Thomas Dierks
email: thomas.dierks@uni-bielefeld.de

Lars Schlotawa

Eva Charlotte Ennemann

Karthikeyan Radhakrishnan

Bernhard Schmidt

Anupam Chakrapani

Hans-Jürgen Christen

Beat Steinmann Hugo Moser

Jutta Gärtner

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

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

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires 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. 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. and later onset beyond the second year of life [START_REF] Schlotawa | Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency[END_REF] . Late infantile forms include the majority of MSD cases; a Saudi variant form was described separately [START_REF] Al Aqeel | Saudi variant of multiple sulfatase deficiency[END_REF] . Up to now, only one juvenile case of MSD has been published with an onset of symptoms in late childhood [START_REF] Blanco-Aguirre | Unusual clinical presentation in two cases of multiple sulfatase deficiency[END_REF] .

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 deficiencies [START_REF] Dierks | Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease -Lysosomal storage disorders caused by defects of non-lysosomal proteins[END_REF][START_REF] Diez-Roux | Sulfatases and human disease[END_REF] . 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 damage [START_REF] Dierks | Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease -Lysosomal storage disorders caused by defects of non-lysosomal proteins[END_REF] .

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 Cαformylglycine (FGly) [START_REF] Dierks | Conversion of cysteine to formylglycine: a protein modification in the endoplasmic reticulum[END_REF][START_REF] Schmidt | A novel amino acid modification in sulfatases that is defective in multiple sulfatase deficiency[END_REF][START_REF] Dierks | Sequence determinants directing conversion of cysteine to formylglycine in eukaryotic sulfatases[END_REF] . 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 341 [START_REF] Dierks | Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycinegenerating enzyme[END_REF][START_REF] Mariappan | ERp44 mediates a thiol-independent retention of formylglycine-generating enzyme in the endoplasmic reticulum[END_REF] . FGE is encoded by the sulfatase modifying factor 1 gene (SUMF1). Patients with MSD carry mutations in this gene [START_REF] Cosma | The multiple sulfatase deficiency gene encodes an essential and limiting factor for the activity of sulfatases[END_REF][START_REF] Dierks | Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme[END_REF] . Null mutations result in the complete absence of sulfatase activities, as shown in a SUMF1 gene trap mouse [START_REF] Settembre | Systemic inflammation and neurodegeneration in a mouse model of multiple sulfatase deficiency[END_REF] . 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 mutations [START_REF] Annunziata | Multiple sulfatase deficiency is due to hypomorphic mutations of the SUMF1 gene[END_REF] . Until now, more than 30 different SUMF1 mutations were found, most of which are missense mutations [START_REF] Diaz-Font | Clinical and mutational characterization of three patients with multiple sulfatase deficiency: report of a new splicing mutation[END_REF][START_REF] Dierks | Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme[END_REF][START_REF] Cosma | Molecular and functional analysis of SUMF1 mutations in multiple sulfatase deficiency[END_REF][START_REF] Dierks | Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycinegenerating enzyme[END_REF][START_REF] Artigalas | Multiple sulfatase deficiency: clinical report and description of two novel mutations in a Brazilian patient[END_REF] .

In a first approach we investigated the consequences of four different MSD causing SUMF1 mutations on FGE [START_REF] Schlotawa | Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency[END_REF] 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.

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 reports [START_REF] Steinmann | A newly recognized cause of low urinary estriol in pregnancy: multiple sulfatase deficiency of the fetus[END_REF] . 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% CO 2 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 earlier [START_REF] Schlotawa | Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency[END_REF][START_REF] Dierks | Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycinegenerating enzyme[END_REF][START_REF] Preusser-Kunze | Molecular characterization of the human Calpha-formylglycine-generating enzyme[END_REF][START_REF] Mariappan | ERp44 mediates a thiol-independent retention of formylglycine-generating enzyme in the endoplasmic reticulum[END_REF] . 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 described [START_REF] Rommerskirch | Multiple sulfatase deficiency: catalytically inactive sulfatases are expressed from retrovirally introduced sulfatase cDNAs[END_REF][START_REF] Voznyi | A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease)[END_REF] .

FGE activity assays were performed with FGE enriched from cells and media using the synthetic ASA-derived peptide P23 as substrate [START_REF] Dierks | Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme[END_REF] . 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 [ 35 S]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 earlier [START_REF] Gieselmann | In vitro mutagenesis of potential Nglycosylation sites of arylsulfatase A. Effects on glycosylation, phosphorylation, and intracellular sorting[END_REF] . 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.

RESULTS

Clinical presentation and mutational analysis of ten patients with MSD

Based on Eto's classification [START_REF] Eto | Pathochemistry, pathogenesis and enzyme replacement in multiple-sulfatase deficiency[END_REF] we distinguished one neonatal (NVS) and nine late infantile patients. The latter were further subdivided into seven severe (LIS) and two milder (LIM) cases [START_REF] Dierks | Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease -Lysosomal storage disorders caused by defects of non-lysosomal proteins[END_REF][START_REF] Schlotawa | Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency[END_REF] . 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 medium [START_REF] Preusser-Kunze | Molecular characterization of the human Calpha-formylglycine-generating enzyme[END_REF][START_REF] Mariappan | The noncatalytic N-terminal extension of formylglycine-generating enzyme is required for its biological activity and retention in the endoplasmic reticulum[END_REF] . 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).

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 predictions [START_REF] Dierks | Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycinegenerating enzyme[END_REF] .

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 arylsulfatase A as substrate (at saturating concentration, 15-times above K M of wt FGE) [START_REF] Dierks | Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme[END_REF] .

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.

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 stability [START_REF] Dierks | Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycinegenerating enzyme[END_REF] . To prove this prediction we metabolically labeled FGE expressing HT-1080 cell clones with [ 35 S]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 expected [START_REF] Schlotawa | Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency[END_REF] , 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 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 study [START_REF] Schlotawa | Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency[END_REF] 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 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 cases [START_REF] Perlmutter-Cremer | Unusual early manifestation of multiple sulfatase deficiency[END_REF][START_REF] Vamos | Multiple sulphatase deficiency with early onset[END_REF][START_REF] Burch | Multiple sulphatase deficiency presenting at birth[END_REF][START_REF] Busche | Neonatal manifestation of multiple sulfatase deficiency[END_REF] . Most symptoms were present at birth and the patient showed a rapidly progressive course of disease. The group of late infantile patients [START_REF] Diaz-Font | Clinical and mutational characterization of three patients with multiple sulfatase deficiency: report of a new splicing mutation[END_REF][START_REF] Mancini | Pitfalls in the diagnosis of multiple sulfatase deficiency[END_REF][START_REF] Loffeld | Mild ichthyosis in a 4-year-old boy with multiple sulphatase deficiency[END_REF] 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 form [START_REF] Blanco-Aguirre | Unusual clinical presentation in two cases of multiple sulfatase deficiency[END_REF] , (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 line [START_REF] Schlotawa | Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency[END_REF][START_REF] Blanco-Aguirre | Unusual clinical presentation in two cases of multiple sulfatase deficiency[END_REF][START_REF] Dierks | Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme[END_REF][START_REF] Dierks | Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycinegenerating enzyme[END_REF][START_REF] Preusser-Kunze | Molecular characterization of the human Calpha-formylglycine-generating enzyme[END_REF] . 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) [START_REF] Dierks | Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycinegenerating enzyme[END_REF] .

