Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum
2007; Springer Nature; Volume: 26; Issue: 10 Linguagem: Inglês
10.1038/sj.emboj.7601695
ISSN1460-2075
AutoresEster Zito, Mario Buono, Stefano Pepe, Carmine Settembre, Ida Annunziata, Enrico Maria Surace, Thomas Dierks, Maria Monti, Marianna Cozzolino, Piero Pucci, Andrea Ballabio, Maria Pia Cosma,
Tópico(s)Trypanosoma species research and implications
ResumoArticle19 April 2007free access Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum Ester Zito Ester Zito Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Mario Buono Mario Buono Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Stefano Pepe Stefano Pepe Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Carmine Settembre Carmine Settembre Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Ida Annunziata Ida Annunziata Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Enrico Maria Surace Enrico Maria Surace Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Thomas Dierks Thomas Dierks Department of Chemistry, Biochemistry I, Bielefeld University, Bielefeld, Germany Search for more papers by this author Maria Monti Maria Monti CEINGE Advanced Biotechnology and Department of Organic Chemistry and Biochemistry, Federico II University, Napoli, Italy Search for more papers by this author Marianna Cozzolino Marianna Cozzolino CEINGE Advanced Biotechnology and Department of Organic Chemistry and Biochemistry, Federico II University, Napoli, Italy Search for more papers by this author Piero Pucci Piero Pucci CEINGE Advanced Biotechnology and Department of Organic Chemistry and Biochemistry, Federico II University, Napoli, Italy Search for more papers by this author Andrea Ballabio Andrea Ballabio Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Medical Genetics, Department of Pediatrics, Faculty of Medicine, Federico II University, Naples, Italy Search for more papers by this author Maria Pia Cosma Corresponding Author Maria Pia Cosma Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Ester Zito Ester Zito Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Mario Buono Mario Buono Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Stefano Pepe Stefano Pepe Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Carmine Settembre Carmine Settembre Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Ida Annunziata Ida Annunziata Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Enrico Maria Surace Enrico Maria Surace Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Thomas Dierks Thomas Dierks Department of Chemistry, Biochemistry I, Bielefeld University, Bielefeld, Germany Search for more papers by this author Maria Monti Maria Monti CEINGE Advanced Biotechnology and Department of Organic Chemistry and Biochemistry, Federico II University, Napoli, Italy Search for more papers by this author Marianna Cozzolino Marianna Cozzolino CEINGE Advanced Biotechnology and Department of Organic Chemistry and Biochemistry, Federico II University, Napoli, Italy Search for more papers by this author Piero Pucci Piero Pucci CEINGE Advanced Biotechnology and Department of Organic Chemistry and Biochemistry, Federico II University, Napoli, Italy Search for more papers by this author Andrea Ballabio Andrea Ballabio Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Medical Genetics, Department of Pediatrics, Faculty of Medicine, Federico II University, Naples, Italy Search for more papers by this author Maria Pia Cosma Corresponding Author Maria Pia Cosma Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy Search for more papers by this author Author Information Ester Zito1, Mario Buono1, Stefano Pepe1, Carmine Settembre1, Ida Annunziata1, Enrico Maria Surace1, Thomas Dierks2, Maria Monti3, Marianna Cozzolino3, Piero Pucci3, Andrea Ballabio1,4 and Maria Pia Cosma 1 1Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy 2Department of Chemistry, Biochemistry I, Bielefeld University, Bielefeld, Germany 3CEINGE Advanced Biotechnology and Department of Organic Chemistry and Biochemistry, Federico II University, Napoli, Italy 4Medical Genetics, Department of Pediatrics, Faculty of Medicine, Federico II