione S-transferase (GST) family are composed of many cytosolic, mitochondrial, and microsomal (now designated as MAPEG) proteins. GSTs are present in eukaryotes and in prokaryotes, where they catalyze a variety of reactions and accept endogenous and xenobiotic substrates.

GSTs can constitute up to 10% of cytosolic protein in some mammalian organs. GSTs catalyse the conjugation of reduced glutathione — via a sulfhydryl group — to electrophilic centers on a wide variety of substrates. This activity detoxifies endogenous compounds such as peroxidised lipids, as well as breakdown of xenobiotics. GSTs may also bind toxins and function as transport proteins, and, therefore, an early term for GSTs was “ligandin”. The mammalian GST super-family consists of cytosolic dimeric isoenzymes of 45–55 kDa size that have been assigned to at least six classes: Alpha, Mu, Pi, Theta, Zeta and Omega.

Most mammalian isoenzymes have affinity for the substrate 1-chloro-2,4-dinitrobenzene (CDNB), and spectrophotometric assays utilising this substrate are commonly used to report GST activity. However, some endogenous compounds, e.g., bilirubin, can inhibit the activity of GSTs. In mammals, GST isoforms have cell specific distributions (e.g., alpha GST in hepatocytes and pi GST in the biliary tract of the human liver).

Family members
The following is a list of human glutathione S-transferases:

Structure
Mammalian cytosolic GSTs are dimeric both subunits being from the same class of GSTs, although not necessarily identical. The monomers are in the range of 22–30 kDa. They are active over a wide variety of substrates with considerable overlap.

GSTs and biotransformation
Glutathione S-transferases are considered, among several others, to contribute to the phase II biotransformation of xenobiotics. Drugs, poisons, and other compounds not traditionally listed in either groups are usually modified by the phase I and/or phase II mechanisms, and finally excreted from the body. GSTs contribute to this type of metabolism by conjugating these compounds (often electrophilic and somewhat lipophilic in nature) with reduced glutathione to facilitate dissolution in the aqueous cellular and extracellular media, and, from there, out of the body.

GST-tags and the GST pull-down assay
Genetic engineers have used glutathione S-transferase to create the GST gene fusion system. This system is used to purify and detect proteins of interest. In a GST gene fusion system, the GST sequence is incorporated into an expression vector alongside the gene sequence encoding the protein of interest. Induction of protein expression from the vector's promoter results in expression of a fusion protein: the protein of interest fused to the GST protein. This GST-fusion protein can then be purified from cells via its high affinity for glutathione.

Fusion proteins offer an important biological assay for direct protein-to-protein interactions. For instance, to demonstrate that protein X binds to protein Y a GST-X fusion protein would be generated. Assay beads, coated with the tripeptide glutathione, strongly bind the GST fusion protein (GST-X). Therefore if X binds Y, then GST-X will also bind Y, and Y will be present on assay beads.

GST is commonly used to create fusion proteins. The tag has the size of 220 amino acids (roughly 26 KDa), which, compared to other tags like the myc- or the FLAG-tag, is quite big. It is fused to the N-terminus of a protein. However, many commercially-available sources of GST-tagged plasmids include a thrombin domain for cleavage of the GST tag during protein purification.

A GST-tag is often used to separate and purify proteins that contain the GST-fusion. GST-fusion proteins can be produced in Escherichia coli, as recombinant proteins. The GST part binds its substrate, glutathione. Agarose beads can be coated with glutathione, and such glutathione-Agarose beads bind GST-proteins. These beads are then washed, to remove contaminating bacterial proteins. Adding free glutathione to beads that bind purified GST-proteins will release the GST-protein in solution.

Role in disease
Genetic polymorphisms in glutathione S-transferase and its altered expression and activity are associated with oxidative DNA damage and also damage of the kidney. Subsequently the individual’s risk of cancer susceptibility increases.