SUMF1 Mutations Affecting Stability and Activity of Formylglycine

SUMF1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in Multiple Sulfatase Deficiency Thomas Dierks, Lars Schlotawa, Eva Charlotte Ennemann, Karthikeyan Radhakrishnan, Bernhard Schmidt, Anupam Chakrapani, Hans-Jürgen Christen, Hugo Moser, Beat Steinmann, Jutta Gärtner

To cite this version:

Thomas Dierks, Lars Schlotawa, Eva Charlotte Ennemann, Karthikeyan Radhakrishnan, Bernhard Schmidt, et al.. SUMF1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in Multiple Sulfatase Deficiency. European Journal of Human Genetics, Nature Publishing Group, 2011, 10.1038/ejhg.2010.219. hal-00608022

HAL Id: hal-00608022 https://hal.archives-ouvertes.fr/hal-00608022 Submitted on 12 Jul 2011

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Dear Dr. van Ommen,

Thank you very much for the thorough reviewing of our manuscript (MS No: 590-10- EJHG) entitled “SUMF1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in Multiple Sulfatase Deficiency”. We are pleased to hear that the manuscript has been well received by the reviewers and in principle has been recommended for publication. Enclosed please find a revised version of the manuscript that we wish to resubmit for publication in EJHG.

Below we would like to respond to the points raised by the reviewers. The indicated change numbers correspond to those given in the margin of the new text.

Reviewer #1

1) One very surprising result of this paper was the finding that a deletion of 25 amino acids results in a hypomorph with some, albeit very low residual activity (0.3% of wildtype). It was technically very demanding from the beginning to determine this reliably. We made this effort to verify or falsify the concept that only hypomorphic mutations (at least one per patient) cause the disease. Hence we consider the wording “thus challenging the concept that only hypomorphic mutations cause MSD” justified at this point of the manuscript, i.e. when describing the objective of the study. In retrospect the data obtained give further proof to the validity of the concept, as is discussed in the text. 2) Changes #1 & 2: Both suggested changes have been made. 3) Changes #3 & 4: Changed as suggested. 4) Change #5: Changed as suggested.

Reviewer #2

1) Changes #6-10: As suggested, in the text we have changed “mild” into “attenuated”, “milder” or “comparatively mild” for describing PATIENT phenotypes, in order to avoid speaking of a mild phenotype. For classification of the CLINICAL disease types (see also tables 1 to 3), we have to use the established term “late infantile mild” (LIM) form in order to discriminate it from the “late infantile severe” (LIS) form. It is an internationally established disease classification for MSD and has been adopted by other authors. 2) Changes #11-19: Wording (“mutated”) changed throughout, mostly into “variant” as suggested. 3) Change #20: “100 µCi” was replaced by “3.7 MBq”. 4) There seems to be a misunderstanding. The two mentioned mutations are not listed among the missense mutations in this paragraph. Rather, they are clearly contrasted from those and mentioned separately in the preceding paragraph. 5) Changes #21-24: (Figure 4): “Relative secretion” is secretion corrected for degradation during the measured time interval and is calculated as defined in the legend, namely: medium FGE/total FGE after 6 h (A) or 1.5 h (C). This definition is now amended by a short explanation in the legend. Thus, for FGE-G247R and -G263V we have 66% and 77% relative secretion, respectively, after 1.5 h (wt only 9%) and we have 100% relative secretion for both mutants after 6 h (wt only 52%). The pulse-chase data clearly demonstrate that FGE-G247R and -G263V show drastically reduced intracellular stability. These variants after 6h have completely disappeared from the cells. Surprisingly, 45% of the initially synthesized G263V (23% for G247R), i.e. EVERYTHING surviving the 6h period, has been secreted (while the remaining 55% or 77%, respectively, was degraded). For other variants (S155P, R327X, A149-179del) we observe total degradation and no (or almost no) secretion. Conclusion: FGE-G247R and -G263V escape to a considerable extent from the ER quality control system and reach the ER exit pathway. Secretion is also observed for wild-type FGE (54% under the conditions of Fig. 4A) and has been described even for endogenous FGE, i.e. at physiological expression levels (see e.g. Zito et. al (2007) EMBO J. 26, 2443-2453). FGE- G247R and G263V are secreted much faster and at higher levels than wild-type FGE, as can be recognized from Fig. 4C: 60% of initial G263V but only 9% of wt FGE leave the cell within 1.5h. So we observe at about similar expression levels an increase of both absolute secretion (60 vs. 9% of what was synthesized initially) and relative secretion (60/78 vs. 9/100, i.e. 77 vs. 9% of what remained undegraded). As G247 and G263 are both surface exposed (see new Fig.S1), substitution of these residues may well interfere with ER retention and hence with delivery for degradation. Importantly, both degradation and secretion lead to loss of FGE from the ER and hence contribute to disease severity.

For clarification we made four amendments to the Discussion and the figure legend (see changes# 21-24).

Reviewer #3

1) Change #25: The requested expression data for patient fibroblasts have been included into a new figure, which is provided as supplemental material (new Fig.S2). Clearly, the expression in patient cells was too low to allow detailed studies. (Please find a further comment below, point 6). A comparison with recombinant expression in HT-1080 cells is not meaningful, as this varies among different clones. For our experiments we selected well expressing clones showing comparable expression for the different mutated FGE variants (with the exception of the low expressing FGE-S155P). Interestingly, like in patient cells ALL MSD-causing FGE variants showed less efficient expression in comparison to wild-type FGE also in HT-1080 cells. This information is included in the legend to the new Fig.S2.

2) Change #26: The requested patient history data have been included into a new table, which is provided as supplemental material (new Tab. S1).

3) Change #27: The suggested crystal structure, onto which the mutations have been mapped, is provided as supplemental material (new Fig.S1). Reference to this figure is given throughout the text.