University, Naples, Italy *Corresponding author. Telethon Institute of Genetics and Medicine (TIGEM), via P Castellino 111, Naples 80131, Italy. Tel.: +39 081 6132226; Fax: +39 081 5609877; E-mail: [email protected] The EMBO Journal (2007)26:2443-2453https://doi.org/10.1038/sj.emboj.7601695 Correction(s) for this article Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum01 December 2016 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Sulfatase modifying factor 1 (SUMF1) is the gene mutated in multiple sulfatase deficiency (MSD) that encodes the formylglycine-generating enzyme, an essential activator of all the sulfatases. SUMF1 is a glycosylated enzyme that is resident in the endoplasmic reticulum (ER), although it is also secreted. Here, we demonstrate that upon secretion, SUMF1 can be taken up from the medium by several cell lines. Furthermore, the in vivo engineering of mice liver to produce SUMF1 shows its secretion into the blood serum and its uptake into different tissues. Additionally, we show that non-glycosylated forms of SUMF1 can still be secreted, while only the glycosylated SUMF1 enters cells, via a receptor-mediated mechanism. Surprisingly, following its uptake, SUMF1 shuttles from the plasma membrane to the ER, a route that has to date only been well characterized for some of the toxins. Remarkably, once taken up and relocalized into the ER, SUMF1 is still active, enhancing the sulfatase activities in both cultured cells and mice tissues. Introduction The sulfatases are a family of enzymes that catalyze the hydrolysis of sulfate esters after they have been post-translationally activated (Diez-Roux and Ballabio, 2005). A consensus sequence in their catalytic domain contains a cysteine that is modified into formylglycine (FGly) within the endoplasmic reticulum (ER). The FGly is essential for sulfatase activity, as in its hydrate form, FGly is able to accept the sulfate group from the substrate and subsequently remove it from the enzyme (Schmidt et al, 1995; Boltes et al, 2001). We and others have identified the gene, sulfatase modifying factor 1 (SUMF1), that encodes the FGly-generating enzyme (Cosma et al, 2003; Dierks et al, 2003). In patients affected by multiple sulfatase deficiency (MSD), all of their sulfatase activities are reduced because SUMF1 is hampered in its function, and mutations in SUMF1 have been found in all MSD patients analyzed to date (Cosma et al, 2004). The crystal structure of SUMF1 has been solved and SUMF1 has been recognized as having an oxygenase function (Dierks et al, 2005). Overexpression of SUMF1 with sulfatases in cultured cells via transfection or viral delivery results in a strong enhancement of the sulfatase activities (Cosma et al, 2003; Fraldi et al, 2007). SUMF1 has been conserved through evolution and has retained a high homology with the bacterial SUMF1 proteins (Sardiello et al, 2005). Sequence comparisons led to the discovery of a paralogue of SUMF1 in the vertebrate genome, known as SUMF2 (Cosma et al, 2003; Dierks et al, 2003). The primary sequences of the SUMF1 and SUMF2 proteins are highly similar. SUMF2 colocalizes with SUMF1 within the ER and inhibits the enhancing effect of SUMF1 on the sulfatases (Zito et al, 2005). SUMF1 is an ER-resident protein. It is glycosylated and its intracellular form contains high mannose-type oligosaccharides. SUMF1 is also secreted and the secreted form contains a family of complex-type oligosaccharides (Preusser-Kunze et al, 2005). Many secreted glycoproteins are cleared from the plasma through the mannose receptor (MR) (Lee et al, 2002). The MR is an endocytic receptor for glycans that is ubiquitously expressed. It contains carbohydrate recognition domains that bind complex-type oligosaccharides such as terminal fucose and acetylglucosamine residues (Taylor and Drickamer, 1993). Sulfatases are also secreted into the plasma and taken up via the mannose-6-phosphate receptor (MPR) (Ni et al, 2006). The MPR has two forms in the cell, MPR 46 and MPR 300, both of which can transport hydrolases from the trans-Golgi network to endosomes and/or lysosomes (Ni et al, 2006). We now show that endogenous SUMF1 is also