Abstract
Formylglycine-generating enzyme (FGE) catalyzes the oxidation of a specific cysteine residue in nascent sulfatase polypeptides to formylglycine (FGly). This FGly is part of the active site of all sulfatases and is required for their catalytic activity. Here we demonstrate that residues 34-68 constitute an N-terminal extension of the FGE catalytic core that is dispensable for in vitro enzymatic activity of FGE but is required for its in vivo activity in the endoplasmic reticulum (ER), i.e. for generation of FGly residues in nascent sulfatases. In addition, this extension is needed for the retention of FGE in the ER. Fusing a KDEL retention signal to the C terminus of FGE is sufficient to mediate retention of an N-terminally truncated FGE but not sufficient to restore its biological activity. Fusion of FGE residues 1-88 to secretory proteins resulted in ER retention of the fusion protein. Moreover, when fused to the paralog of FGE (pFGE), which itself lacks FGly-generating activity, the FGE extension (residues 34-88) of this hybrid construct led to partial restoration of the biological activity of co-expressed N-terminally truncated FGE. Within the FGE N-terminal extension cysteine 52 is critical for the biological activity. We postulate that this N-terminal region of FGE mediates the interaction with an ER component to be identified and that this interaction is required for both the generation of FGly residues in nascent sulfatase polypeptides and for retention of FGE in the ER.
Highlights
Folding [1, 3]
N-terminally Truncated FGly-generating enzyme (FGE) Is Catalytically Active but Lacks Biological Function—FGE is a soluble endoplasmic reticulum (ER)-localized protein, where it converts in newly synthesized sulfatase polypeptides a cysteine into the active site FGly residue that is indispensable for sulfatases to become catalytically active
The generation of active sulfatases depends on the oxidation of a specific cysteine in nascent sulfatase polypeptides to a FGly residue
Summary
Construction of Expression Plasmids—The plasmids used to express galactose-6-sulfatase, steroid sulfatase (STS), and different forms of His6- or HA-tagged FGE and pFGE have been described previously [11, 15, 17]. For co-expression of FGE-HA, ⌬34 – 68FGE-HA, or ⌬34 – 68FGE-HA-KDEL with steroid sulfatase, the pBI vector (BD Biosciences) was used, which allows expression of two cDNAs from a bidirectional tetracycline/ doxycycline-responsive promoter [15]. In the second step cloning of steroid sulfatase into multiple cloning site I of pBI vector was performed as described earlier [15]. The best clones were rescreened through Western blotting for doxycycline-dependent pFGE production after transient transfection with pBI-pFGE [17]. Indirect Immunofluorescence—For recombinant expression of pBI-⌬34 – 68FGE-HA, pBI-⌬34 – 68FGE-HA-KDEL, pBIpFGE, pBI-pFGE⌬PGEL, and pBI-FGE1– 88-pFGE⌬PGEL, the MSDi Tet-On or HT1080 Tet-On cells were grown on coverslips for 1 day and transiently transfected as described above. For Western blot analysis, polyclonal antisera against FGE, pFGE, or steroid sulfatase and a monoclonal anti-galactose-6-sulfatase antibody were used as primary antibodies [11, 15]. Relative specific sulfatase activities were calculated, i.e. catalytic activity divided by the Western blot signal (arbitrary units) and referred to that of cells expressing the sulfatase only (relative specific sulfatase activity ϭ 1)
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