Biochemical signatures of disease severity in multiple sulfatase deficiency.

Sulfatases catalyze essential cellular reactions, including degradation of glycosaminoglycans (GAGs). All sulfatases are post-translationally activated by the formylglycine generating enzyme (FGE) which is deficient in multiple sulfatase deficiency (MSD), a neurodegenerative lysosomal storage disease. Historically, patients were presumed to be deficient of all sulfatase activities; however, a more nuanced relationship is emerging. Each sulfatase may differ in their degree of post-translational modification by FGE, which may influence the phenotypic spectrum of MSD. Here, we evaluate if residual sulfatase activity and accumulating GAG patterns distinguish cases from controls and stratify clinical severity groups in MSD. We quantify sulfatase activities and GAG accumulation using three complementary methods in MSD participants. Sulfatases differed greatly in their tolerance of reduction in FGE-mediated activation. Enzymes that degrade heparan sulfate (HS) demonstrated lower residual activities than those that act on other GAGs. Similarly, HS-derived urinary GAG subspecies preferentially accumulated, distinguished cases from controls, and correlated with disease severity. Accumulation patterns of specific sulfatase substrates in MSD provide fundamental insights into sulfatase regulation and will serve as much-needed biomakers for upcoming clinical trials. This work highlights that biomarker investigation of an ultra-rare disease can simultaneously inform our understanding of fundamental biology and advance clinical trial readiness efforts.

New mouse models with hypomorphic SUMF1 variants mimic attenuated forms of multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultrarare lysosomal storage disorder due to deficiency of all known sulfatases. MSD is caused by mutations in the Sulfatase Modifying Factor 1 (SUMF1) gene encoding the enzyme responsible for the post-translational modification and activation of all sulfatases. Most MSD patients carry hypomorph SUMF1 variants resulting in variable degrees of residual sulfatase activities. In contrast, Sumf1 null mice with complete deficiency in all sulfatase enzyme activities, have very short lifespan with significant pre-wean lethality, owing to a challenging preclinical model. To overcome this limitation, we genetically engineered and characterized in mice two commonly identified patient-based SUMF1 pathogenic variants, namely p.Ser153Pro and p.Ala277Val. These pathogenic missense variants correspond to variants detected in patients with attenuated MSD presenting with partial-enzyme deficiency and relatively less severe disease. These novel MSD mouse models have a longer lifespan and show biochemical and pathological abnormalities observed in humans. In conclusion, mice harboring the p.Ser153Pro or the p.Ala277Val variant mimic the attenuated MSD and are attractive preclinical models for investigation of pathogenesis and treatments for MSD.

Natural history of multiple sulfatase deficiency: Retrospective phenotyping and functional variant analysis to characterize an ultra-rare disease.

Multiple sulfatase deficiency (MSD) is an ultra-rare neurodegenerative disorder caused by pathogenic variants in SUMF1. This gene encodes formylglycine-generating enzyme (FGE), a protein required for sulfatase activation. The clinical course of MSD results from additive effect of each sulfatase deficiency, including metachromatic leukodystrophy (MLD), several mucopolysaccharidoses (MPS II, IIIA, IIID, IIIE, IVA, VI), chondrodysplasia punctata, and X-linked ichthyosis. While it is known that affected individuals demonstrate a complex and severe phenotype, the genotype-phenotype relationship and detailed clinical course is unknown. We report on 35 cases enrolled in our retrospective natural history study, n = 32 with detailed histories. Neurologic function was longitudinally assessed with retrospective scales. Biochemical and computational modeling of novel SUMF1 variants was performed. Genotypes were classified based on predicted functional change, and each individual was assigned a genotype severity score. The median age at symptom onset was 0.25 years; median age at diagnosis was 2.7 years; and median age at death was 13 years. All individuals demonstrated developmental delay, and only a subset of individuals attained ambulation and verbal communication. All subjects experienced an accumulating systemic symptom burden. Earlier age at symptom onset and severe variant pathogenicity correlated with poor neurologic outcomes. Using retrospective deep phenotyping and detailed variant analysis, we defined the natural history of MSD. We found that attenuated cases can be distinguished from severe cases by age of onset, attainment of ambulation, and genotype. Results from this study can help inform prognosis and facilitate future study design.

Multiple Sulfatase Deficiency: A Disease Comprising Mucopolysaccharidosis, Sphingolipidosis, and More Caused by a Defect in Posttranslational Modification.

Multiple sulfatase deficiency (MSD, MIM #272200) is an ultra-rare disease comprising pathophysiology and clinical features of mucopolysaccharidosis, sphingolipidosis and other sulfatase deficiencies. MSD is caused by impaired posttranslational activation of sulfatases through the formylglycine generating enzyme (FGE) encoded by the sulfatase modifying factor 1 (SUMF1) gene, which is mutated in MSD. FGE is a highly conserved, non-redundant ER protein that activates all cellular sulfatases by oxidizing a conserved cysteine in the active site of sulfatases that is necessary for full catalytic activity. SUMF1 mutations result in unstable, degradation-prone FGE that demonstrates reduced or absent catalytic activity, leading to decreased activity of all sulfatases. As the majority of sulfatases are localized to the lysosome, loss of sulfatase activity induces lysosomal storage of glycosaminoglycans and sulfatides and subsequent cellular pathology. MSD patients combine clinical features of all single sulfatase deficiencies in a systemic disease. Disease severity classifications distinguish cases based on age of onset and disease progression. A genotype- phenotype correlation has been proposed, biomarkers like excreted storage material and residual sulfatase activities do not correlate well with disease severity. The diagnosis of MSD is based on reduced sulfatase activities and detection of mutations in SUMF1. No therapy exists for MSD yet. This review summarizes the unique FGE/ sulfatase physiology, pathophysiology and clinical aspects in patients and their care and outlines future perspectives in MSD.

Comparative Study of Antifungal Efficacy of Various Endodontic Irrigants with and without Clotrimazole in Extracted Teeth Inoculated with Candida albicans.

AIM AND OBJECTIVE: To assess the application of clotrimazole (1%) as a complementary antifungal agent along with sodium hypochlorite (5.25%), chlorhexidine gluconate (2%), and doxycycline hydrochloride (5%) against Candida albicans. MATERIALS AND METHODS: Seventy freshly extracted single-rooted premolars with matured apices were collected, stored, and handled according to the Occupational Safety and Health Administration (OSHA) and the Center for Disease Control and Prevention (CDC) guidelines and recommendations. These were divided into three groups (two tests and one control group) depending on irrigants used. The efficacy of each irrigant group was compared. The observations were statistically analyzed by the multiple intergroup comparisons using ANOVA and Scheffe multiple comparisons (p < 0.001). RESULTS: The sodium hypochlorite (group IA-mean 129.6) has shown a statistically significant decrease in colony-forming units (CFUs) (p < 0.01) on comparison with chlorhexidine [(IB) mean 190.2]. A similar result was obtained in comparison with the sodium hypochlorite group (IA) and doxycycline HCl group [(IC) mean 318.4] and also between the sodium hypochlorite group (IA) and the control group [(III) mean 554.2]. The intragroup comparison of group II, group IIA (mean 63.3), and group IIB (mean 73.8) showed no statistically significant difference. Group III (mean 554.2) was the least effective of all the subgroups. CONCLUSION: Sodium hypochlorite showed better antifungal efficacy than chlorhexidine and doxycycline when used alone. The addition of clotrimazole increased the efficiency of doxycycline also, but it was less compared to sodium hypochlorite and chlorhexidine. Within the limitations of this study, the inclusion of 1% clotrimazole increased the antifungal efficacy of all the three irrigants. CLINICAL SIGNIFICANCE: Our study compared the efficacy of the various endodontic irrigants and also determined their efficiency with the addition of the antifungal agent. Clotrimazole (1%) addition in irrigating solutions showed better results and promoted faster healing.

Expanding the genetic cause of multiple sulfatase deficiency: A novel SUMF1 variant in a patient displaying a severe late infantile form of the disease.

Multiple sulfatase deficiency (MSD) is a rare inherited metabolic disease caused by defective cellular sulfatases. Activity of sulfatases depends on post-translational modification catalyzed by formylglycine-generating enzyme (FGE), encoded by the SUMF1 gene. SUMF1 pathologic variants cause MSD, a syndrome presenting with a complex phenotype. We describe the first Polish patient with MSD caused by a yet undescribed pathologic variant c.337G>A [p.Glu113Lys] (i.e. p.E113K) in heterozygous combination with the known deletion allele c.519+5_519+8del [p.Ala149_Ala173del]. The clinical picture of the patient initially suggested late infantile metachromatic leukodystrophy, with developmental delay followed by regression of visual, hearing and motor abilities as the most apparent clinical symptoms. Transient signs of ichthyosis and minor dysmorphic features guided the laboratory workup towards MSD. Since MSD is a rare disease and there is a variable clinical spectrum, we thoroughly describe the clinical outcome of our patient. The FGE-E113K variant, expressed in cell culture, correctly localized to the endoplasmic reticulum but was retained intracellularly in contrast to the wild type FGE. Analysis of FGE-mediated activation of steroid sulfatase in immortalized MSD cells revealed that FGE-E113K exhibited only approx. 15% of the activity of wild type FGE. Based on the crystal structure we predict that the exchange of glutamate-113 against lysine should induce a strong destabilization of the secondary structure, possibly affecting the folding for correct disulfide bridging between C235-C346 as well as distortion of the active site groove that could affect both the intracellular stability as well as the activity of FGE. Thus, the novel variant of the SUMF1 gene obviously results in functionally impaired FGE protein leading to a severe late infantile type of MSD.

σ1B adaptin regulates adipogenesis by mediating the sorting of sortilin in adipose tissue.

Here, we describe altered sorting of sortilin in adipocytes deficient for the σ1B-containing AP-1 complex, leading to the inhibition of adipogenesis. The AP-1 complex mediates protein sorting between the trans-Golgi network and endosomes. Vertebrates express three AP1 σ1 subunit isoforms - σ1A, σ1B and σ1C (also known as AP1S1, AP1S2 and AP1S3, respectively). σ1B-deficient mice display impaired recycling of synaptic vesicles and lipodystrophy. Here, we show that sortilin is overexpressed in adipose tissue from σ1B(-/-) mice, and that its overexpression in wild-type cells is sufficient to suppress adipogenesis. σ1B-specific binding of sortilin requires the sortilin DxxD-x12-DSxxxL motif. σ1B deficiency does not lead to a block of sortilin transport out of a specific organelle, but the fraction that reaches lysosomes is reduced. Sortilin binds to the receptor DLK1, an inhibitor of adipocyte differentiation, and the overexpression of sortilin prevents DLK1 downregulation, leading to enhanced inhibition of adipogenesis. DLK1 and sortilin expression are not increased in the brain tissue of σ1B(-/-) mice, although this is the tissue with the highest expression of σ1B and sortilin. Thus, adipose-tissue-specific and σ1B-dependent routes for the transport of sortilin exist and are involved in the regulation of adipogenesis and adipose-tissue mass.

Trans-Golgi network morphology and sorting is regulated by prolyl-oligopeptidase-like protein PREPL and the AP-1 complex subunit µ1A.

The AP-1 complex recycles between membranes and the cytoplasm and dissociates from membranes during clathrin-coated-vesicle uncoating, but also independently of vesicular transport. The µ1A N-terminal 70 amino acids are involved in regulating AP-1 recycling. In a yeast two-hybrid library screen we identified the cytoplasmic prolyl-oligopeptidase-like protein PREPL as an interaction partner of this domain. PREPL overexpression leads to reduced AP-1 membrane binding, whereas reduced PREPL expression increases membrane binding and impairs AP-1 recycling. Altered AP-1 membrane binding in PREPL-deficient cells mirrors the membrane binding of the mutant AP-1* complex, which is not able to bind PREPL. Colocalisation of PREPL with residual membrane-bound AP-1 can be demonstrated. Patient cell lines deficient in PREPL have an expanded trans-Golgi network, which could be rescued by PREPL expression. These data demonstrate PREPL as an AP-1 effector that takes part in the regulation of AP-1 membrane binding. PREPL is highly expressed in brain and at lower levels in muscle and kidney. Its deficiency causes hypotonia and growth hormone hyposecretion, supporting essential PREPL functions in AP-1-dependent secretory pathways.

Proprotein convertases process and thereby inactivate formylglycine-generating enzyme.

Formylglycine-generating enzyme (FGE) post-translationally converts a specific cysteine in newly synthesized sulfatases to formylglycine (FGly). FGly is the key catalytic residue of the sulfatase family, comprising 17 nonredundant enzymes in human that play essential roles in development and homeostasis. FGE, a resident protein of the endoplasmic reticulum, is also secreted. A major fraction of secreted FGE is N-terminally truncated, lacking residues 34-72. Here we demonstrate that this truncated form is generated intracellularly by limited proteolysis mediated by proprotein convertase(s) (PCs) along the secretory pathway. The cleavage site is represented by the sequence RYSR(72)↓, a motif that is conserved in higher eukaryotic FGEs, implying important functionality. Residues Arg-69 and Arg-72 are critical because their mutation abolishes FGE processing. Furthermore, residues Tyr-70 and Ser-71 confer an unusual property to the cleavage motif such that endogenous as well as overexpressed FGE is only partially processed. FGE is cleaved by furin, PACE4, and PC5a. Processing is disabled in furin-deficient cells but fully restored upon transient furin expression, indicating that furin is the major protease cleaving FGE. Processing by endogenous furin occurs mostly intracellularly, although also extracellular processing is observed in HEK293 cells. Interestingly, the truncated form of secreted FGE no longer possesses FGly-generating activity, whereas the unprocessed form of secreted FGE is active. As always both forms are secreted, we postulate that furin-mediated processing of FGE during secretion is a physiological means of higher eukaryotic cells to regulate FGE activity upon exit from the endoplasmic reticulum.

Effective Gene Therapy for Metachromatic Leukodystrophy Achieved with Minimal Lentiviral Genomic Integrations.

Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease (LSD) characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplant (HSCT) with lentiviral (LV) based gene therapy has great potential to treat MLD. However, if enzyme production is inadequate, this could result in continued loss of motor function, implying a high vector copy number (VCN) requirement for optimal enzymatic output. This may place children at increased risk for genomic toxicity due to higher VCN. We increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression, and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human HSC with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4X more ARSA activity than the currently EU-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-old Arsa -KO MLD mice transplanted with Arsa -KO BM cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching WT mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating early symptomatic MLD patients.

Drug screening identifies tazarotene and bexarotene as therapeutic agents in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD, MIM #272200) results from pathogenic variants in the SUMF1 gene that impair proper function of the formylglycine-generating enzyme (FGE). FGE is essential for the posttranslational activation of cellular sulfatases. MSD patients display reduced or absent sulfatase activities and, as a result, clinical signs of single sulfatase disorders in a unique combination. Up to date therapeutic options for MSD are limited and mostly palliative. We performed a screen of FDA-approved drugs using immortalized MSD patient fibroblasts. Recovery of arylsulfatase A activity served as the primary readout. Subsequent analysis confirmed that treatment of primary MSD fibroblasts with tazarotene and bexarotene, two retinoids, led to a correction of MSD pathophysiology. Upon treatment, sulfatase activities increased in a dose- and time-dependent manner, reduced glycosaminoglycan content decreased and lysosomal position and size normalized. Treatment of MSD patient derived induced pluripotent stem cells (iPSC) differentiated into neuronal progenitor cells (NPC) resulted in a positive treatment response. Tazarotene and bexarotene act to ultimately increase the stability of FGE variants. The results lay the basis for future research on the development of a first therapeutic option for MSD patients.

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.

A homozygous missense variant of SUMF1 in the Bedouin population extends the clinical spectrum in ultrarare neonatal multiple sulfatase deficiency.

BACKGROUND: Multiple sulfatase deficiency (MSD, MIM #272200) is an ultrarare congenital disorder caused by SUMF1 mutation and often misdiagnosed due to its complex clinical presentation. Impeded by a lack of natural history, knowledge gained from individual case studies forms the source for a reliable diagnosis and consultation of patients and parents. METHODS: We collected clinical records as well as genetic and metabolic test results from two MSD patients. The functional properties of a novel SUMF1 variant were analyzed after expression in a cell culture model. RESULTS: We report on two MSD patients-the first neonatal type reported in Israel-both presenting with this most severe manifestation of MSD. Our patients showed uniform clinical symptoms with persistent pulmonary hypertension, hypotonia, and dysmorphism at birth. Both patients were homozygous for the same novel SUMF1 mutation (c.1043C>T, p.A348V). Functional analysis revealed that the SUMF1-encoded variant of formylglycine-generating enzyme is highly instable and lacks catalytic function. CONCLUSION: The obtained results confirm genotype-phenotype correlation in MSD, expand the spectrum of clinical presentation and are relevant for diagnosis including the extremely rare neonatal severe type of MSD.

Recognition and ER Quality Control of Misfolded Formylglycine-Generating Enzyme by Protein Disulfide Isomerase.

Multiple sulfatase deficiency (MSD) is a fatal, inherited lysosomal storage disorder characterized by reduced activities of all sulfatases in patients. Sulfatases require a unique post-translational modification of an active-site cysteine to formylglycine that is catalyzed by the formylglycine-generating enzyme (FGE). FGE mutations that affect intracellular protein stability determine residual enzyme activity and disease severity in MSD patients. Here, we show that protein disulfide isomerase (PDI) plays a pivotal role in the recognition and quality control of MSD-causing FGE variants. Overexpression of PDI reduces the residual activity of unstable FGE variants, whereas inhibition of PDI function rescues the residual activity of sulfatases in MSD fibroblasts. Mass spectrometric analysis of a PDI+FGE variant covalent complex allowed determination of the molecular signature for FGE recognition by PDI. Our findings highlight the role of PDI as a disease modifier in MSD, which may also be relevant for other ER-associated protein folding pathologies.

Eukaryotic formylglycine-generating enzyme catalyses a monooxygenase type of reaction.

C α-formylglycine (FGly) is the catalytic residue of sulfatases in eukaryotes. It is generated by a unique post-translational modification catalysed by the FGly-generating enzyme (FGE) in the endoplasmic reticulum. FGE oxidizes a cysteine residue within the conserved CxPxR sequence motif of nascent sulfatase polypeptides to FGly. Here we show that this oxidation is strictly dependent on molecular oxygen (O2) and consumes 1 mol O2 per mol FGly formed. For maximal activity FGE requires an O2 concentration of 9% (105 µM). Sustained FGE activity further requires the presence of a thiol-based reductant such as DTT. FGly is also formed in the absence of DTT, but its formation ceases rapidly. Thus inactivated FGE accumulates in which the cysteine pair Cys336/Cys341 in the catalytic site is oxidized to form disulfide bridges between either Cys336 and Cys341 or Cys341 and the CxPxR cysteine of the sulfatase. These results strongly suggest that the Cys336/Cys341 pair is directly involved in the O2 -dependent conversion of the CxPxR cysteine to FGly. The available data characterize eukaryotic FGE as a monooxygenase, in which Cys336/Cys341 disulfide bridge formation donates the electrons required to reduce one oxygen atom of O2 to water while the other oxygen atom oxidizes the CxPxR cysteine to FGly. Regeneration of a reduced Cys336/Cys341 pair is accomplished in vivo by a yet unknown reductant of the endoplasmic reticulum or in vitro by DTT. Remarkably, this monooxygenase reaction utilizes O2 without involvement of any activating cofactor.

Rapid degradation of an active formylglycine generating enzyme variant leads to a late infantile severe form of multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE's catalytic capacity is impaired resulting in reduced cellular sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms.

SUMF1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in multiple sulfatase deficiency.

Multiple Sulfatase Deficiency (MSD) is caused by mutations in the sulfatase-modifying factor 1 gene encoding the formylglycine-generating enzyme (FGE). FGE post translationally activates all newly synthesized sulfatases by generating the catalytic residue formylglycine. Impaired FGE function leads to reduced sulfatase activities. Patients display combined clinical symptoms of single sulfatase deficiencies. For ten MSD patients, we determined the clinical phenotype, FGE expression, localization and stability, as well as residual FGE and sulfatase activities. A neonatal, very severe clinical phenotype resulted from a combination of two nonsense mutations leading to almost fully abrogated FGE activity, highly unstable FGE protein and nearly undetectable sulfatase activities. A late infantile mild phenotype resulted from FGE G263V leading to unstable protein but high residual FGE activity. Other missense mutations resulted in a late infantile severe phenotype because of unstable protein with low residual FGE activity. Patients with identical mutations displayed comparable clinical phenotypes. These data confirm the hypothesis that the phenotypic outcome in MSD depends on both residual FGE activity as well as protein stability. Predicting the clinical course in case of molecularly characterized mutations seems feasible, which will be helpful for genetic counseling and developing therapeutic strategies aiming at enhancement of FGE.

ERp44 mediates a thiol-independent retention of formylglycine-generating enzyme in the endoplasmic reticulum.

Inside the endoplasmic reticulum (ER) formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the post-translational oxidation of a specific cysteine. Thereby formylglycine is generated, which is essential for sulfatase activity. Here we show that ERp44 interacts with FGE forming heterodimeric and, to a lesser extent, also heterotetrameric and octameric complexes, which are stabilized through disulfide bonding between cysteine 29 of ERp44 and cysteines 50 and 52 in the N-terminal region of FGE. ERp44 mediates FGE retrieval to the ER via its C-terminal RDEL signal. Increasing ERp44 levels by overexpression enhances and decreasing ERp44 levels by silencing reduces ER retention of FGE. Suppressing disulfide bonding by mutating the critical cysteines neither abrogates ERp44.FGE complex formation nor interferes with ERp44-mediated retention of FGE, indicating that noncovalent interactions between ERp44 and FGE are sufficient to mediate ER retention. The N-terminal region of FGE harboring Cys(50) and Cys(52) is dispensible for catalytic activity in vitro but required for FGE-mediated activation of sulfatases in vivo. This in vivo activity is affected neither by overexpression nor by silencing of ERp44, indicating that a further ER component interacting with the N-terminal extension of FGE is critical for sulfatase activation.

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.

AP-1 membrane-cytoplasm recycling regulated by mu1A-adaptin.

The adaptor protein complex AP-1 mediates vesicular protein sorting between the trans Golgi network and endosomes. AP-1 recycles between membranes and the cytoplasm together with clathrin during transport vesicle formation and vesicle uncoating. AP-1 recycles independent of clathrin, indicating binding to unproductive membrane domains and premature termination of vesicle budding. Membrane recruitment requires ADP ribosylation factor-1-GTP, a transmembrane protein containing an AP-1-binding motif and phosphatidyl-inositol phosphate (PI-4-P). Little is known about the regulation of AP-1 membrane-cytoplasm recycling. We identified the N-terminal domain of micro1A-adaptin as being involved in the regulation of AP-1 membrane-cytoplasm recycling by constructing chimeras of micro1A and its homologue micro2. The AP-1* complex containing this mu2-micro1A chimera had slowed down recycling kinetics, resulting in missorting of mannose 6-phosphate receptors. The N-terminal domain is only accessible from the cytoplasmic AP-1 surface. None of the proteins known to influence AP-1 membrane recycling bound to this micro1A domain, indicating the regulation of AP-1 membrane-cytoplasm recycling by an yet unidentified cytoplasmic protein.