secreted. Furthermore, we demonstrate that both overexpressed and endogenous SUMF1 can be taken up from the medium by different cells and from the plasma in tissues of mice engineered to produce SUMF1 in the liver. The uptake of SUMF1 is principally mediated by the MR, and to some extent by the MPR. The secretion and activity of SUMF1 are independent of its glycosylation; in contrast, its uptake in cells is impaired when the protein lacks sugars. Finally, we show here that once SUMF1 has been taken up, it relocalizes to the ER in both cultured cells and mice tissues, where it is enzymatically active, as it can enhance the sulfatase activities. Results SUMF1 is secreted and taken up into the ER SUMF1 is localized in the ER, where it activates all newly synthesized sulfatases during or shortly after cotranslational import (Cosma et al, 2003; Dierks et al, 2003). We have produced a stable HeLa cell clone that expresses SUMF1-3xFlag that we have named the HL3xFS1 clone. Three specific bands of about 42, 39 and 33 kDa that correspond to three forms of the SUMF1 protein were detected in cellular extracts from the HL3xFS1 clone using an anti-Flag antibody (Figure 1A). These different bands of SUMF1 correspond to its differently glycosylated and/or proteolytically processed forms, as has been previously demonstrated (Preusser-Kunze et al, 2005; Zito et al, 2005), and as shown by mass spectrometry analysis of protein extracts of the HL3xFS1 clone (Supplementary Figure 1). SUMF1 was predicted to be secreted through large-scale, bioinformatics, high-throughput screening (Clark et al, 2003). Recently, it was demonstrated that SUMF1 is secreted when it is overexpressed in HT1080 cells (Preusser-Kunze et al, 2005). As expected, Western blotting of conditioned medium collected from the HL3xFS1 clone showed secretion of SUMF1-3xFlag into the medium (Supplementary Figure 2A). In addition, by decorating the filter with antibodies against several different intracellular and secreted proteins, we have demonstrated that SUMF1-3xFlag is actively secreted into the medium, and have excluded that cell breakage was causing a leakage of SUMF1 into the medium. The overexpression of SUMF1-3xFlag also does not result in the nonspecific secretion of other endogenous proteins (Supplementary Figure 2A). Figure 1.Secretion and uptake of SUMF1. (A) SUMF1 expression in the HL3xFS1 clone. Three bands of about 42, 39 and 33 kDa were detected by Western blotting with an anti-Flag antibody. (B) SUMF1 is secreted from the HL3xFS1 clone and taken up into Cos7 and HeLa cells. Lanes 1–4: cellular extracts from recipient Cos7 and HeLa cells incubated with conditioned medium (HL3xFS1M) collected from the HL3xFS1 clone or with control HeLa cell medium, as analyzed by Western blotting. Lanes 5 and 6: 5% of SUMF1-Flag-conditioned and control medium used to culture the recipient cells. Lane 7: 10% of the total SUMF1-Flag-conditioned medium. Western blotting was carried out with anti-Flag and anti--tubulin antibodies. (C) SUMF1 is secreted from the HL3xFS1 clone and taken up in wild-type MEFs and Sumf1−/− MEFs. Lanes 1–4: cellular extracts from recipient cells cultured in SUMF1-Flag-conditioned and control medium were immunoblotted with anti-Flag and anti--tubulin antibodies. (D) Endogenous SUMF1 is secreted from HepG2 cells and taken up by MSD fibroblasts. HepG2 cells were cultured for 24, 36 and 48 h. Protein extracts and concentrated media were analyzed by Western blotting with an anti-human-SUMF1 antibody. MSD1 (p.A149-A173del+p.S359X) and MSD2 (p.M1R+rfs) fibroblasts were cultured for 24 h in concentrated HepG2 medium collected from two T75 flasks of confluent HepG2 cells. The protein extracts were analyzed using anti-SUMF1 and anti--tubulin antibodies. Download figure Download PowerPoint The sulfatases are secreted proteins that can be taken up into all cells of the body via the ubiquitously expressed MPR. Starting from this observation, we asked whether SUMF1 itself can also be taken up into cells from the medium. We have in the laboratory, mouse embryonic fibroblasts (MEFs) from a Sumf1−/− mouse model that we recently generated (Settembre et al, 