However, the consequences on FGE activity remained difficult to predict, because the substitutions did not directly affect the substrate binding groove, the oxygen binding pocket or even the catalytic site close to the redox active cysteines [START_REF] Roeser | A general binding mechanism for all human sulfatases by the formylglycine-generating enzyme[END_REF] .

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 ER [START_REF] Dierks | Conversion of cysteine to formylglycine: a protein modification in the endoplasmic reticulum[END_REF][START_REF] Fey | Characterization of posttranslational formylglycine formation by luminal components of the endoplasmic reticulum[END_REF] . 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 ER [START_REF] Vembar | One step at a time: endoplasmic reticulum-associated degradation[END_REF][START_REF] Christis | Protein folding includes oligomerization -examples from the endoplasmic reticulum and cytosol[END_REF] . 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 levels [START_REF] Preusser-Kunze | Molecular characterization of the human Calpha-formylglycine-generating enzyme[END_REF][START_REF] Fraldi | Multistep, sequential control of the trafficking and function of the multiple sulfatase deficiency gene product, SUMF1 by PDI, ERGIC-53 and ERp44[END_REF][START_REF] Zito | Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum[END_REF] .

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 peptide [START_REF] Dierks | Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme[END_REF][START_REF] Preusser-Kunze | Molecular characterization of the human Calpha-formylglycine-generating enzyme[END_REF][START_REF] Peng | Identification of formylglycine in sulfatases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[END_REF] , 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 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 [START_REF] Dierks | Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycinegenerating enzyme[END_REF] . 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-compartments [START_REF] Mariappan | ERp44 mediates a thiol-independent retention of formylglycine-generating enzyme in the endoplasmic reticulum[END_REF][START_REF] Fraldi | Multistep, sequential control of the trafficking and function of the multiple sulfatase deficiency gene product, SUMF1 by PDI, ERGIC-53 and ERp44[END_REF] . 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 levels [START_REF] Zito | Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum[END_REF] , we conclude that impaired retention contributes to disease 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 activity [START_REF] Schlotawa | Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency[END_REF] .

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 Such factors can be expected to have an impact on the clinical presentation of patients [START_REF] Conary | Synthesis and stability of steroid sulfatase in fibroblasts from multiple sulfatase deficiency[END_REF][START_REF] Steckel | Multiple sulfatase deficiency: degradation of arylsulfatase A and B after endocytosis in fibroblasts[END_REF][START_REF] Steckel | Synthesis and stability of arylsulfatase A and B in fibroblasts from multiple sulfatase deficiency[END_REF] .

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 approaches [START_REF] Dierks | Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease -Lysosomal storage disorders caused by defects of non-lysosomal proteins[END_REF][START_REF] Zito | Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum[END_REF] , 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 MSD [START_REF] Sawkar | Gaucher disease-associated glucocerebrosidases show mutation-dependent chemical chaperoning profiles[END_REF][START_REF] Ulloa-Aguirre | Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease[END_REF] . A: Stable cell lines were labeled with [START_REF] Fey | Characterization of posttranslational formylglycine formation by luminal components of the endoplasmic reticulum[END_REF] S methionine/cysteine for 1 h and chased for 6 h in label-free medium. FGE was immunoprecipitated from cells and medium and quantified by 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). 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.

Figure 1 :

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

Figure 2 :

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

Figure 3 :

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

Figure 4 :

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

Table 1 :

 1 Clinical characterization and mutational analysis of 10 patients with MSD a The 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.

Table 3 :

 3 Fig. 1!

  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.

  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 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).

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.), 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) 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 earlier [START_REF] Dierks | Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme[END_REF] . 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 before [START_REF] Dierks | Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme[END_REF] . 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 earlier [START_REF] Schlotawa | Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency[END_REF][START_REF] Preusser-Kunze | Molecular characterization of the human Calpha-formylglycine-generating enzyme[END_REF] . The PCR product was cloned into the pSB4.7pA expression vector [START_REF] Dierks | Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme[END_REF] any PCR error. HT-1080 cells were stably transfected with these plasmids as described earlier [START_REF] Schlotawa | Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency[END_REF][START_REF] Preusser-Kunze | Molecular characterization of the human Calpha-formylglycine-generating enzyme[END_REF] . 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 described [START_REF] Preusser-Kunze | Molecular characterization of the human Calpha-formylglycine-generating enzyme[END_REF] . Cells were grown on coverslips for 24 h and colocalization 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 described [START_REF] Rommerskirch | Multiple sulfatase deficiency: catalytically inactive sulfatases are expressed from retrovirally introduced sulfatase cDNAs[END_REF][START_REF] Voznyi | A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease)[END_REF] .

FGE activity assays were performed with FGE enriched from cells and media using the synthetic ASA-derived peptide P23 (200 nM) as substrate [START_REF] Dierks | Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme[END_REF] . 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. (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.

SUPPLEMENTARY TABLE (Schlotawa et al.)

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.


Guido Heine

Gennadiy Drozdenko

Annika Lahl

Nadine Unterwalder

Henrik Mei

Hans-Dieter Volk

Thomas Dörner

Andreas Radbruch

Prof. Dr Margitta Worm
email: margitta.worm@charite.de

Efficient tetanus toxoid immunization upon vitamin D supplementation

Keywords: vitamin D, supplementation, tetanus toxoid, immunization, humoral recall response TT, tetanus toxoid, rTT-C, recombinant tetanus toxoid C-fragment, calcitriol, 1α,25-dihydroxyvitamin D3, SEB, Staphylococcus enterotoxin B

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The vitamin D system, besides its well-described function in calcium homeostasis, is involved in the fine tuning of immunity [START_REF] Adams | Unexpected actions of vitamin D: new perspectives on the regulation of innate and adaptive immunity[END_REF][START_REF] Hayes | The immunological functions of the vitamin D endocrine system[END_REF][START_REF] Holick | Vitamin D deficiency[END_REF].

