The Non-catalytic N-terminal Extension of Formylglycine-generating Enzyme Is Required for Its Biological Activity and Retention in the Endoplasmic Reticulum

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. 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. Sulfatases carry in their active site a unique amino acid, Cα-formylglycine (FGly), 2The abbreviations used are: FGly, Cα-formylglycine; ER, endoplasmic reticulum; FGE, formylglycine-generating enzyme; pFGE, paralog of FGE; MSD, multiple sulfatase deficiency; STS, steroid sulfatase; HA, hemagglutinin. 2The abbreviations used are: FGly, Cα-formylglycine; ER, endoplasmic reticulum; FGE, formylglycine-generating enzyme; pFGE, paralog of FGE; MSD, multiple sulfatase deficiency; STS, steroid sulfatase; HA, hemagglutinin. which is crucial for their catalytic activity (1von Figura K. Schmidt B. Selmer T. Dierks T. BioEssays. 1998; 20: 505-510Crossref PubMed Scopus (80) Google Scholar, 2Schmidt B. Selmer T. Ingendoh A. von Figura K. Cell. 1995; 82: 271-278Abstract Full Text PDF PubMed Scopus (293) Google Scholar). In most eukaryotic and prokaryotic sulfatases, FGly is generated from a cysteine residue (3Dierks T. Schmidt B. von Figura K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11963-11968Crossref PubMed Scopus (109) Google Scholar, 4Dierks T. Lecca M.R. Schlotterhose P. Schmidt B. von Figura K. EMBO J. 1999; 18: 2084-2091Crossref PubMed Scopus (118) Google Scholar, 5Dierks T. Miech C. Hummerjohann J. Schmidt B. Kertesz M.A. von Figura K. J. Biol. Chem. 1998; 273: 25560-25564Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). This posttranslational protein modification in eukaryotes occurs in the endoplasmic reticulum (ER) during or shortly after translocation of sulfatase polypeptides into the lumen of the ER and before their folding (1von Figura K. Schmidt B. Selmer T. Dierks T. BioEssays. 1998; 20: 505-510Crossref PubMed Scopus (80) Google Scholar, 3Dierks T. Schmidt B. von Figura K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11963-11968Crossref PubMed Scopus (109) Google Scholar). The oxidation of cysteine to FGly is catalyzed by the FGly-generating enzyme (FGE) (6Cosma M.P. Pepe S. Annunziata I. Trott D.A. Parenti G. Ballabio A. Cell. 2003; 113: 445-456Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 7Dierks T. Schmidt B. Borissenko L.V. Peng J. Preusser A. Mariappan M. von Figura K. Cell. 2003; 113: 435-444Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), which uses molecular oxygen as the terminal electron acceptor. 3J. Peng, T. Dierks, A. Preusser-Kunze, M. Mariappan, K. von Figura, and B. Schmidt, unpublished results. 3J. Peng, T. Dierks, A. Preusser-Kunze, M. Mariappan, K. von Figura, and B. Schmidt, unpublished results. The critical role of FGE for the synthesis of sulfatases is highlighted by multiple sulfatase deficiency (MSD), a rare inherited disorder caused by mutations in the gene encoding FGE and characterized by the synthesis of catalytically inactive sulfatases (6Cosma M.P. Pepe S. Annunziata I. Trott D.A. Parenti G. Ballabio A. Cell. 2003; 113: 445-456Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 7Dierks T. Schmidt B. Borissenko L.V. Peng J. Preusser A. Mariappan M. von Figura K. Cell. 2003; 113: 435-444Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 8Cosma M.P. Pepe S. Parenti G. Settembre C. Annunziata I. Wade-Martins R. Di Domenico C. Di Natale P. Mankad A. Cox B. Uziel G. Mancini G.M. Zammarchi E. Donati M.A. Kleijer W.J. Filocamo M. Carrozzo R. Carella M. Ballabio A. Hum. Mutat. 2004; 23: 576-581Crossref PubMed Scopus (54) Google Scholar, 9Annunziata I. Bouche V. Lombardi A. Settembre C. Ballabio A. Hum. Mutat. 2007; 28: 928Crossref PubMed Scopus (32) Google Scholar, 10Schlotawa L. Steinfeld R. von Figura K. Dierks T. Gärtner J. Hum. Mutat. 