Human GGT2 does not autocleave into a functional enzyme: A cautionary tale for interpretation of microarray data on redox signaling

Abstract

Aims: Human γ-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 γ-glutamyl substrates. Substituting the coding sequence of hGGT1 to conform to alterations in a CX3C 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.

FIG. 1.

Cartoon of the structural components of mature hGGT1. hGGT1 is a type II membrane glycoprotein that undergoes autocatalytic maturation to form a heterodimeric enzyme composed of a large (a.a. 1–380, dark circles) and a small (a.a. 381–569, light circles) subunit. The transmembrane domain of the mature heterodimer is located at the N-terminus of the large subunit (a.a 5–26). The catalytic threonine residue (T381) forms the N-terminus of the small subunit. hGGT2 is predicted (www.uniprot.org/uniprot/P36268) to possess the same compositional features and structural organization as hGGT1. hGGT1, human γ-glutamyltranspeptidase 1.

FIG. 2.

Alignment of hGGT1 with the deduced amino-acid sequences of hGGT2. The amino-acid sequence of hGGT1 was aligned with the predicted amino-acid sequences from the XM_001129425.3 (hGGT2-1), XM_001129377.3 (hGGT2-2), and ENST00000405188 (hGGT2-3) mRNA variants of hGGT2. The conserved single-pass transmembrane domain is underlined. Cysteine residues (C50, C74, C192, and C196) that have been shown to form disulfide bonds in mammalian GGT1 are boxed. The conserved CX₃C motif in eukaryotic orthologs of hGGT1, which has been altered in hGGT2, is shaded in gray. The seven N-glycosylation sites in hGGT1 are highlighted with a black box containing a white “N.” The catalytic threonine residue that forms the N-terminus of the small subunit (T381) after autocleavage of the hGGT1 propeptide into the mature heterodimeric enzyme is highlighted with a black box containing a white “T” with an arrow above. The epitope recognized by the GGT129 antibody is underlined with a bracket.

FIG. 3.

hGGT2 is expressed as an inactive propeptide that fails to autocleave. (A) Whole cell lysates from HEK293T cells transiently transfected with either empty vector (lanes 1 and 7), hGGT1 cDNA (lanes 2 and 8), hGGT2-1 cDNA (lanes 3 and 9), hGGT2-2 cDNA (lanes 4 and 10), or hGGT2-3c DNA (lanes 5 and 11) expression constructs were incubated in the presence (lanes 7–11) or absence (lanes 1–5) of PNGaseF and resolved on an 8% SDS-polyacrylamide gel. Resolved proteins were electroblotted onto nitrocellulose and then subjected to immunoblotting, using the GGT129 antibody, which recognizes a common epitope in hGGT1 and each of the hGGT2 alleles. MW, molecular-weight markers. The antibody identifies both the propeptide and large subunit of hGGT1. hGGT2-1,2,3 are present only as a propeptide. (B) Reverse transcription–polymerase chain reaction amplification of hGGT1 and hGGT2 (206 bp) or β-actin (512 bp) from transiently transfected HEK293T cells.

FIG. 4.

Intracellular localization of hGGT1 and hGGT2. HEK293T cells transfected with hGGT1 or hGGT2 cDNA expression constructs and grown on coverslips were fixed and processed for indirect immunofluorescence, using the GGT129 antibody (GGT, green) and an antibody against the ER marker, calnexin (CNX, red). Nuclei were stained with DAPI (blue). Areas of overlapping GGT129 and calnexin immunofluorescence appear yellow in the overlay panels. Exposure times for GGT images differ among the samples due to the lower expression levels of the hGGT2 proteins. hGGT1 is localized to the cell membrane, and hGGT2 proteins co-localize with calnexin in the ER. Scale bar=20 μm and applies to all images shown. ER, endoplasmic reticulum.

FIG. 5.