The biologically active metabolite 1α,25-dihydroxyvitamin D 3 (calcitriol) signals following binding to the nuclear vitamin D receptor and transcriptional regulation of target gene expression [START_REF] Hayes | The immunological functions of the vitamin D endocrine system[END_REF]. Calcitriol-synthesis is catalyzed enzymatically from the otherwise inert storage metabolite 25-hydroxvitamin D 3 [START_REF] Adams | Unexpected actions of vitamin D: new perspectives on the regulation of innate and adaptive immunity[END_REF]Hewison 2008, Hayes et al 2003). Thus, the precursor serum concentration determines the vitamin D status [START_REF] Holick | Vitamin D deficiency[END_REF]) [START_REF] Vieth | Vitamin D toxicity, policy, and science[END_REF]) and 25-hydroxyvitamin D serum concentrations below 50 nM (20 ng/ml) are considered as insufficient [START_REF] Holick | Vitamin D deficiency[END_REF]. Acquired vitamin D deficiency has been correlated with the prevalence and/or increased severity of autoimmune diseases, but also infectious diseases and cancer [START_REF] Adams | Unexpected actions of vitamin D: new perspectives on the regulation of innate and adaptive immunity[END_REF][START_REF] Adorini | Control of autoimmune diseases by the vitamin D endocrine system[END_REF][START_REF] Hayes | The immunological functions of the vitamin D endocrine system[END_REF][START_REF] Holick | Vitamin D deficiency[END_REF]. Direct modulation of immune cells by vitamin D is most likely since different immune cell types can produce and perceive calcitriol including monocytes, dendritic cells, T helper cells and activated B cells [START_REF] Adorini | Control of autoimmune diseases by the vitamin D endocrine system[END_REF]Penna 2008, Hayes et al 2003). Previously, we demonstrated that activated human B cells can synthesize calcitriol from its precursor, which mediates enhanced expression of interleukin (IL) -10 [START_REF] Heine | 1,25dihydroxyvitamin D(3) promotes IL-10 production in human B cells[END_REF] and inhibited IgE production [START_REF] Heine | 1alpha,25-dihydroxyvitamin D3 inhibits anti-CD40 plus IL-4-mediated IgE production in vitro[END_REF]. In newborns, cord blood levels of 25hydroxyvitamin D and the ratio of IL-10 to IgE are both higher in infants born in summer compared to winter [START_REF] Zittermann | Low vitamin D status is associated with low cord blood levels of the immunosuppressive cytokine interleukin-10[END_REF]. Other vitamin D receptor mediated functions are enhanced innate anti-microbial defence, increased tolerance of adaptive immune reactions and altered lymphocyte trafficking [START_REF] Adams | Unexpected actions of vitamin D: new perspectives on the regulation of innate and adaptive immunity[END_REF][START_REF] Adorini | Control of autoimmune diseases by the vitamin D endocrine system[END_REF][START_REF] Hayes | The immunological functions of the vitamin D endocrine system[END_REF]. calcium mobilization reflecting hypercalcaemic toxicity of endogenously synthesized calcitriol [START_REF] Vieth | Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety[END_REF]) and the no-observed-adverse-event-level (NOAEL) for daily vitamin D supplementation was set at 2000 I.U. (50 µg) [START_REF] Vieth | Vitamin D toxicity, policy, and science[END_REF]. Still, no data on immunologic safety, e.g. regarding intact recall responses are available.

Materials and Methods

Study population and design

Individuals were recruited at the Department of Dermatology and Allergy, Charité -Universitätsmedizin Berlin, Germany. The inclusion criteria were scheduled tetanus/diphtheria immunization due to a previous vaccination that dated back at least 5 years. The exclusion criteria were a history for sarcoidosis, hypercalcemia, creatinine serum concentration >1 mg/dL, nephrolithiasis, any chronic disease and planned exposition to UVlight including tanning or travelling to countries at lower latitude (<40°). The study was approved by the Ethical Board of the Charité and written informed consent was obtained from all participants. Individuals were randomly assigned in a double-blind manner to the vitamin D group receiving orally 2000 I.U. vitamin D 3 oil per day (50 µg, n=20) or to the placebo group (equal volume neutral oil; n=12). The daily calcium intake of 1200 mg by each participant was guaranteed by supplementation. Compliance was assessed by determining the 25hydroxyvitamin D serum levels and consumed study medication amount at the end of the supplementation period of 10 weeks. An intramuscular booster immunization with a combined tetanus/diphtheria toxoid vaccine (Td-Merieux ® , Sanofi Pasteur MSD, Strasbourg, France) was performed after 9 weeks supplementation period.

Biochemical analysis

25-hydroxyvitamin D was measured by enzyme immunoassay (EIA) (Immundiagnostik AG, Bensheim, Germany). TT-specific IgG and IgA were measured using an EIA from serially diluted sera (DRG Diagnostics, Marburg, Germany). A long-term protective immunity (5-10 yrs) is assumed >1.0 I.U./ml anti-tetanus IgG. TT-specific IgA concentrations were validated by serially dilution of strongly positive sera and units were calculated based on the absorption at 405 nm. Tetanus-specific IgE was deteremined using the ImmunoCAP system (Phadia, Freiburg, Germany). All other parameters were determined by the Charité routine laboratory facility.

Flow cytometry

Peripheral T-and B lymphocytes were quantified from full blood after surface-staining using TruCount beads and fluorochrome-conjugated monoclonal antibodies against CD4 or CD8 and CD19, CD20, CD27 (all from BD, Heidelberg, Germany) and CD38 (Beckman Coulter, Krefeld, Germany), respectively. Tetanus-specific B cells and plasmablasts were stained from isolated peripheral blood mononuclear cells (PBMCs) after density gradient centrifugation (Lymphocyte separation medium, PAA, Cölbe, Germany) exactly 7 days after TTimmunization by intracellular binding to the fluorescence labelled recombinant C-fragment of tetanus toxin (rTT.C), as described [START_REF] Mei | Blood-borne human plasma cells in steady-state are derived from mucosal immune responses[END_REF][START_REF] Odendahl | Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response[END_REF] following CD38, CD3 and CD14 surface staining. T cell activation was monitored following stimulation of full blood with 2 µg/ml Staphylococcus Enterotoxin B (SEB, Sigma Dreieich, Germany) in the presence of 1 µg/ml Brefeldin A (Sigma) [START_REF] Frentsch | Direct access to CD4+ T cells specific for defined antigens according to CD154 expression[END_REF]. Flow cytometric analyses were performed using a LSRII cytometer equipped with an additional UV laser or FACS Canto II and DivaSoft operation system (all from BD) and FlowJo7 software (TreeStar, Ashland, OR).