2008; 29: 205Crossref PubMed Scopus (36) Google Scholar). FGE is localized in the ER and generates the FGly residue by oxidation of the cysteine in the C(T/S/C/A)PSR pentapeptide conserved in all human sulfatases. The N-terminal 33 residues of human FGE represent a cleavable signal peptide. The mature form of FGE is composed of a compact N-glycosylated monomeric core with a novel fold and little secondary structure that is stabilized by two Ca2+ ions and two intramolecular disulfide bonds (11Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12Dierks T. Dickmanns A. Preusser-Kunze A. Schmidt B. Mariappan M. von Figura K. Ficner R. Rudolph M.G. Cell. 2005; 121: 541-552Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 13Roeser D. Preusser-Kunze A. Schmidt B. Gasow K. Wittmann J.G. Dierks T. von Figura K. Rudolph M.G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 81-86Crossref PubMed Scopus (84) Google Scholar). The N-terminal 55 residues of mature FGE form a domain of unknown structure, which obviously extends from the compact fold of the core protein, as indicated by very (11Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12Dierks T. Dickmanns A. Preusser-Kunze A. Schmidt B. Mariappan M. von Figura K. Ficner R. Rudolph M.G. Cell. 2005; 121: 541-552Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). In the active site two crucial in and oxidation of the cysteine to a novel T. Dickmanns A. Preusser-Kunze A. Schmidt B. Mariappan M. von Figura K. Ficner R. Rudolph M.G. Cell. 2005; 121: 541-552Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 13Roeser D. Preusser-Kunze A. Schmidt B. Gasow K. Wittmann J.G. Dierks T. von Figura K. Rudolph M.G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 81-86Crossref PubMed Scopus (84) Google Scholar). paralog of FGE is in most from to eukaryotes (6Cosma M.P. Pepe S. Annunziata I. Trott D.A. Parenti G. Ballabio A. Cell. 2003; 113: 445-456Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 7Dierks T. Schmidt B. Borissenko L.V. Peng J. Preusser A. Mariappan M. von Figura K. Cell. 2003; 113: 435-444Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). with FGE a and but lacks FGly-generating activity J. Dierks T. Schmidt B. von Figura K. 2003; PubMed Scopus Google Scholar, M. Preusser-Kunze A. M. Schmidt B. Gande S.L. Wenzel D. Dierks T. von Figura K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). FGE and the N-terminal extension of 55 residues in human and a of a residues both in FGE T. Dickmanns A. Preusser-Kunze A. Schmidt B. Mariappan M. von Figura K. Ficner R. Rudolph M.G. Cell. 2005; 121: 541-552Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, A. Schmidt B. Rudolph M.G. Mariappan M. Dierks T. von Figura K. Ficner R. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google a is in pFGE, as an ER retention signal S.L. Mariappan M. Schmidt B. von Figura K. Dierks T. J. 2008; PubMed Scopus Google Scholar). of FGE, pFGE, and sulfatases and to ER retention to FGE S.L. Mariappan M. Schmidt B. von Figura K. Dierks T. J. 2008; PubMed Scopus Google Scholar, E. A. Pepe S. Annunziata I. G. Di Natale P. Ballabio A. M.P. EMBO 2005; PubMed Scopus Google Scholar). FGE of the and by E. M. Pepe S. Settembre C. Annunziata I. Dierks T. M. M. P. Ballabio A. M.P. EMBO J. 2007; PubMed Scopus Google Scholar). the of FGE that retention in the ER is as an N-terminally truncated form with for this that a the of FGE during Mariappan, unpublished results. Mariappan, unpublished results. In for residues and of FGE. that a truncated FGE, residues is catalytically active T. Schmidt B. Borissenko L.V. Peng J. Preusser A. Mariappan M. von Figura K. Cell. 2003; 113: 435-444Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). This that the N-terminal extension of FGE, a that is conserved in all eukaryotic from eukaryotes to a that is of its FGly-generating activity. Here we that the N-terminal extension of FGE is required for the of sulfatases in the N-terminal extension is required for FGE retention in the ER and a of N-terminally truncated FGE in the ER, is not sufficient to restore the of sulfatases. of a fusion protein the N-terminal extension of FGE the in vivo of sulfatases by N-terminally truncated FGE. We from that the N-terminal extension of FGE mediates the interaction with an ER component and both the retention of FGE in the ER and the generation of FGly residues in nascent sulfatase of used to steroid sulfatase and of or FGE and (11Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, M. Preusser-Kunze A. M. Schmidt B. Gande S.L. Wenzel D. Dierks T. von Figura K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar, S.L. Mariappan M. Schmidt B. von Figura K. Dierks T. J. 2008; PubMed Scopus Google Scholar). of or with steroid the which of two from a M. Preusser-Kunze A. M. Schmidt B. Gande S.L. Wenzel D. Dierks T. von Figura K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). In the of or with a or site and a by a by KDEL