Human GGT2 Does Not Autocleave into a Functional Enzyme: A Cautionary Tale for Interpretation of Microarray Data on Redox Signaling

Abstract

Aims: Human c-glutamyltranspeptidase 1 (hGGT1) is a cell-surface enzyme that is a regulator of redox adaptation and drug resistance due to its glutathionase activity. The human GGT2 gene encodes a protein that is 94% identical to the amino-acid sequence of hGGT1. Transcriptional profiling analyses in a series of recent publications have implicated the hGGT2 enzyme as a modulator of disease processes. However, hGGT2 has never been shown to encode a protein with enzymatic activity. The aim of this study was to express the protein encoded by hGGT2 and each of its known variants and to assess their stability, cellular localization, and enzymatic activity. Results: We discovered that the proteins encoded by hGGT2 and its variants are inactive propeptides. We show that hGGT2 cDNAs are transcribed with a similar efficiency to hGGT1, and the expressed propeptides are N-glycosylated. However, they do not autocleave into heterodimers, fail to localize to the plasma membrane, and do not metabolize c-glutamyl substrates. Substituting the coding sequence of hGGT1 to conform to alterations in a CX 3 C motif encoded by hGGT2 mRNAs disrupted autocleavage of the hGGT1 propeptide into a heterodimer, resulting in loss of plasma membrane localization and catalytic activity. Innovation and Conclusions: This is the first study to evaluate hGGT2 protein. The data show that hGGT2 does not encode a functional enzyme. Microarray data which have reported induction of hGGT2 mRNA should not be interpreted as induction of a protein that has a role in the metabolism of extracellular glutathione and in maintaining the redox status of the cell.

Non enzymatic upregulation of tissue factor expression by gamma-glutamyl transferase in human peripheral blood mononuclear cells

“…Perhaps, some role may play a chemokine-like CX3C motif contained in the GGT molecule [33] or exogenous GGT might mimick cytokine-like activities of GGT-related proteins produced by genes different from the GGT1 [34]. For example, the GGT2 gene located close to the GGT1 chromosomal region seems to encode for a full length, enzymatically inactive protein, not localized to the plasma membrane [35] and, according to some Authors, involved in redox modulation [36]. It might also be possible to envisage protein-protein interactions leading to the expression of other procoagulant cytokines.…”

Gamma-Glutamyl Transpeptidase

“…GGT2 mRNA has been detected and this has led to the assumption that GGT2 encodes an enzymatically active protein (Auman et al, 2008; Moon et al, 2012). However, we have recently shown that GGT2 propeptides encoded by all three isoforms of GGT2 failed to autocleave, were enzymatically inactive, did not localize to the plasma membrane, and were rapidly degraded within the cell (West, Wickham, Parks, Sherry, & Hanigan, 2013). …”

“…GGT1) and GGT2, and 94% amino acid identity. The GGT2 gene is transcribed and GGT2 protein has been detected, but the protein has no enzymatic activity and is rapidly degraded within the cell (West, Wickham, et al, 2013). Some microarray platforms have probes that distinguish between GGT1 and GGT2, while others do not creating confusion as to which mRNA is being measured.…”

Novel Insights into Eukaryotic γ-Glutamyltranspeptidase 1 from the Crystal Structure of the Glutamate-bound Human Enzyme

“…It was fully intact in the 4GG2 model of hGGT1, but it was a mix of intact and broken bonds in the 4DGX model, perhaps attributable to x-ray radiation damage. Site-directed mutagenesis studies with rat and human GGT1 demonstrated that both disulfide bonds are critical for optimal autocleavage of mammalian GGT1 into active heterodimers (36,38,39). The formation of the Cys-50/Cys-74 and Cys-192/Cys-196 disulfide bridges may contribute to the proper folding of hGGT1 and, thereby, facilitate autocleavage into the activated heterodimer.…”

“…The bacterial GGTs with crystal structures lack N-glycosylation, yet their propeptides are properly folded and undergo autocleavage (27)(28)(29)37). Two cysteines (Cys-50 and Cys-74) are conserved in the large subunit of all eukaryotic GGT1s and are required for efficient autocleavage of hGGT1 (38,39). Both of these sites are alanines in the bacterial GGTs.…”