Molecular Characterization of pFGE, the Paralog of the C-α-Formylglycine-generating Enzyme

Abstract

C-α formylglycine (FGly) is the catalytic residue in the active site of sulfatases. In eukaryotes, it is generated in the endoplasmic reticulum by post-translational modification of a conserved cysteine residue. The enzymatic activity of sulfatases depends on this posttranslational modification which is mediated by a recently identified unique enzyme named as formylglycine generating enzyme (FGE). The genetic defect of FGE causes multiple sulfatase deficiency (MSD), a lysosomal storage disorder. In the genomes of deuterostomia, including vertebrates and echinodermata, a paralog of FGE, designated as pFGE, is exist. The human pFGE shares 47.1% sequence identity and 62.1% similarity with human FGE. The function of the pFGE is unknown. The goal of this study was to identify and analyze the structural and functional properties of pFGE. In comparison with FGE, pFGE shares a tissue specific expression pattern and the localization in the lumen of the endoplasmic reticulum. Upon overexpression, both FGE and pFGE escape from the endoplasmic reticulum and get secreted. However a constant level of pFGE or FGE is maintained in the cells. Although both lack endoplasmic reticulum retention signal, they are still retained by an unknown saturable mechanism. pFGE was purified to homogeneity from the secretions in a two-step protocol on Ni-NTA agarose followed by anion exchange chromatography and polyclonal and monoclonal antibodies were raised. Limited proteolytic cleavage at similar sites indicates that both also share a similar three-dimensional structure. pFGE, however, is lacking the formylglycinegenerating activity of FGE towards peptidic substrates. This observation holds true for peptidic substrates derived from all 16 sulfatases known or predicted from the human genome. Although overexpression of FGE stimulates the generation of catalytically active sulfatases, overexpression of pFGE has an inhibitory effect. In vitro pFGE interacts with sulfatase-derived peptides but not with FGE. The inhibitory effect of pFGE on the generation of active sulfatases may therefore be caused by a competition of pFGE and FGE for newly synthesized sulfatase polypeptides.In an independent study, we examined the utility of FGE for overexpression of catalytically active sulfatases in a way to improve the efficacy of sulfatase replacement therapy. Our data demonstrating that FGE coexpression is indispensable for efficient synthesis of functional sulfatases. HT1080 cells were stably transfected with galactose-6-sulfatase and FGE. The specific activity of galactose-6-sulfatase increased up to 100 fold in cell lysates by coexpression of FGE. Interestingly, stimulation of sulfatase activity is more pronounced when FGE and sulfatase are concomitantly expressed in the HT1080 cells. Moreover, stable FGE-sulfatase complexes are formed in the endoplasmic reticulum but its physiological significance remains elusive. However, this complex formation may aid in the retention of FGE as a substrate-mediated phenomenon; substantiated by the observation that the secretion of FGE is reduced markedly when coexpressed with sulfatase.