and and a site by M. Preusser-Kunze A. M. Schmidt B. Gande S.L. Wenzel D. Dierks T. von Figura K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google as The with or by the site with and with The into the multiple site of the that by as and with the or site of and site of In the of steroid sulfatase into multiple site of as M. Preusser-Kunze A. M. Schmidt B. Gande S.L. Wenzel D. Dierks T. von Figura K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). construct for of with or or pFGE, the encoding residues 1–88 of FGE with a site and a site as and the and The with and and into the of multiple site with multiple site or or or the encoding residues of generated with a site and site S.L. Mariappan M. Schmidt B. von Figura K. Dierks T. J. 2008; PubMed Scopus Google as and the and The into the that with be that after the site the cysteine of by the as and The of and with The by of to and from an which FGE activity to a in the gene M.P. Pepe S. Parenti G. Settembre C. Annunziata I. Wade-Martins R. Di Domenico C. Di Natale P. Mankad A. Cox B. Uziel G. Mancini G.M. Zammarchi E. Donati M.A. Kleijer W.J. Filocamo M. Carrozzo R. Carella M. Ballabio A. Hum. Mutat. 2004; 23: 576-581Crossref PubMed Scopus (54) Google Scholar), by Ballabio of and and in by with by encoding the S. U. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and (11Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google or in a into and in with from to or from to The for after with a The for after with S.L. Mariappan M. Schmidt B. von Figura K. Dierks T. J. 2008; PubMed Scopus Google Scholar). The of or and with as M. Preusser-Kunze A. M. Schmidt B. Gande S.L. Wenzel D. Dierks T. von Figura K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). with as by or the or with of or two from a the and the with a of two of steroid sulfatase and of or two as indicated in the the after with and the and after for 52 is critical for biological activity of FGE. in STS, FGE, and FGE and in the indicated the The specific activity of to that in cysteine 52 is critical for of by as in A. of the indicated of and the or for and as of the by as M. Preusser-Kunze A. M. Schmidt B. Gande S.L. Wenzel D. Dierks T. von Figura K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). FGE (11Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google or M. Preusser-Kunze A. M. Schmidt B. Gande S.L. Wenzel D. Dierks T. von Figura K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google and proteins protein disulfide and used as The with secondary to or a and or and or or to in of with for and after for in of FGE or from and with an FGE or to a V. Schmidt B. von Figura K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and to and of FGE or the for Sulfatases and of and steroid sulfatase as von Figura K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). FGE, pFGE, or steroid sulfatase and a used as (11Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, M. Preusser-Kunze A. M. Schmidt B. Gande S.L. Wenzel D. Dierks T. von Figura K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). or used as secondary the FGE the of of FGE of and steroid sulfatase as i.e. to signal in or steroid sulfatase specific sulfatase i.e. catalytic activity by the signal and to that of the sulfatase specific sulfatase activity N-terminally FGE but is a protein, in sulfatase polypeptides a cysteine into the active site FGly residue that is for sulfatases to catalytically FGE is as a of amino with a cleavable signal of 33 residues its terminus (11Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). of mature FGE retention in the ER and is The of FGE lacks the N-terminal amino of mature FGE (residues but catalytic activity. a form of FGE N-terminal residues 34–68 we demonstrate catalytic activity in vitro when a as a that the C(T/S/C/A)PSR of human sulfatases (11Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). truncated FGE biological activity in we co-expressed encoding FGE or with encoding sulfatases in two and the catalytic activity of the sulfatases. The activity of the sulfatases generation of the FGly residue as an for the modification of the sulfatases by FGE. In the FGE in human that co-expressed as a We an to a in activity to FGE The not to the of FGE of truncated FGE with that of FGE not activity the that the terminus of FGE is required for the of sulfatases. In a we for from a with multiple sulfatase deficiency which FGE activity to a in the gene M.P. Pepe S. Parenti G. Settembre C. Annunziata I. Wade-Martins R. Di Domenico C. Di Natale P. Mankad A. Cox B. Uziel G. Mancini G.M. Zammarchi E. Donati M.A. Kleijer W.J. Filocamo M. Carrozzo R. Carella M. Ballabio A. Hum. Mutat. 