Tetanus toxoid-induced cytokine release

The TT-specific T cell cytokine profile was monitored before and exactly 7 days after immunization, at the peak of TT-specific T cell circulation (De [START_REF] Rosa | Vaccination in humans generates broad T cell cytokine responses[END_REF]. Isolated PBMCs (4x10 5 cells in 200 µl) were cultured in RPMI1640 containing 10% FCS as described [START_REF] Heine | 1alpha,25-dihydroxyvitamin D3 inhibits anti-CD40 plus IL-4-mediated IgE production in vitro[END_REF], in 96-well round bottom culture plates in the presence or absence of 0.5 µg/ml SEB (Sigma, Dreieich, Germany) or 1% tetanus toxoid (Tetanus-Merieux ® , Sanofi Pasteur MSD, Strasbourg, France) in triplicates. After 24 hours, the supernatants were stored at -80°C until analysis using the Th1/Th2 cytokine CBA kit (BD), which allows the simultaneous quantification of secreted IL-2, IL-4, IL-5, IL-10, TNF-α (all sensitivity thresholds <3 pg/ml), and IFN-γ (< 8 pg/ml).

Statistical methods

Statistical

Results

Efficient increase of 25-hydroxyvitamin D serum concentrations by daily intake of 2000 I.U. cholecalciferol

At study entry, the distribution of age, gender and body-mass-index (Suppl.Tab.1) and vitamin D status did not differ between the two groups (Suppl.Tab.2; Fig. 1B). The median 25hydroxyvitamin D concentrations significantly increased upon daily intake of 2000 I.U. (50 µg) vitamin D from 28.4 nM to 80.3 nM after 10 weeks (P=0.0001, Fig. 1B). As expected, in the placebo group the median 25-hydroxyvitamin D serum concentration decreased by 13.1 nM to 29.1 nM after the study period (P=0.0007). Consequently, the 25-hydroxyvitamin D serum concentrations were strongly different between both groups after the supplementation period (P=0.0001). During 10 weeks of daily intake of 2000 I.U. vitamin D (cholecalciferol), the calcium, phosphate and IgA, IgG, and IgM serum concentrations were not altered in any individual (Suppl.Tab.2). The differential blood count was comparable in both groups except the absolute monocyte numbers which decreased in the placebo group (-0.04/nl) and remained constant in the vitamin D group (P=0.04, Suppl.Tab.2). Adverse events during the study period occurred in both groups in comparable frequencies (vitamin D group: 30.0% common colds, n=6; placebo: 33.3% common colds, n=4, data not shown). No treatment-related adverse event occurred during the study.

Efficient immunization-triggered tetanus toxoid-specific antibody induction upon vitamin D supplementation

Before immunization, the tetanus toxoid (TT)-specific IgG concentrations were comparable between both groups (Fig. 1B). After 9 weeks of supplementation, a tetanus/diphtheria toxoid immunization was performed intramuscular and the TT-specific IgG concentrations were strongly boosted by 29.8 I.U. ± 14.8 upon vitamin D intake and 21.0 I.U. ± 13.3 with placebo (both intragroup changes P<0.0001). The TT-specific IgG boost efficiency was marginally higher in the vitamin D group (P=0.04). The baseline and immunization-induced TT-specific IgA serum concentrations were independent of vitamin D or placebo treatment (P=0.70; data not shown). Herein, TT-specific IgE was present in 9 participants and comparable changes upon immunization were observed in both groups (vitamin D group: 3 up, 3 down; placebo 2 up, 1 down; data not shown). Additionally, circulating tetanus toxoid-specific antibody secreting plasmablasts (Fig. 1C) were analyzed exactly 7 days after immunization, as these cells are only present in the peripheral blood in a very limited time frame [START_REF] Odendahl | Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response[END_REF]. As shown in Fig. 1D, most recombinant tetanus toxoid C-fragment (rTT.C) -specific plasmablasts were of IgG isotype in both groups (intergroup difference P=0.57) followed by rTT.C-specific IgA plasmablasts (P=0.53). The numbers of rTTC-specific plasmablasts and relative frequency of all plasmablasts showed no statistically significant differences in both groups (P=0.51-0.26, data not shown). The frequencies of peripheral naïve B cells (CD27 -- CD19 + ), memory B cells (CD27 + CD19 + ), or terminally differentiated plasmablasts (CD27 ++ CD20 -CD38 hi CD19 + ) were comparable between the vitamin D or placebo group (data not shown). Interestingly, a significant correlation between the increased 25hydroxyvitamin D concentration and TT-IgG serum concentration was observed (P=0.042, R=0.351, Fig. 2).

Stable tetanus toxoid induced cytokine secretion by peripheral immune cells

Whether the tetanus toxoid (TT) -specific and/or polyclonal cytokine response is modulated upon vitamin D intake was analyzed from ex vivo stimulated peripheral blood cells before and exactly 7 days after immunization, at the peak of specific T cell circulation (De [START_REF] Rosa | Vaccination in humans generates broad T cell cytokine responses[END_REF]. Unstimulated PBMCs served as negative control and no significant spontaneous cytokine release was observed, otherwise the individual data set was excluded due to subclinical concomitant infection (vitamin D group: n=5; placebo n=4). As positive control and reference for the circulating T cell pool polyclonal T cell stimulation by SEB resulted in activation CD4 + T helper cells in the vitamin D or placebo group in comparable frequencies (4.7% ± 0.8 or 3.9% ± 0.5 of all lymphocytes, data not shown) as determined by CD154upregulation [START_REF] Frentsch | Direct access to CD4+ T cells specific for defined antigens according to CD154 expression[END_REF]. These data were paralleled by SEB-induced IFN-γ, TNFα, IL-10, IL-2, IL-4 and IL-5 secretion (intergroup differences P-value range 0.84-0.11, Tab.1).

Tetanus toxoid stimulation of PBMCs for 24 h before and one week after immunization induced IL-2, IL-10, IFN-γ, or TNF-α secretion and no significant differences between both groups regarding the respective TT-induced cytokine secretion were observed (P-values 0.76-0.11, Tab.1). TT-induced IL-4 or IL-5 secretion was below the detection threshold in all samples (data not shown).