Oxidative stress does not induce cleavage of hGGT2 propeptides into heterodimers. (A) Nuclear lysates (10 μg total protein) from vehicle-treated HEK293T cells (lane 1), HEK293T cells cultured for 4 h in 250 μM H₂O₂ (lane 2) or 50 μM tBHQ (lane 3) were subjected to SDS-PAGE and immunoblotted with an antibody specific for Nrf2. Presence of Nrf2 in the nucleus of the H₂O₂ and tBHQ-treated cells confirms the induction of oxidative stress under these treatment conditions. (B) HEK293T cells were transiently transfected with empty vector (lanes 1–3), hGGT1 cDNA (lanes 4–6), hGGT2-1 cDNA (lanes 7–9), hGGT2-2 cDNA (lanes 10–12), or hGGT2-3 cDNA (lanes 13–15) expression constructs. Twenty-four hours after transfection, vehicle (ethanol), 250 μM H₂O₂, or 50 μM tBHQ was added to the cells. After 24 h, whole cell lysates were resolved on an 8% SDS-polyacrylamide gel. Resolved proteins were electroblotted onto nitrocellulose and then subjected to immunoblotting, using the GGT129 antibody, which recognizes a common epitope in hGGT1 and each of the hGGT2 alleles. The antibody identifies both the propeptide and large subunit of hGGT1. hGGT2-1,2,3 are expressed but present only as a propeptide under each of these culture conditions. The weak signal observed in lane 3 is carried over from lane 4 as confirmed by independent blots and activity assays. H₂O₂, hydrogen peroxide; tBHQ, tert-butylhydroquinone.

FIG. 6.

hGGT1 CX₃C mutants do not autocleave and mislocalize to the ER. (A) Whole cell lysates from HEK293T cells transfected with either empty vector (lanes 1 and 5) or cDNA expression constructs for wild-type hGGT1 (WT, lanes 2 and 6), hGGT1(C192W;E193Y) (lanes 3 and 7), or hGGT1(C192_E193insPLCPG) (lanes 4 and 8). Five micrograms of protein from each whole cell lysate were subjected to SDS-PAGE and immunoblotting, using antibodies specific for either the large (lanes 1–4) or small (lanes 5–8) subunits of hGGT1. The introduced mutations were in the large subunit of hGGT1 and were outside the epitope recognized by the large subunit antibody. The small subunit in all constructs was identical to wild-type hGGT1 (B) Lysates from (A) were incubated with PNGaseF and subjected to SDS-PAGE and immunoblotting against either the large (lanes 1–4) or small (lanes 5–8) subunits of hGGT1. (C) HEK293T cells transfected with either empty vector, wild-type hGGT1 cDNA, or hGGT1 CX₃C mutant cDNA expression constructs were grown on coverslips and fixed and processed for indirect immunofluorescence, using the GGT129 antibody (GGT, green) and an antibody against the ER marker, calnexin (CNX, red). Nuclei were stained with DAPI (blue). Areas of overlapping GGT129 and calnexin immunofluorescence appear yellow in the overlay panels shown here. Exposure times for GGT images differed among the samples due to the lower expression levels of the mutant hGGT1 proteins. hGGT1 is localized to the cell membrane, and hGGT1 CX₃C mutant proteins co-localize with calnexin in the ER. Scale bars=20 μm. For the full complement of images used to compose the overlay panels, see Supplementary Figure S1.

FIG. 7.

Targeted replacement of hGGT2 residues with wild-type hGGT1 amino acids does not restore autocatalytic maturation. Whole cell lysates from HEK293T cells transfected with either empty vector (lane1), wild-type hGGT1 cDNA (lane 2), GGT2-1 cDNA (lane 3), or mutant GGT2-1 alleles (lanes 4–7) were resolved by SDS-PAGE and electroblotted onto nitrocellulose. Immunoblotting was carried out using the GGT129 antibody. For labeling the hGGT2-1 lanes: hGGT2-1, wild-type hGGT2-1; hGGT2-1(C/E), hGGT2-1(W192C;Y193E); hGGT2-1(C/E/D/D), hGGT2-1(W192C;Y193E;T422D;T423D); hGGT2-1(C/E/S), hGGT2-1(W192C;Y193E;L451S); and hGGT2-1 (C/E/D/D/S), hGGT2-1(W192C;Y193E;T422D;T423D;L451S).