2004; 23: 576-581Crossref PubMed Scopus (54) Google Scholar). of steroid sulfatase with FGE the activity of steroid sulfatase with steroid sulfatase of truncated FGE with steroid sulfatase to the activity of the sulfatase in this N-terminally FGE for the of to the of catalytically active sulfatases be that the N-terminal is required for the retention of FGE in the ER catalyzes the generation of FGly residues in nascent sulfatase the role of the N-terminal residues 34–68 for the retention of FGE in the ER, or and the of the FGE during a for to The that is with after for after the of the FGE is The of FGE to be for FGE and for of FGE in the ER of we fused to its C terminus the KDEL an ER signal used by ER The of the KDEL resulted in the retention of N-terminally truncated FGE in The of and that of FGE. of a and with the ER protein disulfide of the with steroid sulfatase steroid sulfatase activity but for FGE and that ER retention is not sufficient to restore the biological activity of of FGE a ER that the N-terminal residues 34–68 of FGE required for the biological activity and the retention of FGE in the ER. the terminus of FGE a ER retention we fused to secretory a protein we used a secretory form of is a paralog of FGE that lacks FGE activity. a unique from residue to of FGE and in the ER. is in the ER a S.L. Mariappan M. Schmidt B. von Figura K. Dierks T. J. 2008; PubMed Scopus Google Scholar), which is in FGE of the into a secretory protein S.L. Mariappan M. Schmidt B. von Figura K. Dierks T. J. 2008; PubMed Scopus Google Scholar). the two proteins is the N-terminal region of FGE (residues with residues by signal which is in We that residues 1–88 of FGE to the terminus of mature i.e. to to of the fused FGE residues to the of the with FGE. The retention after of pFGE, or the fusion by the of during a for to for of and of the secretory of the Fusing FGE residues 1–88 to the retention to that residues of FGE a ER retention that the of with the by FGE residues 1–88 to its terminus into a pFGE, with the ER protein disulfide The of a ER retention signal FGE residues by FGE residues 1–88 to a secretory protein. The of from for to for the terminus of FGE not of and in and pFGE, and in and with with and the or the ER protein disulfide with a The a of the with in or with as indicated and by a FGE ER or as in and Fusion of FGE in the of N-terminally truncated we the residues 1–88 of FGE its terminus restore the biological activity of N-terminally truncated FGE. of steroid sulfatase with FGE, but not with truncated FGE, to the synthesis of active steroid sulfatase and of steroid sulfatase with the N-terminal 1–88 of FGE fused to the secretory form of steroid sulfatase activity when in addition, truncated FGE, of steroid sulfatase as in of with truncated FGE not restore the of steroid sulfatase that the restoration of FGE activity by the fusion protein is to its of the fusion protein with FGE the of steroid sulfatase as for FGE and Fusing FGE residues 1–88 to proteins as or protein and the fusion proteins with N-terminally truncated FGE and steroid sulfatase not to an of steroid sulfatase not of the biological activity of the of the N-terminal residues by a with FGE. In an to the interaction of and N-terminally truncated FGE, the of the to the we is to ER retention of in the of a KDEL in the ER and the of is the This not when the two that their interaction is not to ER be that itself is in the ER, i.e. to the the to in the ER by a KDEL signal and to its retention of The that but retention of from to after of this a of ER retention of by the of KDEL this that both in the ER. 52 for the of we that FGE residues 34–68 mediate of FGE and that the stabilized by disulfide (11Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The N-terminal extension of FGE a conserved with the two and We cysteine cysteine 52 to and co-expressed the and FGE with steroid sulfatase in of cysteine 52 with the of steroid sulfatase as the of both cysteine of cysteine the biological activity of FGE, but of the sulfatase as as by FGE. that cysteine 52 is critical for the