Discussion

Herein, we show that the daily intake of 2000 I.U. vitamin D over 10 weeks was well tolerated and sufficient to restore the vitamin D status as determined by a median 25hydroxvitamin D concentration of 80.3 nM which is considered as sufficient for optimal vitamin D function [START_REF] Adams | Unexpected actions of vitamin D: new perspectives on the regulation of innate and adaptive immunity[END_REF]Hewison 2008, Holick 2007). The tetanus toxoid boost immunization is fully operational upon vitamin D intake with regard to TT-specific immunoglobulin induction and cytokine expression and more importantly, is not negatively altered in a placebo-controlled setting. In contrast, the data indicate that induction of TTspecific IgG was marginally more efficient in vitamin D compared to the placebo group (P=0.04). These data are in line with previous studies [START_REF] Enioutina | The induction of systemic and mucosal immune responses following the subcutaneous immunization of mature adult mice: characterization of the antibodies in mucosal secretions of animals immunized with antigen formulations containing a vitamin D3 adjuvant[END_REF][START_REF] Enioutina | TLR ligands that stimulate the metabolism of vitamin D3 in activated murine dendritic cells can function as effective mucosal adjuvants to subcutaneously administered vaccines[END_REF][START_REF] Ivanov | 1,25-dihydroxyvitamin d3 enhances systemic and mucosal immune responses to inactivated poliovirus vaccine in mice[END_REF] [START_REF] Van Der Stede | Enhanced induction of the IgA response in pigs by calcitriol after intramuscular immunization[END_REF] and attributed most likely to induction of specific IgG1, which is the dominant subclass induced by the vaccination used herein (>92% of the circulating specific plasmablasts) [START_REF] Frolich | Secondary immunization generates clonally related antigen-specific plasma cells and memory B cells[END_REF]. That vitamin D supplementation did not alter the specific IgG-subclass composition is also suggested by our data that vaccination-induced boosted anti-tetanus antibodies of IgA or IgE isotype was independent of vitamin D supplementation, although common cytokines were involved, and further supported by previous studies in animals [START_REF] Van Der Stede | Enhanced induction of the IgA response in pigs by calcitriol after intramuscular immunization[END_REF]. However, our data should be validated by investigation of a larger study cohort, due to the limited group size.

The reports on vitamin D-mediated modulation of T helper cell polarization in mice and human are heterogeneous [START_REF] Hayes | The immunological functions of the vitamin D endocrine system[END_REF] and include altered Th1/IFN-γ [START_REF] Daniel | 22ene-25-oxa-vitamin D: a new vitamin D analogue with profound immunosuppressive capacities[END_REF], Th2/IL-4- [START_REF] Hayes | The immunological functions of the vitamin D endocrine system[END_REF], but also tolerogenic responses [START_REF] Adorini | Control of autoimmune diseases by the vitamin D endocrine system[END_REF]Penna 2008, Schleithoff et al 2006). Herein, we observed a stable recall T helper cell response upon vitamin D regarding the secretion of TT-specific or polyclonally induced IL-2, IL-4, IL-5, IL-10, IFN-γ or TNFα compared to placebo. Interestingly, the blood monocytes counts were higher in the vitamin D group after the supplementation period, compared to placebo. As differentiation of monocytes/macrophages from progenitor cells is promoted by calcitriol [START_REF] Adams | Unexpected actions of vitamin D: new perspectives on the regulation of innate and adaptive immunity[END_REF]Hewison 2008, Hayes et al 2003), our findings indicate that supplemented vitamin D metabolized to calcitriol modulated cellular immunity. However, whether and how vitamin D-induced monocytes contributed to the enhanced TT-IgG boost efficiency, e.g. by altered antigen presentation IgG promoting costimulation of T and B lymphocytes, is not known yet.

An advantage in our study setting during the winter months was the lack of UV-derived vitamin D synthesis allowing a correlation between vitamin D provided by supplementation with biological effects. As prerequisite, a high prevalence vitamin D deficiency in December was observed herein and confirms a recent epidemiological report from Germany [START_REF] Hintzpeter | Vitamin D status and health correlates among German adults[END_REF]. As vitamin D supplementation was efficient, differing averagely less than 2% from a calculation by Heaney et al. [START_REF] Heaney | Human serum 25hydroxycholecalciferol response to extended oral dosing with cholecalciferol[END_REF], and the 25-hydroxyvitamin D serum levels further decreased in the placebo group during the study period, strongly different 25hydroxyvitamin D serum concentrations between both groups were observed (P=0.0001). In further placebo controlled studies during the winter months, the efficacy of immunological functions of endogenously produced calcitriol following vitamin D supplementation can be examined in different disease settings, e.g. allergy or autoimmunity.

Legends for Figures

  evaluations and graphic display were performed with Prism software (GraphPad Software, Inc., CA, USA) and SPSS15.0 (Spss, Inc. Chicago, IL, USA). Normal distribution was tested by the Kolmogorov-Smirnov test and the D'Agostino & Pearson omnibus normality test. Normally distributed values were tested by the Student-T test and the mean ± standard deviation (SD) are shown. Whitney-Mann-U test or Wilcoxon Test was applied for non-normally distributed values for independent or dependent parameters, respectively and median and interquartile ranges are shown.

Figure 1 :

 1 Figure 1: Efficient immunization-triggered tetanus toxoid-specific antibody induction

Figure 2 :

 2 Figure 2: Correlation between vaccination-induced anti-tetanus-IgG. (concentrations

Acknowledgements

We thank Dr. Helmut Orawa and Ramona Scheuffele (both Institut für Biometrie und klinische Epidemiologie, Charité Campus Mitte) for statistical assistance and Dennis Ernst for outstanding assistance. This work was supported by the Deutsche Forschungsgesellschaft (DFG -SFB650/TP5 and TP16) and by the Charité -Universitätsmedizin Berlin. G.H. and G.D. were supported by the DFG -SFB650 TP5 and H.M by the DFG -SFB650/TP16.

This work was supported by the Deutsche Forschungsgesellschaft (DFG -SFB650/TP5 and TP16) and by the Charité -Universitätsmedizin Berlin.

Conflicts of Interest

All authors declare no financial or personal conflict of interest.

Supplementary information is available at The European Journal of Clinical Nutrition's website.


Hessam Wolfgang Gruber
email: wolfgang.gruber@tu-clausthal.de

Hadjiamini Najafabadi

Udo Geckle

Michael Bruns

Harald Schmidt

Crystallization of Magnetron Sputtered Amorphous Si1-xCx Films (x = 1/3) Studied by Grazing Incidence X-ray Diffractometry

Keywords: amorphous semiconductors, thin films, crystallization magnetron sputtered SiC films, crystallization kinetics magnetron sputtered SiC films, crystallization kinetics

come

Thin films of silicon carbide attract much attention for applications in various branches of technology [1][2][3][4][5][6]. Silicon carbide can be used in high temperature electronics [7] and as wide band gap device in optoelectronics [8]. Thin films can be produced by various techniques like plasma-enhanced chemical vapor deposition (PE-CVD), hot wire CVD (HW-CVD), hot wire plasma-assisted CVD (HWPA-CVD) processes [9] or magnetron sputtering. Using magnetron sputtering, films of high quality with reproducible properties can be deposited on different substrates. After deposition the films are amorphous. Annealing at elevated temperatures leads to crystallization. Nucleation and growth process during crystallization affect the microstructure of the polycrystalline films [10]. For a tailored production of films for applications detailed information of the crystallization kinetics is important. In Ref. [11] crystallization kinetics of amorphous Si 1-x C x films with a stoichiometric composition (x = 1/2) deposited on silicon wafers and glassy carbon, respectively, was investigated by X-ray diffractometry and transmission electron microscopy. Crystallization could be described by a modified JMAK model (according to the formalism developed by Johnson, Mehl [12], Avrami [13][14][15] and Kolmogorov [16]).