4) The suggested experiments using proteasome inhibitors in order to test for a possible secretion of unstable FGE mutants (as an indication of prolonged association with the ER folding machinery) are very interesting from a cell biological point of view. [We do have some preliminary data for a few other mutants (published earlier) showing that proteasome inhibitors increase intracellular lifetime of the mutants without much effect on secretion. Cells, however, suffered from the treatment.] Due to limited space (editorial obligation: 25% reduction of the text, 4 figures maximum) and time (four weeks deadline for resubmission), however, we find that these experiments are beyond the scope of this part of the manuscript, which aims at quantifying FGE mutant stability as one important parameter with regard to the clinical outcome in MSD. Here we feel that the data presented on steady-state FGE levels and, in particular, the detailed pulse-chase data are adequate and conclusive. Given the deadline for resubmission, we see no chance to perform reliable proteasome inhibitor experiments.

5) Change #28: We agree with the reviewer's point of view. Hence in the Perspective at the end of the Discussion we have put slightly more emphasis on a possible correction of FGE function by small molecule chaperones.

6) This is a very relevant question. Earlier we have characterized the A279V mutation showing even 23% residual activity and leading also to a LIM phenotype. (Please note that these activities are measured for purified protein, which is very stable in the test tube.) Here as well as in case of all other FGE variants with high residual activity it is the very low in vivo stability of the protein (inside the ER) that brings the actual in vivo activity below the threshold. At present there is no reliable method to determine in vivo activities and thereby to define this threshold. (This problem also applies to determination of FGE in vivo activities in patients’ parents cells.) This threshold, as judged from in vitro co-expression experiments with reporter sulfatases, certainly is much lower than 16%. Since we have to reduce the manuscript length by 25%, we see no chance to include these considerations into the text. On the other hand, the contribution of both activity and stability losses to disease severity is broadly discussed.

Further we have reduced the manuscript in length (from 22 to 17 pages (i.e. by 23%), not counting legends, tables and supplementary information pages). We hope that the manuscript is now acceptable for publication.

Sincerely,

Thomas Dierks, PhD SUMF1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in Multiple Sulfatase Deficiency

Lars Schlotawa1, Eva Charlotte Ennemann2, Karthikeyan Radhakrishnan2, Bernhard Schmidt3, Anupam Chakrapani4, Hans-Jürgen Christen5, Hugo Moser6§, Beat Steinmann7, Thomas Dierks2*, Jutta Gärtner1

1Department of Pediatrics and Pediatric Neurology, Georg August University Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany; 2Department of Chemistry, Biochemistry I, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany; 3Department of Biochemistry II, Georg August University Göttingen, Humboldtallee 23, 37073 Göttingen, Germany; 4Department of Clinical Inherited Metabolic Disorders, Birmingham Children’s Hospital, NHS Foundation Trust, Steelhouse Lane, Birmingham B4 6NH, United Kingdom; 5Department of Pediatrics II, Children’s Hospital Auf der Bult, Janusz-Korczak-Allee 12, 30173 Hannover, Germany; 6Neurogenetics Research Center Kennedy Krieger Institute, Johns Hopkins University, Kennedy Krieger Institute, 707 North Broadway, Baltimore, MD 21205, United States of America; 7Division of Metabolism, University Children’s Hospital, Steinwiesstr. 75, 8032 Zürich, Switzerland

§died in 2007

*Corresponding author: Thomas Dierks Department of Chemistry, Biochemistry I, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany; Tel.: +49 521 106 2092; Fax: +49 521 106 6014; E-mail: [email protected]

Running title: Genotype-phenotype correlations in MSD

1 ABSTRACT

Multiple Sulfatase Deficiency (MSD) is caused by mutations in the SUMF1 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 due to 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.

Keywords: Formylglycine-Generating Enzyme/ genotype-phenotype correlation/ lysosomal storage disorders/ Multiple Sulfatase Deficiency/ sulfatase/ SUMF1

INTRODUCTION

Multiple Sulfatase Deficiency (MSD, MIM# 272200) is a rare autosomal recessive inborn error of metabolism. Worldwide less than 50 cases have been published so far1. The clinical picture of MSD combines symptoms of the different sulfatase deficiencies. Patients show neurological deterioration and a neurodegenerative course of disease similar to metachromatic leukodystrophy. Developmental delay, dysmorphism, and organomegaly are present as found

2 in various mucopolysaccharidoses. Skeletal abnormalities remind one of Chondrodysplasia punctata type I, and skin changes of X-linked ichthyosis1-3. According to the age of onset neonatal, late infantile and juvenile disease subtypes can be distinguished4. Neonatal MSD is the most severe form with postnatal onset and a broad range of mucopolysaccharidosis-like clinical symptoms. According to the few published cases all neonatal MSD patients died within the first year of life5-8. Late infantile MSD resembles late infantile metachromatic leukodystrophy with progressive loss of mental and motor abilities; these clinical features are combined with other symptoms of single sulfatase deficiencies like dysmorphism, skeletal changes and ichthyosis2,3,9-12. Late infantile MSD can be subdivided into a severe (LIS) and an attenuated (late infantile mild, LIM) form, the latter showing a reduced number of symptoms and later onset beyond the second year of life13. Late infantile forms include the majority of

MSD cases; a Saudi variant form was described separately14. Up to now, only one juvenile case of MSD has been published with an onset of symptoms in late childhood15.

In MSD, enzymatic activity of every sulfatase is reduced or even absent. 17 sulfatases are encoded in the human genome, 13 of them have been characterized biochemically and eight are associated with diseases due to single sulfatase deficiencies10,16. At least five sulfatases are necessary for non-redundant desulfation of glycosaminoglycans in the lysosome. Defective sulfatase function in MSD thus leads to intralysosomal storage and cellular damage10.

The primary defect in MSD, causing deficiency of a whole enzyme family, affects a specific post-translational modification of sulfatases that is strictly required for catalytic activity. All sulfatases contain in their active site the so-called sulfatase signature I, comprising the linear amino acid sequence CxPxR. The cysteine within this motif has to be converted to Cα- formylglycine (FGly)17-19. This modification occurs late co- or early post-translationally in the endoplasmic reticulum (ER) and is mediated by the FGly-generating enzyme (FGE), an unusual mono-oxygenase using molecular oxygen for oxidation of Cβ of the substrate cysteine through its catalytic cysteines 336 and 34125,29. FGE is encoded by the sulfatase

3 modifying factor 1 gene (SUMF1). Patients with MSD carry mutations in this gene20,21. Null mutations result in the complete absence of sulfatase activities, as shown in a SUMF1 gene trap mouse22. Expression of FGE carrying MSD patient mutations in SUMF1 deficient mouse fibroblasts results in detectable, albeit extremely low sulfatase activities, underlining that

MSD is a monogenetic disease caused by hypomorphic mutations23. Until now, more than 30 different SUMF1 mutations were found, most of which are missense mutations9,21,24-26.

In a first approach we investigated the consequences of four different MSD causing SUMF1 mutations on FGE13 expression, localization, stability and activity in either an over-expressing cell culture model or in patient fibroblasts. We found that all four variant FGE proteins could be expressed in HT-1080 cells, correctly localizing to the ER. Expressing FGE A177P and

W179S led to stable proteins, whereas FGE A279V and R349W both were unstable. The specific enzyme activity of the purified FGE proteins varied between 1 and 23% as compared to wild-type (wt) FGE. All three patients with both unstable FGE protein and low residual activity expressed FGE R349W and presented a LIS clinical phenotype. All other patients exhibited a LIM phenotype and expressed either unstable FGE with high residual activity or stable FGE with low residual activity. Intracellular sulfatase activities were massively reduced in fibroblasts from patients with the LIS phenotype. However, when measured in cultured fibroblasts of more mildly affected patients these sulfatase activities were surprisingly variable. Therefore, the molecular analysis of FGE activity and stability seems to provide a more convincing correlation with the clinical presentation of patients of a given genotype.

In this study we further tested this hypothesis. We extended our previous clinical and biochemical characterization of MSD patients to confirm a potential genotype/phenotype correlation. We included another ten patients showing different clinical presentations and various MSD causing SUMF1 missense and also nonsense mutations. For the first time, a neonatal very severe case with nonsense mutations on both alleles is studied at the molecular level, thus challenging the concept that only hypomorphic mutations cause MSD.

4 MATERIALS AND METHODS

Patients and patient cells. Clinical data were obtained from patients’ relatives by personal communication, patient data sheets and from already published case reports27. For ethnic origin and family history of the nine homozygous patients see Tab.S1. Patient skin fibroblasts were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (Invitrogen, Karlsruhe, Deutschland) under 5% CO2 at 37 °C.

Western blot analysis. Equivalent aliquots of medium (40 µl) or cell lysate (50 or 100 µg cell protein) were analyzed by Western blotting using FGE antiserum or a Hexa-His antibody

(Sigma) as described earlier13,25,28,29. ECL signals were quantified using the AIDA software package (Raytest, Straubenhardt, Germany). Endogenous FGE in MSD fibroblasts was detected using ECL femto substrate and enhancer diluted 1:10 in ECL solution (Pierce).

Enzyme assays. Sulfatase activities were measured from whole cell lysates as described30,31.

FGE activity assays were performed with FGE enriched from cells and media using the synthetic ASA-derived peptide P23 as substrate21. See also supplementary informations.

Metabolic labeling and immunoprecipitation. Cells stably expressing FGE proteins were grown on 3 cm plates to 80-90% confluence, starved for 1h in medium depleted of methionine and cysteine and pulsed with 3.7 MBq/ml [35S]methionine/cysteine also for 1 h. Medium was changed to 2 ml medium with 10% fetal calf serum, supplemented with 200 µg/ml methionine and 100 µg/ml cysteine, for 20 min. Cells and medium were harvested either directly or after another change of medium and growth for 1.5, 3 h or 6 h (chase). Immunoprecipitation of

FGE was performed with FGE antiserum according to a protocol described earlier32. Proteins were solubilized and subjected to SDS-PAGE. Dried gels were analyzed by phosphorimaging

(BAS 1000, Fuji) and densitometric quantification using the MacBAS software (Raytest).

Molecular analysis of SUMF1 mutations, FGE expression, FGE purification, immunofluorescence microscopy. See supplementary informations.

5 RESULTS

Clinical presentation and mutational analysis of ten patients with MSD

Based on Eto’s classification4 we distinguished one neonatal (NVS) and nine late infantile patients. The latter were further subdivided into seven severe (LIS) and two milder (LIM) cases 10,13. For classification we considered ten frequent clinical symptoms and also the time point of manifestation of every single symptom (Tab.1). In NVS cases every symptom appears in the first months of life, as holds true for patient 1; (skeletal changes had not been defined). The patient was compound heterozygous for two mutations: one in-frame deletion resulting in a loss of 25 amino acids (p.A149_A173del; Fig.S1) and one mutation leading to a premature stop thereby disrupting the active site of FGE (p.R327X; Fig.S1). All other patients, homozygous for four different SUMF1 missense mutations, did not show all of the ten symptoms and their onset varied between LIS and LIM patients, as the latter displayed less symptoms or a later onset of symptoms. Accordingly, the two patients with FGE G263V were assigned to the LIM group, whereas all other patients, expressing FGE S155P (3 patients), G247R (3), or R345C (1) (Fig.S1), belong to the LIS group (Tab.1).

Expression of recombinant FGE mutant proteins

All FGE proteins, resulting from the individual SUMF1 mutations, were stably expressed in

HT-1080 fibrosarcoma cells. Expressed protein was detected in cell lysates by Western blotting using either an anti-hexahistidine or a polyclonal FGE antibody (Fig.1). Expression levels were comparable among different cell lines; only FGE S155P was expressed at a severely reduced level. Even the two nonsense mutations, p.R327X and p.A149_A173del, showed intracellular protein expression, resulting in smaller versions of FGE (Fig.1, upper panel). Wild-type (wt) FGE is secreted and processed in the Golgi leading to an N-terminally truncated form in the medium28,33. Only three of the FGE variants under study also showed secretion into the cell culture medium (p.G247R, p.G263V, p.R345C). All other variants, despite their comparable intracellular expression, were not secreted (Fig.1, lower panel).

6 Functionally impaired FGE protein, which is not secreted, is retained intracellularly (and degraded, see below). Among the retained proteins are those resulting from the two nonsense mutations probably leading to a drastically impaired FGE structure (see Fig.S1). For all secreted FGE variants it is concluded that they are able to pass quality control mechanisms of the ER in spite of protein instability predictions25.

Subcellular localization of mutant FGE proteins

Immunofluorescence analysis of HT-1080 cell clones revealed a correct localization of each

FGE variant in the ER, as revealed by co-localization with the ER marker PDI (Fig.2). In addition, the secreted FGE proteins (p.G247R, p.G263V, p.R345C) were also visible in the

Golgi (Fig.2 R, S, U; red staining) showing co-localization with a Golgi marker (not shown).

No co-staining was visible with a lysosomal marker (data not shown). The ER localization could be confirmed for the endogenous FGE proteins in MSD patient fibroblasts even though protein expression was extremely reduced and material cross-reacting with the anti-FGE antibody was hardly detectable (Fig.3). Localization of patient FGE protein in the ER is one necessary precondition for residual FGly generation in newly synthesized sulfatases.

Residual catalytic activity of FGE protein variants

The enzymatic activity of FGE variants was measured using a synthetic peptide derived from

21 arylsulfatase A as substrate (at saturating concentration, 15-times above KM of wt FGE) .

FGE was partially purified from cell lysates or conditioned medium. The activities of variant proteins, relative to wt, ranged from 0 to 15.9% (Fig.4B). FGE R327X, with a disrupted active site, displayed no activity; p.A149_A173del with a 25 amino acid deletion at the surface of the FGE core domain (Fig.S1), showed detectable, albeit very low residual activity of 0.3±0.04%. FGE S155P and R345C showed slightly, but significantly higher residual activities of 1.6±0.4% and 2.0±0.2%, respectively. The highest residual activity was measured for p.G263V (15.9±1.2%). Thus, apart from the catalytically fully impaired FGE R327X all investigated FGE proteins were able to catalyze at least some FGly generation in vitro.

7 Stability and intracellular retention of FGE protein variants

Based on the crystal structure of wt FGE, all chosen SUMF1 mutations were expected to impair protein stability25. To prove this prediction we metabolically labeled FGE expressing

HT-1080 cell clones with [35S]methionine/cysteine. Labeled FGE protein was determined before and after 6 h of chase time, as detected by immunoprecipitation of intracellular and secreted FGE, followed by SDS-PAGE and phosphorimaging (Fig.4A). As expected13, wt

FGE was fully stable during the 6 h chase with ~50% of initial protein remaining inside the cells and ~50% being secreted (Fig.4A). FGE variants A149_A173del and R327X both were unstable with only about 1% of cross-reacting material detectable after 6 h; also FGE S155P was unstable (about 2%). These three variants likewise could not be detected in the secretions

(Fig.4A). The other FGE variants (G247R, G263V, R345C) showed clearly reduced stability as well. In fact, intracellular protein was hardly detectable after the chase also here (0-2% of the initial protein amount); however, considerable amounts of protein were secreted indicating the passing of intracellular protein quality control.

To further analyze degradation and secretion kinetics, we repeated the experiment with chase times of 1.5 and 3 h (Fig.4C and data not shown). FGE R327X showed the lowest (12% residual protein after 1.5 h) and R345C the highest (44%) stability (at 91% stability observed for wt FGE) (Fig.4C). For FGE G247R and G263V rather low levels of intracellular protein were detected (~20%), which in part is due to intense secretion. These results confirm the data of Fig.4A. Taken together, all investigated mutations severely affect intracellular FGE protein levels. Apart from degradation, in some cases also increased secretion, i.e. impaired protein retention, contributes to reduced intracellular FGE levels.

Expression level of endogenous FGE protein in MSD patient fibroblasts

We analyzed the amount of endogenous FGE protein in whole cell lysates from patient fibroblasts by immunofluorescence detection (Fig.3) and also by Western blotting (Fig.S2).

Fibroblasts from healthy persons served as a positive control. FGE amounts were extremely

8 reduced for all studied patients. No or only faint bands of cross-reacting material were detectable in Western blots (Fig.S2). The extremely reduced levels of defective protein in the patient cells agree with the results from the pulse-chase experiments showing that all analyzed

MSD causing mutations lead to unstable proteins that are degraded inside the cells and, in some cases, also secreted (see above).

Residual sulfatase activities in MSD patient fibroblasts

To determine the functional consequences of MSD mutations on endogenous sulfatase activities we measured the residual activities of three sulfatases, namely arylsulfatase A

(ASA), arylsulfatase C (ASC) and galactose 6-sulfatase (Gal6S) in cell lysates from patient fibroblasts (Tab.2). For FGE G263V we used a fetal fibroblast cell line, as no other patient cells were available. The lowest sulfatase activities were found in the fibroblasts from the

NVS patient displaying no detectable activity for ASA, less than 1% for ASC and 1.4% for

Gal6S (relative to wt activities, Tab.2). The highest activities were measured in the fetal fibroblasts expressing p.G263V found also in the LIM patients 9 and 10 (Tab.1). Residual activities of these cells were 17% for ASA, 61% for ASC and 39% for Gal6S (Tab.2).

All other cell lines originated from patients with the LIS form of MSD and displayed reduced sulfatase activities, with 2-12% residual activity measured for ASA and 1-22% for Gal6S.

ASC activities were also clearly reduced, but even more variable for the different mutations.

Whereas only <1 to 5% residual ASC activity was measured for fibroblasts expressing FGE

R345C and G247R, ASC activity in FGE S155P fibroblasts reached values between 14% and

31% of wt activity. It should be noted that patient 2, from whom the fibroblasts with highest

ASC activity were derived, developed ichthyosis like all other patients of this study (Tab.1).

DISCUSSION

In a first study13 we were able to show a correlation of both residual activity and stability of

FGE variants with the clinical presentation of selected MSD patients. As this study included

9 only four different SUMF1 mutations (homozygous in the patients), it remained open to discussion if the observed genotype/phenotype correlation holds true also for other mutations and for MSD in general. Here we extended our analysis including another set of clinically thoroughly characterized and genetically defined patients with different forms of disease.

Robust phenotype classification based on the clinical presentation of MSD patients

We investigated ten MSD patients, either historical or actual cases, and classified the patients according to their clinical presentation into groups of NVS, LIM or LIS cases. Our neonatal patient exhibited the whole range of symptoms possible, which agrees with all published neonatal cases5-8. Most symptoms were present at birth and the patient showed a rapidly progressive course of disease. The group of late infantile patients9,11,12 included in this study could be clearly distinguished from the neonatal patient, as symptoms varied in their age of onset with some features presenting only in later life or others lacking completely. Two of the late infantile patients could be sub-grouped as LIM versions of MSD (Tab.1), and differentiated from the very rare juvenile form15, (which is not included in our patient collection). Importantly, we can infer from this classification that independent patients with an identical mutation (in homozygous form) display a comparable clinical phenotype.

Molecular phenotype of MSD: differential functional impairments of recombinant FGE

For extending our pathobiochemical understanding of MSD we expressed all patient SUMF1 mutants in the established HT-1080 cell line13,15,21,25,28. Our approach was substantiated by mapping the amino acid substitutions on the crystal structure of wt FGE (see Fig.S1), which allowed a solid prediction of structural consequences. Without exception, all missense mutations affected strictly conserved residues and were expected to impair FGE protein stability due to unfavorable side chain interactions resulting in clashes with other residues

(p.S155P, p.G247R, p.G263V) and/or losses of specific hydrogen bonds (p.R345C)25.

However, the consequences on FGE activity remained difficult to predict, because the

10 substitutions did not directly affect the substrate binding groove, the oxygen binding pocket or even the catalytic site close to the redox active cysteines34.

All FGE variants could be expressed, including even the variants resulting from nonsense/ deletion mutations. Thus, translation of these mRNAs is not attenuated. All FGE proteins localized to the ER (Tab.3), thereby fulfilling one important precondition for function, as generation of FGly is a post-translational modification of nascent, yet unfolded, sulfatase proteins inside the ER17,35. Nevertheless, we cannot exclude that the ER localization of defective FGE proteins just mimics a correct localization and in fact is mainly caused by the quality control mechanism inside the ER36,37. Importantly, the four studied FGE variants with single substitutions were more (p.G247R, p.G263V, p.R345C) or less (p.S155P) efficient in passing this quality control mechanism, as these proteins were also secreted - just like wt FGE

(Figs. 1, 2 and 4), which is secreted even at normal expression levels28,38,43.

In fact, all FGE variants except p.R327X and p.A149_A173del (see below) were able to catalyze a significant substrate turnover in vitro, as determined after isolation from the medium or cell lysates. The highest residual activity (16 % of wt) was exhibited by FGE

G263V and the lowest by FGE S155P (1.6%). These data, obtained by measuring directly the

FGly formation in a sulfatase substrate peptide21,28,39, are in accordance with observations made by Annunziata et al (ref. 23), who expressed different MSD causing SUMF1 mutations together with reporter sulfatases in FGE deficient cells and observed detectable, albeit reduced sulfatase activities in all cases. Thus, all MSD causing mutations analyzed so far are hypomorphic – at least one per patient. In conclusion, a combination of two null mutations should be lethal. This concept, however, was challenged by our NVS patient, who carried two alleles with nonsense mutations, one of which (p.R327X) definitely disrupting the active site

(Fig.S1), as confirmed in our experiments by the total absence of activity. The other mutation, p.A149_A173del, leads to an internal in-frame deletion of 25 amino acids. Interestingly, this defective FGE at high concentrations of purified protein indeed led to detectable substrate

11 turnover corresponding to a residual activity of 0.3%. Thus, the protein obviously adopts a conformation allowing substrate turnover, albeit with strong impairments. Notably, the deleted 25 residues (pink colored in Fig.S1) are located on the surface of the FGE core domain. Residues 161-176 form a loop that is easily cleaved off by proteases 25. The deletion further removes β-strand 5 (residues 153-158, pink), which normally pairs with the adjacent

β-strand 6 (residues 180-184, blue) to form an antiparallel two-stranded sheet on the surface of the molecule. The residues forming β-strand 6 are also present in FGE A149_A173del.

Upon losing the anti-parallel interaction partner β-strand 5, β-strand 6 is predicted to change at least its orientation, which obviously allows bridging the distance to the deletion breakpoint at position 148 without complete disruption of the catalytic core domain architecture.

Taken together, we found extensive instability for all FGE variants, thereby confirming the predictions from mapping the amino acid substitutions onto the FGE crystal structure. The degradation kinetics, however, varied among the different proteins (Fig.4). Furthermore, we found different secretion properties of FGE variants. About 50% of wt FGE protein is secreted after 6 h. Three impaired FGE proteins, among them the two expressed nonsense mutations, were completely or almost completely (p.S155P) retained inside the cells and degraded. The other three (p.G247R, p.G263V, p.R345C) were partially secreted, two of them

(p.G247R and, in particular, p.G263V) even much more rapidly than wt FGE (Fig.4C). Since expression was comparable or even reduced, the enhanced secretion is not caused by simple saturation of retention mechanisms inside the cells. Rather the interaction with the retention machinery might be affected, which is compatible with the surface exposure of both glycine residues G247 and G263 (Fig.S1). For wt FGE an interaction with ERp44, protein disulfide isomerase and ERGIC-53 is already known; these components act in different ER and Golgi sub-compartments29,38. The exact way of retention of defective FGE variants as well as their delivery for degradation is still unknown. As secretion of wt FGE was described also at physiological expression levels43, we conclude that impaired retention contributes to disease

12 severity in patients with p.G247R and p.G263V mutations. Specific interactions with impact on the degradation and secretion kinetics may be affected. Whatever mechanism applies, impaired retention in addition to impaired stability, observed for all FGE variants tested, both lead to reduced intracellular levels of FGE.

Cellular phenotype in MSD fibroblasts: impairments of endogenous FGE and sulfatases

The consequences of impaired FGE structure and function, as observed in overexpressing cells, could be confirmed in patient fibroblasts. FGE was hardly detectable here, probably due to degradation. Also no intracellular mislocalization was visible. The activities of endogenous sulfatases were differentially reduced, roughly mirroring residual FGE functionality. Lowest activities were found in fibroblasts combining the two FGE nonsense/deletion mutations, i.e. in the NVS case. Highest sulfatase activities were found in LIM fibroblasts homozygous for the p.G263V mutation leading to FGE that is unstable but shows considerable residual activity (as high as 16 %). This observation confirms our previous results of high sulfatase activities in fibroblasts expressing unstable mutant FGE with high residual activity13.

In LIS fibroblasts residual sulfatase activities can vary considerably. Residual ASC activities in the three p.S155P patients reached 14-32 %, whereas only 0.3-3 % were measured for the other three patients of this phenotypic group. Interestingly, the variability was clearly less pronounced for the other two sulfatases tested. This might indicate that individual sulfatases exhibit different affinities to the various compromised FGE proteins. The resulting differential residual activities of specific sulfatases, however, can not be correlated with the onset of specific clinical symptoms during manifestation of the MSD syndrome. Nevertheless, the severity of the disease is reflected by the total residual sulfatase activity (Tab.3).

Perspective: Genotype/phenotype correlation and clinical predictions in MSD patients

Comparing the biochemical impairments of FGE caused by individual SUMF1 mutations with the clinical phenotype of the corresponding MSD patients, we find clear correlations (see

Tab.3). Patients with drastic impairments of both FGE stability and residual enzyme activity

13 displayed the most severe clinical phenotype. In our present study this was the case for the

NVS MSD patient. The mildest phenotype presented in the patient with FGE G263V, which is associated with the highest residual FGE activity among the studied variants; further FGE

G263V, though present at only rather low intracellular levels, can be assumed to truly perform this residual activity, as it efficiently passed intracellular quality control mechanisms.

Taking all available molecular, biochemical and clinical data into account it is now possible to establish a genotype/ phenotype correlation for MSD and to roughly predict the clinical course for patients with the studied SUMF1 mutations. Missense mutations affecting crucial functional or structural residues in FGE as well as nonsense mutations will cause severe forms of the disease, whereas missense mutations not fully abrogating a functional conformation of the FGE protein will lead to attenuated forms. This does not exclude that other factors (mostly putative as yet) act as disease modifier, e. g. proteins interacting with FGE for ER retention, intracellular trafficking and degradation, redox protection, substrate presentation or co-factor regeneration as well as unknown factors regulating expression levels of FGE (and sulfatases).

Such factors can be expected to have an impact on the clinical presentation of patients40-42.

Identification and characterization of these interacting factors as well as understanding their role in the molecular and cell biological functioning of FGE will be the basis for designing therapeutic targets. Likely these targets will be specific for different groups of mutations with similar functional properties. Clear hints towards the existence of such mutant groups come from the observation that despite early degradation of most protein variants, some of them, at comparable expression levels, are rapidly secreted, thereby escaping quality control. Beside enzyme replacement and gene therapy approaches10,43, a promising therapeutic approach would thus be to enhance the stability and/or the intracellular retention, and in turn the total residual FGE activity inside the ER, which represents the bottleneck in MSD patients. The use of small molecule chaperones, meanwhile tested in the therapy of some inborn errors of metabolism, are therefore likely to be beneficial also in MSD44,45.

14 ACKNOWLEDGMENTS

We would like to thank N. Hollstein, N. Eiselt, A. Winter, M. Balleininger, J. Kaiser, T.

Wilke, M.-A. Frese and E. Wiegmann for excellent technical assistance. We are grateful to M.

Henneke, H. Rosewich, P. Huppke, A. Ohlenbusch, M. Mariappan and B. Mueksch for helpful discussions and K. von Figura for his support to implement the project. This work was supported by the Research program of the Faculty of Medicine, Georg August University

Göttingen (L.S.), by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen

Industrie and Shire Human Genetic Therapies, Inc. (Cambridge, MA).

CONFLICT OF INTEREST STATEMENT: The authors declare no conflict of interest.

Supplementary Information accompanies the paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)

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FIGURE LEGENDS

Figure 1: Expression and secretion of mutant FGE in HT-1080 cells.

Cells stably overexpressing His-tagged forms of wt FGE and FGE variants were grown to confluence and incubated with fresh medium for 24 h. Equal amounts of cell lysate protein and corresponding medium aliquots were analyzed by Western blotting using a polyclonal

FGE antibody. The bands visible in the cell lysates (upper panel) represent full length FGE at

42 kDa. The lower bands labeled with asterisks result from N-terminal processing of wt FGE in the secretory route and are not relevant for intracellular FGE function. FGE A149_A173del and R327X were reduced in size due to the deletion/ truncation resulting from the respective mutation. In the medium (lower panel) secreted FGE, detectable almost exclusively as an N- terminally trimmed 38 kDa form, was only visible for wt FGE as well as for FGE G247R,

G263V and R345C. Expression levels varied among the stable cell lines. The data shown were representative for three independent experiments. For endogenous expression of FGE variants in patient fibroblasts see Fig.S2.

Figure 2: Subcellular localization of FGE variants expressed in HT-1080 cells.

Cultured cells, after permeabilization, were stained with a polyclonal FGE antibody (red, A-

G) and monoclonal antibodies against organelle markers. Only the ER marker PDI is shown

(green, H-N). The merge (yellow, O-U) reveals co-localization of each protein expressed.

Figure 3: Subcellular localization of endogenous FGE in MSD fibroblasts.

Endogenous FGE was stained in permeabilized MSD patient fibroblasts and wt control fibroblast cells using a polyclonal FGE antibody (red) and a monoclonal antibody detecting the ER marker PDI (green). The merge (yellow) indicates co-localization inside the ER.

Figure 4: Stability, retention and residual activity of FGE variants.

A: Stable cell lines were labeled with 35S methionine/cysteine for 1 h and chased for 6 h in label-free medium. FGE was immunoprecipitated from cells and medium and quantified by

18 densitometry after SDS-PAGE and phosphorimaging. The results shown are representative for three independent experiments. All variant FGE proteins were unstable. Relative secretion

(calculated as medium FGE/total FGE after 6 h to correct for degradation within the 6 h) varied, as indicated; n.d., cannot be determined due to massive FGE degradation after 6 h. B:

His-tagged FGE was purified from secretions or cells, respectively. FGE amounts were adjusted according to Western blot quantification. Its activity was measured as FGly modification of the synthetic arylsulfatase A substrate peptide P23 using mass spectrometry

(see Methods). Specific activities were calculated and expressed as percentage of wt FGE activity. Mean values and SD were calculated from three independent analyses. C: For a better resolution of FGE secretion and degradation, the pulse chase experiment was repeated, as above (see A), this time with a chase period of only 1.5 h; (relative secretion: medium

FGE/total FGE after 1.5 h).

Table 1: Clinical characterization and mutational analysis of 10 patients with MSD aThe patients were grouped into neonatal very severe (NVS), late infantile severe (LIS) and late infantile mild (LIM) forms of MSD according to manifestation and time point of appearance of each single symptom. b +++, age of onset between 0–2 years of life; ++, 2–4 years; +, after 4 years; -, symptom is missing; nd, not defined.

Table 2: Residual activities of selected sulfatases in MSD fibroblasts a The results and SD were calculated from three independent experiments using total cell

lysates of MSD fibroblasts and an unaffected fibroblast cell line as a control. b Clinical data were analyzed in patients with the same mutation.

Table 3: Synopsis (summary of all results shown in figures 1-4 and tables 1-2) a NVS, neonatal very severe; LIS, late infantile severe; LIM, late infantile mild. b For the NVS case the results for recombinant FGE are given referring to the two different

missense mutations detected in this heterozygous patient (p.R327X/p.A149_A173del). c ER, endoplasmic reticulum.

22 Fig. 1! A149_A173del ! WT ! S155P ! G247R ! Control ! G263V ! R327X ! R345C !

42 kDa! * 37 kDa! Cells! *

38 kDa! Medium!

Fig. 4 A G263V Control WT S155P G247R WT A149_ A173del R327X R345C

Cells (0h)

Cells (6h)

% - 50 2 0 0 39 1 1 2

Medium (6h)

% - 54 3 23 45 52 0 0 16 Total % (6h) - 104 5 23 45 91 1 1 18 Intracellular

- WT WT

Stability ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓

↓↓ ↓ Relative ↓↓ - WT n.d. WT n.d. n.d. Secretion

B % Activity 0 100 1.6 6.7 15.9 0.3 0.0 2.0 (±SD) (±0.4) (± 0.8) (±1.2) (± 0.04) (±0) (±0.2)

C G247R G263V Control WT A149_ A173del S155P R327X R345C

Cells (0h)

Cells (1.5h)

% - 91 22 37 22 18 12 44 Medium (1.5h)

% - 9 0 1 43 60 0 7 Total % (1.5h) - 100 22 38 65 78 12 51 Intracellular

- WT

Stability ↓↓ ↓ ↓↓ ↓↓ ↓↓↓ ↓

↓ ↓↓ ↓↓ ) Relative ( - WT Secretion ↓↓ ↓↓ ↓↓ SUPPLEMENTARY INFORMATION (Schlotawa et al.)

SUPPLEMENTARY MATERIALS AND METHODS

Molecular analysis of SUMF1 mutations. mRNA was extracted from patient fibroblast cell lines using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s procedure, transformed into SUMF1 specific cDNA, and completely sequenced as described earlier21. Genomic DNA was extracted using the DNeasy blood & tissue kit (Qiagen) following the procedure suggested in the kit manual. Nine exons and all intron/exon regions of the SUMF1 gene were fully sequenced as described before21. For mutational analysis we used the SeqMan II software (DNAstar, Madison, USA).

FGE expression. FGE cDNA served as template for an add-on PCR reaction using primers

FGE_SP_EcoRI (5’-GGAATTCGGGACAACATGGCTGCG-3’) and FGE_XbaI_nc (5’-

GCTCTAGACCTTGGTTGTCAGTCCATGG-3’), creating FGE-His cDNA as described earlier13,28. The PCR product was cloned into the pSB4.7pA expression vector21 downstream of its CMV promotor. The pSB-FGE-His plasmid was used as template for site-directed mutagenesis according to the QuikChange Protocol (Stratagene, La Jolla, USA) with Pfu polymerase and complementary primer pairs (coding sequence only): FGE_S155P-His_c