biological activity of FGE. cysteine 52 is critical for by that this cysteine residue a crucial role in the of the FGE catalytic core retention of FGE not by cysteine cysteine 52 to The the of two to the N-terminal extension of FGE. the retention of FGE in the the an in the of sulfatase that obviously is in The generation of active sulfatases the oxidation of a specific cysteine in nascent sulfatase polypeptides to a FGly the FGly-generating activity of FGE in its catalytic core domain residues this that the in vivo generation of FGly residues in nascent sulfatase polypeptides not the catalytic core domain of FGE but the N-terminal extension of FGE. This extension is in the of eukaryotic and an that be from and to eukaryotes We that FGE residues 34–68 required for the in vivo of FGE, residues sufficient to restore the generation of active sulfatases by N-terminally truncated FGE. This required the fusion of FGE residues 1–88 residues by signal to the Fusion of FGE residues 1–88 to as or protein or of the not restore the of sulfatases when co-expressed with the catalytic core domain of FGE not We from that the N-terminal extension of mature FGE, i.e. residues and that fusion to a is required to an active to FGE residues this active of a fusion is to enzymatic the catalytic core in the truncated FGE This obviously as by an retention of to of a form of FGE residues 34–68 required for the retention of FGE in the ER, and residues sufficient to mediate the retention of proteins in the ER. the as residues 34–68 as a ER retention signal to be The of ER retention of FGE is not The structure of the of FGE indicated that FGE is to which to of the residues of the to (11Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google but not to of the of which the of the in in the 1997; PubMed Scopus Google Scholar). This the that the N-terminal extension of FGE mediates its retention in the ER or its to the ER a to the is to that the retention of N-terminally truncated FGE is not sufficient to the generation of FGly residues in nascent sulfatase the of this we that the N-terminal extension of FGE mediates interaction with an the retention of FGE in the ER as as the generation of FGly residues in nascent sulfatase The of this component in the ER to be FGE biological activity be to the of nascent sulfatase or to cysteine to the that in vitro by or T. Schmidt B. Borissenko L.V. Peng J. Preusser A. Mariappan M. von Figura K. Cell. 2003; 113: 435-444Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). the N-terminal extension two conserved cysteine residues in and 52 as part of a bonds in to be J. Biol. Chem. Full Text PDF PubMed Google and to to In a disulfide bonds in the ER, the is part of a the cysteine of the to be and that in the active site of the to oxygen E. A. D. Biol. PubMed Scopus Google Scholar, A. J. Biol. 2005; PubMed Scopus Google Scholar). FGE in part is as a stabilized by disulfide cysteine 52 (11Preusser-Kunze A. Mariappan M. Schmidt B. Gande S.L. Mutenda K. Wenzel D. von Figura K. Dierks T. J. Biol. Chem. 2005; 280: 14900-14910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The disulfide of the is when the with as before Here we that cysteine but not is required for the in vivo activity of FGE. cysteine 52 cysteine to be required for retention of FGE in the ER. This to the that FGE in the ER and the in vivo activity of FGE two by the N-terminal extension of FGE. be that FGE is a ER protein that an retention cysteine to of an retention In we as a protein, which with FGE in a that the N-terminal extension of FGE. is a ER protein with a domain and a ER signal of the KDEL with a of proteins a that protein disulfide oxygen as a terminal electron acceptor. to to the the Ca2+ from the ER in and to a secretory protein of T. M. A. T. A. R. EMBO J. PubMed Scopus Google Scholar, T. M. T. T. T. K. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, T. A. Cell. Biol. 2007; PubMed Scopus Google Scholar, T. S. L. M. S. C. R. EMBO J. 2007; PubMed Scopus Google Scholar). of of proteins ER protein and activity. be to the interaction of FGE with is critical for the oxidation of a cysteine residue to FGly in nascent sulfatase polypeptides and mediates the retention of FGE in the ER. be that the of as an ER retention for FGE and is by Mariappan M. K. Dierks T. Schmidt B. von Figura K. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar).