SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies

Sulfatases are enzymes that hydrolyse a diverse range of sulfate esters. Deficiency of lysosomal sulfatases leads to human diseases characterized by the accumulation of either GAGs (glycosaminoglycans) or sulfolipids. The catalytic activity of sulfatases resides in a unique formylglycine residue in their active site generated by the post-translational modification of a highly conserved cysteine residue. This modification is performed by SUMF1 (sulfatase-modifying factor 1), which is an essential factor for sulfatase activities. Mutations in the SUMF1 gene cause MSD (multiple sulfatase deficiency), an autosomal recessive disease in which the activities of all sulfatases are profoundly reduced. In previous studies, we have shown that SUMF1 has an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells. In the present study, we demonstrate that SUMF1 displays an enhancing effect on sulfatases activity when co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by five different diseases owing to sulfatase deficiencies or from murine models of the same diseases [i.e. MLD (metachromatic leukodystrophy), CDPX (X-linked dominant chondrodysplasia punctata) and MPS (mucopolysaccharidosis) II, IIIA and VI]. The SUMF1-enhancing effect on sulfatase activity resulted in an improved clearance of the intracellular GAG or sulfolipid accumulation. Moreover, we demonstrate that the SUMF1-enhancing effect is also present in vivo after AAV-mediated delivery of the sulfamidase gene to the muscle of MPSIIIA mice, resulting in a more efficient rescue of the phenotype. These results indicate that co-delivery of SUMF1 may enhance the efficacy of gene therapy in several sulfatase deficiencies.     

Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum

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.     

Sulphatase activities are regulated by the interaction of sulphatase-modifying factor 1 with SUMF2

Sulphatases undergo a unique post-translational modification that converts a highly conserved cysteine located within their active site into formylglycine. This modification is necessary for the catalytic activities of the sulphatases, and it is generated by the protein product of sulphatase-modifying factor 1 (SUMF1), the gene mutated in multiple sulphatase deficiency (MSD). A paralogous gene, SUMF2, was discovered through its sequence similarity to SUMF1. We present evidence that SUMF2 colocalizes with SUMF1 within the endoplasmic reticulum and that the two proteins form heterodimers. SUMF1 and SUMF2 also form homodimers. In addition, SUMF2 is able to associate with the sulphatases with and without SUMF1. We have previously shown that co-transfection of SUMF1 with the sulphatase complementary DNAs greatly enhances the activities of the overexpressed sulphatases. Here, we show that SUMF2 inhibits the enhancing effects of SUMF1 on sulphatases, suggesting that the SUMF1-SUMF2 interaction represents a further level of control of these sulphatase activities.     

Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme

C(alpha)-formylglycine (FGly) is the catalytic residue in the active site of eukaryotic sulfatases. It is posttranslationally generated from a cysteine in the endoplasmic reticulum. The genetic defect of FGly formation causes multiple sulfatase deficiency (MSD), a lysosomal storage disorder. We purified the FGly generating enzyme (FGE) and identified its gene and nine mutations in seven MSD patients. In patient fibroblasts, the activity of sulfatases is partially restored by transduction of FGE encoding cDNA, but not by cDNA carrying an MSD mutation. The gene encoding FGE is highly conserved among pro- and eukaryotes and has a paralog of unknown function in vertebrates. FGE is localized in the endoplasmic reticulum and is predicted to have a tripartite domain structure.     

A major step on the road to understanding a unique posttranslational modification and its role in a genetic disease

The posttranslational conversion of cysteine to C(alpha)-formylglycine in the catalytic site of mammalian sulfatases is deficient in the rare but devastating disorder multiple sulfatase deficiency (MSD). Two papers in this issue of Cell report the cloning of a gene responsible for this activity.     

Complementation of multiple sulfatase deficiency in somatic cell hybrids

Multiple sulfatase deficiency (MSD) is an inherited disorder characterized by deficient activity of seven different sulfatases. Genetic complementation for steroid sulfatase (STS), arylsulfatase A, and N-acetylgalactosamine 6-SO4 sulfatase was demonstrated in somatic cell hybrids between MSD fibroblasts and mouse cells ( LA9 ) or Chinese hamster cells ( CHW ). In an electrophoretic system that separates human and rodent STS isozymes, enzyme from hybrids migrated as human enzyme. We concluded that the rodent cell complemented the MSD deficiency and allowed normal expression of the STS structural gene. Some MSD- LA9 hybrids showed significant levels of human arylsulfatase A activity, as shown by the immunoprecipitation of active enzyme by human-specific antiserum. Complementation was also suggested for N-acetylgalactosamine 6- sulfatate sulfatase (GalNAc-6S sulfatase) in several MSD- LA9 hybrids by the demonstration of a significant increase in activity (10-fold) over that of the GalNAc-6S sulfatase-deficient parental mouse and MSD cells. Thus, it was possible to demonstrate complementation for more than one sulfatase in a single MSD-rodent hybrid. Normal levels of sulfatase activity in hybrids indicate that the sulfatase structural genes are intact in MSD cells.     

Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycine-generating enzyme

Sulfatases are enzymes essential for degradation and remodeling of sulfate esters. Formylglycine (FGly), the key catalytic residue in the active site, is unique to sulfatases. In higher eukaryotes, FGly is generated from a cysteine precursor by the FGly-generating enzyme (FGE). Inactivity of FGE results in multiple sulfatase deficiency (MSD), a fatal autosomal recessive syndrome. Based on the crystal structure, we report that FGE is a single-domain monomer with a surprising paucity of secondary structure and adopts a unique fold. The effect of all 18 missense mutations found in MSD patients is explained by the FGE structure, providing a molecular basis of MSD. The catalytic mechanism of FGly generation was elucidated by six high-resolution structures of FGE in different redox environments. The structures allow formulation of a novel oxygenase mechanism whereby FGE utilizes molecular oxygen to generate FGly via a cysteine sulfenic acid intermediate.     

Molecular basis of multiple sulfatase deficiency,mucolipidosis II/III and Niemann–Pick C1 disease — Lysosomal storage disorders caused by defects of non-lysosomal proteins

Multiple sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann–Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further, the discovery of FGE as an essential sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single sulfatase deficiencies.     

Expression, localization, structural, and functional characterization of pFGE, the paralog of the Calpha-formylglycine-generating enzyme

pFGE is the paralog of the formylglycine-generating enzyme (FGE), which catalyzes the oxidation of a specific cysteine to Calpha-formylglycine, the catalytic residue in the active site of sulfatases. The enzymatic activity of sulfatases depends on this posttranslational modification, and the genetic defect of FGE causes multiple sulfatase deficiency. The structural and functional properties of pFGE were analyzed. The comparison with FGE demonstrates that both share a tissue-specific expression pattern and the localization in the lumen of the endoplasmic reticulum. Both are retained in the endoplasmic reticulum by a saturable mechanism. Limited proteolytic cleavage at similar sites indicates that both also share a similar three-dimensional structure. pFGE, however, is lacking the formylglycine-generating activity of FGE. 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.     

Inhibition of endogenous miR-23a/miR-377 in CHO cells enhances difficult-to-express recombinant lysosomal sulfatase activity

Difficult-to-express (DTE) recombinant proteins such as multi-specific proteins, DTE monoclonal antibodies, and lysosomal enzymes have seen difficulties in manufacturability using Chinese hamster ovary (CHO) cells or other mammalian cells as production platforms. CHO cells are preferably used for recombinant protein production for their ability to secrete human-like recombinant proteins with posttranslational modification, resistance to viral infection, and familiarity with drug regulators. However, despite huge progress made in engineering CHO cells for high volumetric productivity, DTE proteins like recombinant lysosomal sulfatase represent one of the poorly understood proteins. Furthermore, there is growing interest in the use of microRNA (miRNA) to engineer CHO cells expressing DTE proteins to improve cell performance of relevant bioprocess phenotypes. To our knowledge, no research has been done to improve CHO cell production of DTE recombinant lysosomal sulfatase using miRNA. We identified miR-23a and miR-377 as miRNAs predicted to target SUMF1, an activator of sulfatases, using in silico prediction tools. Transient inhibition of CHO endogenous miR-23a/miR-377 significantly enhanced recombinant sulfatase enzyme-specific activity by ~15–21% compared to scramble without affecting cell growth. Though inhibition of miR-23a/miR-377 had no significant effect on the mRNA and protein levels of SUMF1, overexpression of miR-23a/377 caused ~30% and ~27–29% significant reduction in endogenous SUMF1 protein and mRNA expression levels, respectively. In summary, our data demonstrate the importance of using miRNA to optimize the CHO cell line secreting DTE recombinant lysosomal sulfatase.     

Cloning of a mucin-desulfating sulfatase gene from Prevotella strain RS2 and its expression using a Bacteroides recombinant system

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.     