2007). Thus, Cos7 and HeLa cells, and MEFs from wild-type and Sumf1−/− mice were incubated in conditioned medium collected from the confluent HL3xFS1 clone after 12 h of culture. The protein extracts from the recipient cells were analyzed by Western blotting with an anti-Flag antibody. SUMF1 was seen to be taken up in all three cell types (Figure 1B and C, lanes 1–4). Here, the controls included conditioned and non-conditioned medium (Figure 1B, lanes 5 and 6) and a sample of the conditioned medium that was not applied to recipient cells (Figure 1B, lane 7). Protein loading controls were also performed using an anti--tubulin antibody (Figure 1B and C). To confirm these results, we performed mass spectrometry analysis of SUMF1-Flag following its uptake into recipient HeLa cells incubated in conditioned medium collected from the HL3xFS1 clone. Protein extracts of the recipient cells were immunoprecipitated with an anti-Flag antibody and MALDIMS and LC-MS/MS analysis of the excised, deglycosylated and trypsin-digested bands were carried out. We clearly detected SUMF1 in bands 1 and 2 and did not detect it in band 3 (Supplementary Figure 1). These data clearly show that the overexpressed SUMF1 that is secreted into the medium by the HL3xFS1 clone is taken up into all the cell lines analyzed. By Western blotting, we detected primarily the uptake of the 42-kDa SUMF1 form, although under some conditions (e.g. when more protein is accumulated in the conditioned medium), the 39-kDa SUMF1 form can also be taken up, as shown in the mass spectrometry analysis (Supplementary Figure 1). We then investigated whether secretion and uptake of SUMF1 occurs only when SUMF1 is overexpressed, or whether the endogenous protein can also follow the same route. For this, we used HepG2 cells, which are a human hepatoma cell line that has been described as being highly secretory (Knowles et al, 1980). HepG2 cells were cultured for 8 h and the conditioned medium was analyzed. A specific, albeit faint, band was detected by Western blotting using an anti-human-SUMF1 polyclonal antibody (Supplementary Figure 2B). Thus, we performed time courses by collecting the conditioned medium after 24, 36 and 48 h of HepG2 cultivation. A positive signal for SUMF1 was detected in the cellular pellet and in the concentrated media from these HepG2 cells using an anti-human-SUMF1 polyclonal antibody (Figure 1D, top panels). These data demonstrate that endogenous SUMF1 is secreted from the HepG2 cells. Furthermore, for the uptake of this endogenous SUMF1, confluent recipient MSD fibroblasts were incubated in the concentrated conditioned medium (collected after 8 h of culture of 10 million confluent HepG2 cells). Of note, these two specifically chosen MSD fibroblast cell lines harbor mutations that cause a frameshift or the early truncation of SUMF1, and thus they did not have detectable levels of the endogenous protein. The endogenous SUMF1 of the HepG2 cells was indeed taken up by these MSD cells, as shown by Western blotting (Figure 1D, lower panels). A cellular extract of wild-type fibroblasts was also analyzed to indicate the control levels of SUMF1 in these cells (Figure 1D, lower panels, WT). The total proteins were normalized using an anti--tubulin antibody (Figure 1D, lower panels). Thus, the endogenous SUMF1 in HepG2 cells is secreted into the medium and can be taken up by human fibroblast cell lines. We next analyzed the subcellular localization of SUMF1 after its uptake. Sumf1−/− MEFs, Cos7 cells and MSD fibroblasts were incubated with SUMF1-Flag-conditioned medium. Indirect immunofluorescence using an anti-Flag antibody demonstrated that SUMF1-Flag localized in the perinuclear region of Sumf1−/− MEFs and Cos7 cells (Figure 2A and Supplementary Figure 3), with an ER localization confirmed in Sumf1−/− MEFs, Cos7 cells and MSD cells by its colocalization with the ER marker ERAB using anti-Flag, anti-SUMF1 and anti-ERAB antibodies (Figure 2B; Supplementary Figure 3, and data not shown). In addition, SUMF1 colocalized partly with the endosome markers LAMP1 and LAMP2, indicating that this uptake could be mediated via the endosomal compartment (Figure 2B, and data not shown). The localization of SUMF1 after its uptake resembles the localization of the endogenous SUMF1 protein in wild-type MEFs that colocalizes with the ERAB marker (Figure 2C). Vice versa, a SUMF1-specific signal was not seen in Sumf1−/− MEFs (Figure 2D). These results are striking since they show for the first time that apart from some of the toxins (Sandvig and van Deurs, 2000), other proteins appear to be taken up by cells and localized to the ER. Figure 2.Subcellular localization of SUMF1 taken up into Sumf1−/− MEFs cultured in the conditioned medium for 12 h. (A) SUMF1-Flag is taken up and localizes to the perinuclear region of Sumf1−/− MEFs. Internalized SUMF1-Flag is revealed using an anti-Flag antibody. DAPI is shown as a nuclear marker. (B) Subcellular localization of SUMF1 following its uptake into Sumf1−/− MEFs. SUMF1-Flag uptake in Sumf1−/− MEFs was revealed using an anti-SUMF1 antibody. SUMF1-Flag colocalizes with ERAB (an ER marker) and LAMP1 (an endosome marker), as seen by confocal microscopy. (C, D) Wild-type and Sumf1−/− MEFs were decorated with anti-SUMF1 and anti-ERAB antibodies. Scale bar, 5 μm. Download figure Download PowerPoint SUMF1 is enzymatically active after its uptake To determine if SUMF1 is enzymatically active after its uptake, the activities of different sulfatases were tested in recipient cells. First, we transfected Cos7 cells with IDS (iduronate sulfatase), SGSH (sulfamidase) and ARSA (arylsulfatase A) cDNAs. These cells were then incubated with SUMF1-conditioned medium or with a conditioned medium containing the fully inactive SUMF1C336R mutant (Cosma et al, 2004; Dierks et al, 2005). These conditioned media were recovered from HeLa cells that had been transfected with either wild-type SUMF1 cDNA or the SUMF1C336R mutant cDNA. The sulfatase activities were measured as previously described (Cosma et al, 2004), using specific substrates (Figure 3A). Enhancement of sulfatase activity was detected after the uptake of SUMF1 and not after the uptake of the SUMF1C336R mutant, with respect to the basal levels of the transfected sulfatases. Since IDS, SGSH and other sulfatases are also secreted and taken up, we wanted to be certain that these enhanced sulfatase activities measured in the Cos7 cells were actually due to uptake of SUMF1 and not to the uptake of endogenous sulfatases secreted into the conditioned medium by the HeLa cells. We thus used a lentiviral vector expressing SUMF1-3xFlag to transduce two different human MPSII and MPSIIIA fibroblast lines. MPSII and MPSIIIA are two mucopolysaccharidoses in which the IDS and SGSH enzymes, respectively, are inactive (Neufeld and Muenzer, 2001). The transduced fibroblasts were used as the SUMF1-producing cell lines so as to obtain medium containing active SUMF1 and inactive IDS or SGSH. MSD-recipient cells were incubated with these conditioned media. IDS activity was measured in the cell line cultured in the SUMF1-conditioned medium produced from the MPSII (II-1S1 and II-2S1)-transduced cells, whereas SGSH activity was measured in the MSD cell lines incubated in the SUMF1-conditioned medium produced from the MPSIIIA-transduced cells (IIIA-1S1 and IIIA-2S1). A partial rescue of both enzymatic activities was detected (Figure 3B). The IDS and SGSH activities measured in the MSD cell lines after uptake were higher with respect to the basal levels for both of these enzymes. SUMF1 expression in MPSII and MPSIIIA fibroblasts and its uptake into the MSD cell lines was also investigated by Western blotting (Figure 3B). In addition, to be further sure that the effects seen were due to SUMF1 uptake, we investigated the rescue of a sulfatase activity that is not secreted, ARSC (Ballabio and Shapiro, 1995). We thus incubated the MSD cell lines with SUMF1-conditioned medium from the HL3xFS1 clone. We measured a high level of ARSC activity after uptake in the MSD cell line. The levels detected were also much higher than the endogenous enzymatic ARSC activity of the MSD fibroblasts incubated with control HeLa cell-conditioned medium (Figure 3B). Finally, we performed uptake experiments using MEFs from Sumf1−/− mice as