5’-CTGAGAAGTTTGGCGACCCCTTTGTCTTTGAAGGC-3’, FGE_R224W-His_c

5’-ACTTGGGCAGGGAAGTGGCTGCCCACGGAAG-3’, FGE_G247R-His_c

5’-ATAGACTTTTCCCCTGGCGCAACAAACTGCAGC-3’, FGE_G263V-His_c

5’-TGCCAACATTTGGCAGGTCGAGTTTCCGGTGA-3’, FGE_R345C-His_c

5’-TATTGTTACAGGTATTGCTGTGCTGCTCGGAGCC-3’, FGE_R327X-His_c

5’-CCTTCTGGGAAAGACTGAGTGAAGAAAGGTGG-3’, FGE_A149_A173del His_c

5’-GAACTCAACTGGCTATTTGACAGAGGTTGCAGCTGCTCCCTGGTG-3’. DpnI- treated products were amplified in E. coli. All constructs were fully sequenced to preclude any PCR error. HT-1080 cells were stably transfected with these plasmids as described earlier13,28. Different clones were tested for FGE expression and selected for further studies. Immunofluorescence microscopy. Endogenous FGE in human skin fibroblasts or mutant

FGE in stably transfected HT-1080 cells was detected by indirect immunofluorescence using a procedure previously described28. Cells were grown on coverslips for 24 h and co- localization studies were done using FGE antiserum and monoclonal antibodies against PDI

(Biomol, Hamburg, Germany), LAMP1 (Abcam, Cambridge, United Kingdom) and Golgi protein 58K (Abcam). Confocal images were taken on a Leica TCS SP2 AOBS laser-scanning microscope (Leica, Solms, Germany).

Enzyme assays. Sulfatase activities were measured from whole cell lysates as described30,31.

FGE activity assays were performed with FGE enriched from cells and media using the synthetic ASA-derived peptide P23 (200 nM) as substrate21. After incubation for different time intervals at 37 °C, FGly formation was determined using MALDI-TOF mass spectrometry. The specific activity was calculated in relation to the FGE protein amount determined by Western blotting. FGE amounts were adjusted in order not to exceed 50% turnover of P23 in 30 min at 37 °C.

FGE purification. HT1080 cells stably overexpressing His-tagged FGE proteins were grown to near confluence. Medium was collected every 48 h, cleared by spinning at 1100 g for 10 min, and the supernatant was subjected to ammoniumsulfate precipitation (50% w/v). The precipitate was resuspended in buffer I (50 mM sodium phosphate, 300 mM sodium chloride, pH 8.0) and dialyzed over night at 4 °C against buffer I. The dialysate was cleared by centrifugation and incubated overnight at 4 °C with Ni-NTA agarose (50% suspension,

Qiagen, Hilden, Germany), 100 µl per ml dialysate. The agarose was washed three times with

1 ml of 25 mM imidazole in buffer I. FGE-His was eluted three times with 3 ml 250 mM imidazole in buffer I (each time incubating the Ni-NTA agarose for 20 min at 4 °C). The elution fractions were pooled and concentrated using an ultrathimble (Schleicher & Schuell,

Dassel, Germany). For purification of intracellular FGE-His, cells were harvested, resuspended in buffer I, sonicated in presence of a protease inhibitor cocktail (Sigma Aldrich,

Munich, Germany), centrifuged, incubated with Ni-NTA agarose, and processed following the protocol mentioned above.

SUPPLEMENTARY FIGURE LEGENDS (Schlotawa et al.)

Figure S1: Structural basis for MSD in the patients of this study. The FGE crystal structure is shown in ribbon presentation with blue parts representing areas that are not directly affected by any of the mutations. The MSD causing missense mutations p.S155P, p.G247R, p.G263V and p.R345C were mapped onto this structure, as indicated by olive colored arrowheads. The deletion mutation p.A149_A173del mainly affects β-strand 5

(residues 153-158, pink) and a loop of unknown structure (residues 161-176, pink dotted line). This deletion mutation as well as mutations p.G247R and p.G263V affect surface exposed residues (see also Discussion). The nonsense mutation p.R327X is a null mutant

(Fig.4B), as it lacks the camel colored parts of the structure, thereby disrupting the catalytic core, where FGly in the sulfatase substrate (red colored stick model) is generated close to the oxygen binding pocket (green circular area). See references 25 and 34 for further explanation.

Grey spheres represent calcium ions.

Figure S2: Endogenous expression of FGE variants in patient fibroblasts. Cross-reacting material was detected by Western Blot analysis using FGE antiserum. The bands visible at 41 kDa correspond to intracellular FGE. The other bands visible reflect truncated forms of FGE, probably degradation products. For patient 1, however, truncated forms of FGE represent the encoded variants FGE p.R327X and p.A149_A173del. Expression of FGE is drastically reduced in all patient fibroblasts as compared to control fibroblasts (WT, wild-type). This observation holds true also in case of recombinant expression of FGE variants in HT-1080 cells (see Fig.1). SUPPLEMENTARY TABLE (Schlotawa et al.)

Patient Mutation Origin Consanguinity No. cDNA Protein

c.463C>T p.S155P 2 USA nd c.463C>T p.S155P

c.463C>T p.S155P 3 c.463C>T p.S155P USA nd

c.463C>T p.S155P 4 USA nd c.463C>T p.S155P

c.739G>C p.G247R 5 c.739G>C p.G247R Turkey yes

c.739G>C p.G247R 6 Turkey yes c.739G>C p.G247R c.739G>C p.G247R 7 Turkey yes c.739G>C p.G247R

c.1033C>T p.R345C 8 Switzerland no c.1033C>T p.R345C c.788G>T p.G263V 9 c.788G>T p.G263V Pakistan yes

c.788G>T p.G263V 10 c.788G>T p.G263V Pakistan yes

Table S1: Ethnic origin and family history of the nine patients with homozygous SUMF1 mutations.

Patients with a given mutation belong to the same ethnic origin. Apart from patient 8 all other patients were children of consanguinous parents. No family data are available for patients 2, 3 and 4; nd: not defined.

A149_A173del S155P

G263V

R345C R327X

G247R Patient 1 2 3 4 5 fetus 8

FGE p.G247R p.S155P p.G263V WT p.R327X p.S155P p.S155P p.S155P p.S155P p.S155P p.G247R p.G263V p.R345C p.R345C Variant p.A149_A173del 41 kDa

Expression +++ + + + + + - +

Fig. S2 (Schlotawa et al.)