Genetic manipulation of murine embryonic stem cells with enhanced green fluorescence protein and sulfatase-modifying factor I genes

Background aimsMucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) in which an absence of sulfamidase results in incomplete degradation and subsequent accumulation of its substrate, heparan sulfate. Most neurodegenerative LSD remain untreatable. However, therapy options, such as gene, enzyme end cell therapy, are under investigation. Previously, we have constructed an embryonic stem (ES) cell line (NS21) that over-expresses human sulphamidase as a potential treatment for murine MPS IIIA.MethodsIn the present study the sulfatase-modifying factor I (SUMF1) and enhanced green fluorescence protein (eGFP) genes were co-introduced under a cytomegalovirus (CMV) promoter into NS21 cells, to enhance further sulfamidase activity and provide a marker for in vivo cell tracking, respectively. eGFP was also introduced under the control of the human elongation factor-1α (hEF-1α) promoter to compare the stability of transgene expression.ResultsDuring differentiation of ES cells into glial precursors, SUMF1 was down-regulated and was hardly detectable by day 18 of differentiation. Likewise, eGFP expression was heterogeneous and highly unstable. Use of a human EF-1α promoter resulted in more homogeneous eGFP expression, with ～ 50% of cells eGFP positive following differentiation into glial precursors. Compared with NS21 cells, the outgrowth of eGFP-expressing cells was not as confluent when differentiated into glial precursors.ConclusionsOur data suggest that SUMF1 enhances sulfamidase activity in ES cells, hEF-1α is a stronger promoter than CMV for ES cells and over-expression of eGFP may affect cell growth and contribute to unstable gene expression.     

A novel protein modification generating an aldehyde group in sulfatases: its role in catalysis and disease

In multiple sulfatase deficiency, a rare human lysosomal storage disorder, all known sulfatases are synthesized as catalytically poorly active polypeptides. Analysis of the latter has shown that they lack a protein modification that was detected in all members of the sulfatase family. This novel protein modification generates a 2-amino-3-oxopropanoic acid (Cα-formylglycine) residue by oxidation of the thiol group of a cysteine that is conserved among all eukaryotic sulfatases. The oxidation occurs in the endoplasmic reticulum at a stage when the nascent polypeptide is not yet folded. The aldehyde is part of the catalytic site and is likely to act as an aldehyde hydrate. One of the geminal hydroxyl groups accepts the sulfate during sulfate ester cleavage leading to the formation of a covalently sulfated enzyme intermediate. The other hydroxyl is required for the subsequent elimination of the sulfate and regeneration of the aldehyde group. In some prokaryotic members of the sulfatase gene family, the DNA sequence predicts a serine residue, and not a cysteine. Analysis of one of these prokaryotic sulfatases, however, revealed the presence of the Cα-formylglycine indicating that the aldehyde group is essential for all members of the sulfatase family and that it can be generated from either cysteine or serine. BioEssays 20 :505–510, 1998. © 1998 John Wiley & Sons, Inc.     

A superfamily of metalloenzymes unifies phosphopentomutase and cofactor-independent phosphoglycerate mutase with alkaline phosphatases and sulfatases

Sequence analysis of the probable archaeal phosphoglycerate mutase resulted in the identification of a superfamily of metalloenzymes with similar metal-binding sites and predicted conserved structural fold. This superfamily unites alkaline phosphatase, N-acetylgalactosamine-4-sulfatase, and cerebroside sulfatase, enzymes with known three-dimensional structures, with phosphopentomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, phosphoglycerol transferase, phosphonate monoesterase, streptomycin-6-phosphate phosphatase, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1, and several closely related sulfatases. In addition to the metal-binding motifs, all these enzymes contain a set of conserved amino acid residues that are likely to be required for the enzymatic activity. Mutational changes in the vicinity of these residues in several sulfatases cause mucopolysaccharidosis (Hunter, Maroteaux-Lamy, Morquio, and Sanfilippo syndromes) and metachromatic leucodystrophy.     

Manganese deficiency in Chlamydomonas results in loss of photosystem II and MnSOD function, sensitivity to peroxides, and secondary phosphorus and iron deficiency