The crystallization mechanism was found to be an interface controlled three dimensional growth process from pre-existing small crystallites with dimensions of 10 to 15 nm which are formed in a transient process in the early stage of crystallization.

The rate constants of crystallization and also the corresponding activation enthalpies were found to depend strongly on the substrate. While an activation enthalpy of 8.9 eV is found for crystallization of silicon carbide deposited on glassy carbon an activation enthalpy of about 4.1 eV is found for crystallization of silicon carbide deposited on silicon wafers [11]. Compared to the same temperature, crystallization of silicon carbide is significantly faster for silicon substrates. The samples for the two types of substrates where produced under identical experimental conditions which excludes an influence due to different conditions of preparation. The dependence of the activation enthalpies of crystallization on the type of substrate, therefore, was referred to the importance of point defects for crystallization. Since the temperatures used were close to the melting point of silicon the silicon substrate can possibly act as a source for point defects. Therefore, crystallization of silicon carbide deposited on silicon substrates is faster than crystallization of silicon carbide deposited on glassy carbon if a point defect mediated crystallization process is assumed [11].

For an understanding of crystallization of silicon carbide it is important to know the chemical order of the amorphous system which depends on the composition and on the production method [18]. According to the tetrahedron model for amorphous silicon-carbon alloys given in Ref. [18] three types of chemical order can be considered (a) complete random order where no preferential chemical bonding between Si and C atoms exists, (b) complete chemical order with homogenous dispersion which means that in Si-rich alloys a C atom in the centre of a tetrahedron is surrounded by four Si atoms and a maximum of possible Si-C bonds is realized and (c) complete chemical order with phase separation which means that the Si-C bonds are clustered [18]. Molecular-dynamics simulations revealed [19] that 40% -45% homonuclear bonds are present in stoichiometric amorphous silicon carbide. This means that the system is not complete chemically ordered. The same simulations also revealed that a high degree of short and medium range order exists which means that that the system is not complete randomly ordered. Consequently, the real atomic structure of an as-deposited amorphous Si 1-x C x film can be considered as a mixture of the three types of chemical order. In Ref. [20] structural relaxation of magnetron sputtered amorphous silicon carbide films deposited on silicon substrates was is investigated. It was found that as-deposited films show a certain degree of random order which means that the order is a mixture of type (a) and (b). After annealing at 800 °C the degree of random order converts to complete chemical order. In Ref. [11] crystallization experiments were carried out at 1200 °C and higher temperatures. At these temperatures the amorphous films are expected to be already relaxed to complete chemical order after very short times of annealing. However, if the composition of the films deviates from the stoichiometric composition complete chemical order should be accompanied by a phase separation. If the composition strongly deviates from the stoichiometric composition the resulting phases should be detectable. Therefore, for further insight into this topic, in this work we investigated the crystallization of silicon carbide in magnetron sputtered Si 1-x C x films with x = 1/3 deposited on silicon wafers. For silicon rich Si 1-x C x films a silicon phase can be expected in addition to the silicon carbide phase in the case of complete chemical order with phase separation. If these phases are crystalline they can be detected by XRD. We used grazing incidence X-ray diffractometry (GIXRD) to determine the crystallized fractions and the sizes of the crystallites.

Experimental details

Thin amorphous Si 1-x C x films with x = 1/3 were prepared by r.f. co-sputtering using a 3'' US GUN low profile planar magnetron source (AP&T, Nürtingen, Germany) mounted on a standard DN 150 CF double cross recipient equipped with pre-sputter shutter and sample positioner allowing various distances (100 nm -200 nm) between magnetron and substrate. Deposition rates of 2 -5 nm/min were achieved, using an operation pressure of 1 × 10 -3 mbar, a sputtering power of 80 W and a substrate temperature of 200 °C. Carbon stripes (99.99 %, Goodfellow, Bad Nauheim, Germany) of 5 mm × 25 mm were radially fixed at equal distances on a silicon base target (99.999 %, Norwegian Talc, Bad Soden, Germany). Films were deposited on single crystalline single silicon (111) wafers (CIS, Germany). Composition was determined by Rutherford backscattering spectrometry (RBS). Concentration depth profiles were measured with secondary ion mass spectrometry (SIMS). A CAMECA IMS-3F was used. The thickness of the films was determined measuring the SIMS crater using a surface profilometer (Alphastep 500, TENCOR). The samples were annealed at temperatures between 1200 °C and 1350 °C in an argon atmosphere (99.996 %) at 1000 mbar for times between 0.5 h and 190 h. A glassy carbon crucible was used. The samples were embedded in a fine-grained SiC powder to protect them from contamination. A special mechanical transfer unit was used to place the samples into the hot zone of the furnace within about 5 minutes. During annealing temperature was kept constant within 1 °C using a eurotherm type 815 temperature controller. Since the transfer times were small compared to the annealing times, heating and cooling times were neglected and our experiments can be considered as an ex-situ investigation of isothermal crystallization. The samples were analysed with GIXRD. Using GIXRD the X-ray strikes the sample under a small angle. The detector is moved while the sample is kept fix. Due to the small incident angle maximum information from the film is obtained while Bragg peaks from the substrate can be eliminated. We worked with a Siemens Kristalloflex/D5000 using Co K α radiation (λ = 0.1789 nm, 40 kV, 40 mA). A rocking curve was recorded before each GIXRD measurement to eliminate a possible tilt angle of the sample. The grazing incidence angle was 5°. At this angle the substrate did not contribute to the diffractograms and a maximum count rate from the film was achieved. Since silicon precipitates are expected from composition considerations it is essential to eliminate the contribution of the silicon substrate.