recipient cells. These cells have the main advantage of not having any residual sulfatase activity (Settembre et al, 2007). SUMF1 was produced from the MPSII- and MPSIIIA-producing cells and from the HL3xFS1 clone. A partial rescue of the IDS, SGSH and ARSC activities in the extracts from the recipient cells was seen (Figure 3C). The activities of IDS and SGSH were higher with respect to the basal levels in the Sumf1−/− MEFs; however, they were lower with respect to the levels measured in the wild-type MEFs. Of note, after the uptake of SUMF1, the ARSC activity was higher with respect to ARSC levels measured in both the Sumf1−/− and wild-type MEFs (Figure 3C). Likewise, although we clearly detected a rescue of the activity of different sulfatases in the MSD fibroblasts (Figure 3B), the measured enzymatic levels in the complemented MSD cell lines were lower with respect to the mean activities in wild-type fibroblasts (IDS was about 20% with respect to wild type, SGSH about 27% and ARSC about 80%; data not shown). Thus, to better detect the enhancing activity of internalized SUMF1, we decided to overexpress one sulfatase, IDS, in recipient MSD and Sumf1−/− cell lines using a lentiviral vector. The IDS-transduced MSD cells were incubated with SUMF1-conditioned media from each of the two different MPSII-producing cell lines: the IDS activities were increased by 30 and 76% in the recipient MSD cells after SUMF1 uptake (Figure 3D, II-2S1 and II-1S1, respectively). Similarly, the IDS-transduced Sumf1−/− MEFs were cultured with SUMF1-conditioned medium produced from Ids−/− MEFs transduced with a lentivirus expressing SUMF1. After SUMF1 uptake, the IDS activity was increased by 90% in the recipient Sumf1−/− cells (Figure 3D, Ids−/−S1). Altogether, these data demonstrate that SUMF1 is active after its uptake, as it can still enhance sulfatase activities. Figure 3.SUMF1 is enzymatically active following its uptake. (A) After its uptake, SUMF1 enhances the activity of transfected sulfatases. Cos7 cells were transfected with IDS, SGSH and ARSA cDNAs, and after 24 h, were incubated with medium containing SUMF1 or the inactive SUMF1C336R mutant. Sulfatase activities were measured in cellular extracts and compared with the activities in the extracts prepared from non-transfected cells (NT) and from sulfatase-transfected cells without SUMF1 uptake. (B) SUMF1 is taken up and can partially rescue sulfatase activities in MSD fibroblasts. Two different MPSII (II-1, II-2) and MPSIIIA (IIIA-1, IIIA-2) cell lines were transduced with a lentiviral vector carrying SUMF1-3xFlag cDNA to generate the conditioned-medium-producing II-1S1, II-2S1, IIIA-1S1 and IIIA-2S1 cells. The IDS and SGSH activities were analyzed in cellular pellets of producing cells and of MSD (p.S155P+S155P)-recipient fibroblasts. For ARSC activity, the producer cell line was the HL3xFS1 clone, and ARSC activity was evaluated in recipient cells and in control cells (MSD incubated in HeLa medium). Expression of SUMF1-Flag (producing cells, upper panels) and its uptake (recipient cells, lower panel) was also evaluated by Western blotting using an anti-Flag antibody. Western blotting with an anti--tubulin antibody provided the loading controls. (C) Uptake of SUMF1 in Sumf1−/− MEFs cultured in media collected from II-2S1 (for IDS activity), from IIIA-2S1 (for SGSH activity) and from HL3xFS1 (for ARSC activity) cells. The IDS, SGSH and ARSC activities in Sumf1−/− and wild-type MEFs are shown. (D) Uptake of SUMF1 in MSD fibroblasts and Sumf1−/− MEFs overexpressing IDS. The MSD+IDS-recipient cells were cultured in SUMF1-conditioned medium collected from II-2S1 and II-1S1-producing cells. The Sumf1−/−+IDS-recipient cells were cultured in SUMF1-conditioned medium collected from Ids−/− MEFs transduced with a lentivirus overexpressing SUMF1 cDNA. The IDS activities are expressed as percentages of the increased activities of the recipient cells cultured in the SUMF1-conditioned medium versus the activity measured in the recipient cells cultured in non-conditioned medium. Download figure Download PowerPoint Glycosylation of SUMF1 is not essential for its secretion and function SUMF1 is N-glycosylated on its asparagine 141 (N141; Preusser-Kunze et al, 2005). To analyze the role of the glycosyl residues in the secretion and activity of SUMF1, we mutated N141 to alanine in the SUMF1-3xFlag cDNA. The SUMF1N141A mutant protein was detected in cellular extracts of HeLa cells transfected with the mutated cDNA and showed a profile of migrating bands slower with respect to wild-type SUMF1 (Figure 4A, lanes 1 and 3). SUMF1N141A is not sensitive to EndoH digestion (Figure 4A, lane 4) and the Western blotting showed a pattern of bands equivalent to those of SUMF1 digested with EndoH (Figure 4A, compare lanes 1 and 4), further demonstrating that SUMF1N141A is not glycosylated. In addition, the protein correctly localized in the ER when the mutant cDNA was transfected in HeLa cells (Figure 4B). Surprisingly, SUMF1N141A was still secreted into the medium, and furthermore, the secreted mutant was insensitive to the digestion with PNGase F, as expected (Figure 4C, upper panel). This enzyme cleaves between the innermost GlcNAC and asparagine residues of the glycosylated content and has previously been demonstrated to digest the secreted SUMF1 forms (Preusser-Kunze et al, 2005). Finally, we did not detect uptake of SUMF1N141A in HeLa cells cultured in medium containing the mutant SUMF1 form (Figure 4C, lower panel). Figure 4.SUMF1 secretion and activity is independent of its glycosylation. (A) Glycosylation analysis. HeLa cells were transfected with SUMF1-3xFlag, SUMF1421G>A,422C>A-3xFlag (transduced to SUMF1N141A-3xFlag) and xSumf1-3xFlag. After 48 h, the cells were harvested and the cellular extracts were treated with EndoH, as indicated. The samples were analyzed by Western blotting and the filters were decorated with an anti-Flag antibody. The pattern of bands of SUMF1 in the second lane shows EndoH-sensitive glycosylation. (B) SUMF1N141A localizes to the ER. HeLa cells transfected with SUMF1421G>A,422C>A-3xFlag were processed for indirect immunofluorescence using anti-Flag and anti-ERAB antibodies. SUMF1N141A colocalizes with ERAB (an ER marker), as seen by confocal microscopy. (C) SUMF1N141A is secreted from transfected HeLa cells. Media were collected and concentrated. The conditioned medium containing SUMF1N141A was digested with PNGase F. The samples were analyzed by Western blotting using an anti-Flag antibody (upper panels). For uptake, HeLa cells were cultured for 12 h in concentrated medium collected from one T75 flask of confluent cells transfected with SUMF1-3xFlag or SUMF1421G>A,422C>A-3xFlag. The protein extracts were analyzed using anti-Flag and anti--tubulin antibodies (lower panels). (D) SUMF1 secreted from HL3xFS1 contains PNGase F-sensitive glycosylation. Media collected from the HL3xFS1 clone were treated with PNGase F, as indicated (upper panel). HeLa cells were cultured in media treated and not treated with PNGase F (lower panel). The samples were analyzed by Western blotting. (E) xSumf1 localizes to the ER. HeLa cells transfected with xSumf1-3xFlag were analyzed by indirect immunofluorescence using anti-Flag and anti-ERAB antibodies. xSumf1 colocalized with the ERAB marker, as seen by confocal microscopy. (F) xSumf1 is secreted from transfected HeLa cells. Media were collected, concentrated and analyzed by Western blotting using an anti-Flag antibody (upper panels). For uptake, HeLa cells were cultured for 12 h in concentrated conditioned medium collected from one T75 flask of confluent cells transfected with SUMF1-3xFlag or xSumf1-3xFlag. The protein extracts were analyzed using an anti-Flag antibody (lower panels). (G) SUMF1N141A and xSumf1 retain the enhancing activity on IDS and ARSC. ARSC and IDS were transfected alone or in combination with SUMF1-3xFlag, xSumf1-3xFlag, dSumf1-3xFlag or SUMF1421G>A,422C>A-3xFlag in HeLa cells. The IDS and ARSC activities are expressed as percentages of the increased activities in cells expressing only the sulfatase versus the activities measured in cells expressing the sulfatase plus SUMF1, xSumf1, dSumf1 or SUMF1N141A. Download figure Download PowerPoint To investigate whe
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