For photoheterotrophic growth, a Chlamydomonas reinhardtii cell requires at least 1.7 x 10(7) manganese ions in the medium. At lower manganese ion concentrations (typically <0.5 microm), cells divide more slowly, accumulate less chlorophyll, and the culture reaches stationary phase at lower cell density. Below 0.1 microm supplemental manganese ion in the medium, the cells are photosynthetically defective. This is accompanied by decreased abundance of D1, which binds the Mn(4)Ca cluster, and release of the OEE proteins from the membrane. Assay of Mn superoxide dismutase (MnSOD) indicates loss of activity of two isozymes in proportion to the Mn deficiency. The expression of MSD3 through MSD5, encoding various isoforms of the MnSODs, is up-regulated severalfold in Mn-deficient cells, but neither expression nor activity of the plastid Fe-containing superoxide dismutase is changed, which contrasts with the dramatically increased MSD3 expression and plastid MnSOD activity in Fe-deficient cells. Mn-deficient cells are selectively sensitive to peroxide but not methyl viologen or Rose Bengal, and GPXs, APX, and MSRA2 genes (encoding glutathione peroxidase, ascorbate peroxidase, and methionine sulfoxide reductase 2) are slightly up-regulated. Elemental analysis indicates that the Mn, Fe, and P contents of cells in the Mn-deficient cultures were reduced in proportion to the deficiency. A natural resistance-associated macrophage protein homolog and one of five metal tolerance proteins were induced in Mn-deficient cells but not in Fe-deficient cells, suggesting that the corresponding gene products may be components of a Mn(2+)-selective assimilation pathway.     

SUMF2对炎症的影响

炎症是机体的一种适应性反应,失调时将会对自身机体造成损伤。研究表明许多疾病的发生发展均与炎症的失调有关。硫酸酯酶作为人体不可或缺的蛋白质家族与炎症之间有着密切关联。发热是炎症最主要的表现形式,硫酸酯酶修饰因子2(sulfatase-modifying factor 2,SUMF2)参与硫酸酯酶的激活且与发热密切相关。脂多糖刺激人体后,SUMF2的表达明显降低。SUMF2已被证实与白细胞介素-13(Interleukin-13,IL-13)之间存在相互作用,且能在体外水平抑制IL-13的分泌,IL-13可以通过抑制炎性因子的产生发挥抗炎作用。本文将就硫酸酯酶、SUMF2及其对炎症之间的影响进行综述。     

Synthesis and stability of arylsulfatase A and B in fibroblasts from multiple sulfatase deficiency

Fibroblasts from patients with multiple sulfatase deficiency were analyzed for activities of arylsulfatase A and B, iduronate 2-sulfatase and sulfamatase. A group of patients (group I) severely deficient in all sulfatases (residual activities less than or equal to 10% of control) were differentiated from patients (group II) with residual sulfatase activities of up to 90% of control. The synthesis and stability of arylsulfatase A and B were determined in pulse-chase labelling experiments. The apparent rate of synthesis of arylsulfatase A and B varied from 30% to normal in both fibroblasts from group I and II multiple sulfatase deficiency. In group I the molecular activity of the arylsulfatase A and B was more than 10-fold lower than in control fibroblasts. In group II the molecular activity of the arylsulfatase A was twofold to threefold lower and that of arylsulfatase B half of normal. In fibroblasts of both groups the stability of arylsulfatase A polypeptides was significantly diminished. For arylsulfatase B the instability was restricted to the mature 47000-Mr polypeptide and was variable within both groups. These results demonstrate that multiple sulfatase deficiency is a heterogeneous disorder, in which the primary defects can impair both the catalytic properties and the stability of sulfatases.     

Seed‐specific overexpression of antioxidant genes in Arabidopsis enhances oxidative stress tolerance during germination and early seedling growth

Enzymatic and non‐enzymatic antioxidants play important roles in the tolerance of abiotic stress. To increase the resistance of seeds to oxidative stress, At2S3 promoter from Arabidopsis was used to achieve overexpression of the antioxidants in a seed‐specific manner. This promoter was shown to be capable of driving the target gene to have a high level of expression in seed‐related organs, including siliques, mature seeds, and early seedlings, thus making its molecular farming applications in plants possible. Subsequently, genes encoding Mn‐superoxide dismutase (MSD1), catalase (CAT1), and homogentisate phytyltransferase (HPT1, responsible for the first committed reaction in the tocopherol biosynthesis pathway) were overexpressed in Arabidopsis under the control of the At2S3 promoter. Double overexpressers co‐expressing two enzymes and triple overexpressers were produced by cross pollination. Mn‐SOD and total CAT activities, as well as γ‐tocopherol content, significantly increased in the corresponding overproduction lines. Moreover, single MSD1‐transgene, double, and triple overexpressers displayed remarkably enhanced oxidative stress tolerance compared to wild type during seed germination and early seedling growth. Interestingly, an increase in the total CAT activity was also observed in the single MSD1‐transgenic lines as a result of MSD1 overexpression. Together, the combined increase in Mn‐SOD and CAT activities in seeds plays an essential role in the improvement of antioxidant capacity at early developmental stage in Arabidopsis.