Results

The composition of the as-produced samples was determined by RBS to be Si 66 C 34 . Concentration depth profile measurements with SIMS revealed constant silicon and carbon intensities throughout the film. The thickness of the films was determined to be 1.5 µm. No change in homogeneity in depth profiles could be observed by SIMS after annealing. The scan size of the primary beam during SIMS analysis was 250 µm. Therefore, resolution of lateral homogeneity is limited to 250 µm. From the diffractograms information about the crystallized phases (position of a Bragg peak), the crystallized fraction of a phase (integrated area of a Bragg peak), the size of the grains (width of a Bragg peak) and the micro strain (shift of the line position) can be obtained. An as-deposited sample was measured with GIXRD in the 2Θ range from 10 ° to 130 °. No Bragg peaks were observed. This shows that asdeposited films are X-ray amorphous and the peaks of the silicon substrate are eliminated. After the first annealing step (1 hour at 1200 °C and 0.5 h at higher temperatures) Bragg peaks from β-SiC and silicon are observed. Since no silicon peaks are found in the diffractograms of the as-deposited samples we attribute these silicon peaks to crystalline precipitates formed within the film. Determination of the crystallized fraction proceeds from the idea that the crystallized fraction of a phase is proportional to the integrated area of a certain Bragg peak, A(t).

If the sample is annealed until no change in the integrated peak area is observed the normalization area A(t = ∞) is determined and the crystallized fraction at a time t, x(t), is given by:

x(t) = A(t)/A(t = ∞) (1)
Fig. 1 shows the Bragg peaks at 2Θ = 41.6° for a sample annealed at 1350 °C for different annealing times. The peak can be identified as the (111)-peak of β-SiC. The crystallized fraction of silicon carbide determined from the integrated area under the (111)-peak is shown in Fig. 2 together with the crystallized fraction of silicon determined from the integrated area under the silicon (220)-peak. Surprisingly, the silicon (220)-peak decreases with increasing annealing time. At 1200 °C the silicon (220)-peak can only be resolved up to an annealing time of 15 h. At 1350° C the silicon (220)-peak can no longer be resolved for annealing times higher than 3.5 h. The crystallized fraction of the β-SiC increases with increasing annealing time. An initial fast crystallization step leads to a crystallized fraction of about 0.25 after an annealing time of 1 h at 1200 °C. At higher annealing times the crystallized fraction increases with a slower rate but the data points do not follow a simple sigmoidal curve. This points to a complicate sequence of crystallization steps. The Bragg peaks were numerically fitted with Lorentzian functions. From the full width at half maximum, β(2Θ), the crystallite size can be determined using the Scherrer formula [21]

Θ Θ = cos ) 2 ( 9 . 0 β λ d ( 2
)
where d is the average size of the crystallites, and λ the wavelength of the radiation. However, if micro strain is present, the Scherrer equation does not give reliable values for the average grain size. To overcome this problem average grain size and micro strain can be determined simultaneously using the Williamson-Hall equation [22,[START_REF] Klug | X-ray Procedures for Polycrystalline and Amorphous Material[END_REF]

λ ε λ β Θ + = Θ Θ sin 4 1 cos ) 2 ( d ( 3
)
where ε is the micro strain. From the ordinate section in Eq. 3 the crystallite size can be determined. Unfortunately for β-SiC only three Bragg peaks with sufficient intensity can be resolved in our diffractograms. Therefore the ordinate section in Eq. 3 is associated with large errors. The slope in Eq. 3, however, and hence the micro strain can be determined with sufficient accuracy using three data points. Fig. 3 shows the micro strain for a sample annealed at 1200 °C. Up to an annealing time of 36 h the micro strain is less than 0.001. At higher annealing times the micro strain increases slightly but never exceeds 0.002. The increase of micro strain may be due to impingement since more than 50 % of silicon carbide is crystallized for annealing times higher than 36 h. For short annealing times the influence of micro strain on line widths can be neglected and the Scherrer equation can be used to determine the size of the crystallites. Fig. 4 shows the average size of the crystallites determined according Eq. 2 with respect to the annealing time. The line width of the most intensive Bragg peak, the (111)-peak, was used. At the beginning of crystallization the experimental points follow a straight line. At higher annealing times the growth of the crystallites slows down until the size becomes constant. From a straight line fit at the beginning of crystallization we obtain the rate constant u = dr/dt for the growth of the crystallites (r being the radius of the crystallites), which are plotted in Fig. 5 as a function of reciprocal temperature.

Discussion

As shown by data from Fig. 2 annealing of Si 1-x C x films with x=1/3 leads to crystallization of a silicon and a silicon carbide phase. This can be expected because of the non-stoichiometric composition of the as-deposited sample. Further, formation of crystalline silicon is much faster than formation of crystalline silicon carbide. This is in accordance with results on the crystallization of amorphous silicon [START_REF] Spinella | [END_REF]. The results can be explained with the assumption that chemical ordering and phase separation have already taken place during the first annealing step and our films consist of a silicon phase and silicon carbide phase. In Fig. 2 data reveal unexpected decrease of Si-peak intensity with annealing time until it can no longer be resolved with our experimental equipment. Therefore, in Fig. 2 the crystalline fraction of silicon is 0 for annealing times higher than 20 h. The line width of the silicon (220)-peak, however, remains constant as long as the peak can be resolved. This makes it very unlikely that the silicon grains are transformed continuously into gaseous SiO or a non-crystalline phase like amorphous SiO 2 via internal oxidation. A continuous dissolution of silicon grains is expected to be connected with a significant line broadening. Also, consumption of silicon grains caused by a reaction of Si with free C would lead to a line broadening. At 1200 °C formation of a liquid silicon phase can also be excluded. Another possible explanation for the decrease of the silicon (220)-peak is that Si diffuses to the substrate. During the first annealing step phase separation takes place and silicon grains grow while the silicon carbide phase remains amorphous. When crystallization of silicon carbide sets in the amount of grain boundaries in the film is as large that Si can diffuse to the substrate along these fast diffusion paths. This means that transport of Si from the film to the silicon substrate can take place. If Si grows epitaxially on the silicon substrate it does not longer contribute to the diffractograms measured by GIXRD. Transport of Si from the film to the substrate is not associated with the volume selfdiffusivity of silicon in silicon carbide which is known to be very low (< 10 -21 m 2 /s at 1100 °C [25]). For the transport of silicon through a polycrystalline silicon carbide layer produced by carbonization of silicon wafers Si diffusivities up to 10 -15 m 2 /s are reported [26][27][START_REF] Rimai | The[END_REF] at about 1200 °C. This results in diffusion lengths of some micro meter for annealing times of some hours. Considering these values the decrease of the silicon peak might be explained by a decrease of the phase faction of silicon in the film due to diffusion of Si from the film to the substrate. However, also in this case a line broadening is expected which is not observed. Nevertheless, the experimental method itself, GIXRD, used to detect crystalline silicon can give an explanation for the decrease of the silicon (220)-peak. With GIXRD only those grains contribute to the diffractogram which fulfil the Bragg condition under the given grazing incidence angle since the sample is kept fix. After the first annealing step the silicon grains are more or less randomly orientated so that they are detected by GIXRD. During further annealing, the grains realign and become textured towards an orientation for which the Bragg condition is not fulfilled for the gracing incidence angle used in the experiment. Consequently, grains are no longer detected by GIXRD. In this case the number of silicon grains which contribute to the diffractogram decreases while the line width of the silicon (220)-peak is not affected. Theoretically, XRD in Bragg-Brentano geometry can detect a phase even in the case of texture. Such measurements were not successful since it was not possible to separate the silicon (220)-peak of the film from the contribution of the silicon substrate.

The crystallization curve given in Fig. 2 shows a very complex shape. This is an indication of a change in the mechanism of crystallization during annealing. Additionally, determination of the crystallized fraction using the integrated area under a Bragg-peak measured with GIXRD may lead to a methodical error if the preferential orientation of the crystallites changes with annealing time. Therefore, the statistical error at the data points in Fig. 2 which was calculated from the errors of the integrated areas determined by least-squares fits with Lorentzian lines might be superimposed by a systematic error. Hence, it is not straight forward to analyze the crystallization curve in Fig. 2 using JMAK theory. Information about the activation enthalpy of crystallization, however, can be obtained from the dependence of the average size of the crystallites on annealing time. As already mentioned at the beginning of crystallization the average size of the crystallites with respect to the annealing time can be described with a straight line. The rate constant u for the growth of pre-existing nuclei is given by [29]

                ∆ - -         ∆ - = = T k G T k H u dt dr u B B I 0 exp 1 exp
where r is the radius of the crystallites, ∆H I is the activation enthalpy for the transfer of an atom across the amorphous-crystalline interface and its incorporation into the crystal lattice, ∆G is the Gibbs energy difference between the amorphous and the crystalline state, and u 0 is a pre-exponential factor. Since ∆G is commonly larger than k B T the growth rate can be described by an Arrhenius behaviour

        ∆ - = T k H u u B c exp 0
where ∆H c = ∆H I is the activation enthalpy of crystallization. The growth rates can be obtained from the slopes of the straight line fits in Fig. 4. The temperature dependent growth rates obey the Arrhenius law (Fig. 5). From the slope in the Arrhenius diagram an activation enthalpy ∆H c = 4.0 ± 0.7 eV can be determined. In Ref. [11] two growth rates have been determined for Si 1-x C x with x = 1/2. In that case after a first step crystallization slows down resulting in two crystallization rates u 1 and u 2 . The values from Ref. [11] are included in Fig. 5. The activation enthalpy of 4.1 ± 0.5 eV given in Ref. [11] is, within error limits, in good agreement with the activation enthalpy found in the present work. The absolute values of u found in this work lie in between u 1 and u 2 from Ref. [11]. Extrapolation of the crystallite size given in Fig. 4 to t = 0 leads to an initial size of 12 ± 1 nm. This is also in agreement with Ref. [11] where the size of the pre-existing crystallites is reported to be in the order of 10 to 15 nm. It should be emphasized that the initial crystallite size is not the critical size of the nuclei but the size of the pre-existing crystallites resulting from a transient process in the very early stage of crystallization [11]. The consistence of the growth rates, of the activation enthalpies and of the initial crystallite size in this work compared to Ref. [11] demonstrates that the growth mechanism in the first stage of crystallization is the same for the non-stoichiometric system and for the stoichiometric system. Obviously, this is a consequence of the phase separation in Si 1-x C x with x = 1/3 during the first annealing step. In a later stage the mechanism of crystallization may change. This is suggested by Fig. 2. The main result of our work, however, is that phase separation takes place and is completed before crystallization of silicon carbide begins. It should be pointed out that so far we only investigated silicon rich Si 1-x C x films. Carbon rich Si 1-x C x films will be investigated in future to clarify whether our conclusions are valid also in this case.

Summary

In this work we investigated the crystallization of silicon carbide in magnetron sputtered Si 1-x C x films with x = 1/3 deposited on silicon substrates. The evolution of the crystallized fraction with respect to the annealing time does not show a simple sigmoidal curve which was observed in the case of magnetron sputtered Si 1-x C x films with x = 1/2. For the first stage of crystallization an activation enthalpy of 4.0 ± 0.7 eV for crystallization could be deduced from the growth rates of the crystallites. The activation enthalpy is, within error limits, equal to the activation enthalpies found for crystallization of silicon carbide in magnetron sputtered Si 1-x C x films with x = 1/2 deposited on silicon substrates. The absolute values for the growth rates and the initial size of the crystallite sizes are also in good agreement with the values for the films with a stoichiometric composition. Therefore, we conclude that prior to crystallization of silicon carbide a phase separation has taken place in the Si 1-x C x films with x = 1/3 resulting in an amorphous silicon carbide phase and an amorphous or crystalline silicon phase. For the first stage of crystallization of the amorphous silicon carbide the same crystallization mechanism is active as in the case of the films with a stoichiometric composition. 3). Also shown are the growth rates u 1 (triangles) and u 2 (circles) from Ref. [11]. annealing time (h)

  Crystallization of Magnetron Sputtered Amorphous Si 1-x C x Films (x = 1/3) Studied by Grazing Incidence X-ray Diffractometry 1. Introduction

21 Deleted: 2 Deleted: 4 F

 2124 SurprisinglyDeleted: , the intensity of the Si peak decreases with increasing annealing time Deleted:

Formatted:FormattedFig. 1 :

 1 Figure captions: Fig. 1: (111) peaks of β-SiC for a Si 2 C film deposited on a silicon wafer. Shown are the diffractograms of a sample as-deposited and annealed at 1350 °C for different annealing times.

Fig. 2 :

 2 Fig. 2: Crystallized fractions of β-SiC and Si for a sample annealed at 1200 °C. The error bars at the data points correspond to the errors of the integrated areas determined by least-squares fits with Lorentzian lines.

Fig. 3 :

 3 Fig. 3: Micro strain determined from a Williamson-Hall plot for a sample annealed at 1200 °C. The error bars correspond to the standard deviations of the straight line fits based on three data points, respectively.

Fig. 4 :

 4 Fig. 4: Average crystallite size determined by the Scherrer equation for a sample annealed at 1200 °C. The error bars correspond to standard deviations.

Fig. 5 :

 5 Fig.5: Arrhenius representation of the growth rates u (squares). The error bars correspond to the standard deviations of the straight line fits (Fig.3). Also shown are the growth rates u 1 (triangles) and u 2 (circles) from Ref.[11].

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Acknowledgements

We thank E. Ebeling for the sample surface preparation and M. Rudolphi and H. Baumann for the RBS measurements. Funding by the Deutsche Forschungsgemeinschaft (DFG) is gratefully acknowledged.