FGE and its salient features

The gene encoding human FGE, designated asSUMF1(sulfatase modifying factor 1) is located on chromosome 3p26. It belongs to a new gene family with orthologous members in pro- and eukaryotic sulfatase expressing organisms. Some distant FGE-related homologs seem to have distinct oxygenase and other functions [Landgrebe et al.,2003].

1.2.1 Expression and localization of endogenous and recombi-nant FGE

FGE is ubiquitously expressed. Northern blot analysis of total RNA from skin fibrob-lasts and polyA⁺ RNA from various tissues (Fig. 1.4B) shows a single transcript of 2.1 kb. Relative to actin RNA, the abundance varies by one order of magnitude and is highest in pancreas, kidney and lowest in brain.

Studies on localization of FGE by indirect immunofluorescence and

immuno-kb

Figure 1.4: FGE and pFGE expression profile in various tissues. Northern blot analysis of poly A⁺RNA from human tissues for expression of pFGE (upper), FGE (middle) and β-actin (lower)[Dierks et al.,2003;Mariappan et al.,2005].

electron microscopy in human fibroblasts have shown the expression of endogenous FGE to be largely localized in the lumen of the endoplasmic reticulum (Fig. 1.5A) [Preusser-Kunze et al.,2005]. Even variously tagged forms of FGE colocalize with PDI, a luminal protein of the endoplasmic reticulum (Fig. 1.5B) [Dierks et al., 2003].

Immunofluorescence analysis with Golgi marker GM130 or the endosomal marker LAMP1 showed no co-localization, while by electron microscopy some FGE

1.2. FGE and its salient features 5

PDI FGE Merge

A

30µm 100µm

B

Figure 1.5: Subcellular localization of endogenous or C-terminally tagged FGE.Indirect immunofluorescence of FGE (red) and PDI (green). The merge reveals the co-localization of PDI and endogenous FGE (A) or C-terminally tagged FGE-HA (B). (A: Mariappan M., unpublished data), B: Dierks et al., 2003.

was detected in Golgi, endosomes and lysosomes. Upon overexpression FGE is secreted into the medium [Preusser-Kunze et al.,2005].

1.2.2 Structural properties of FGE

Derived from the gene SUMF1, human FGE is predicted to have 374 residues.

The protein contains a cleavable signal sequence of 33 residues, which indicates translocation of FGE into the endoplasmic reticulum, and contains a single N-glycosylation site at asparagine 141. FGE contains eight cysteines, six of them forming three disulfide bridges (50-52, 218-365, 235-346) (Fig. 1.6). The cysteines 50 and 52 of the first bridge however, are partially reduced (about 30%) or engaged in an intermolecular disulfide bond resulting in FGE homodimers. Cysteines 336 and 341, are found in reduced form but also disulfide bonded (see below).

Residues 87-367 of FGE are listed in the PFAM protein motif database as a domain of unknown function (PFAM: DUF323). Phylogenetic sequence con-servation analysis has revealed that FGE is composed of three highly conserved subdomains (I, II and III in Fig. 1.6). In human FGE the generally non-conserved N-terminal region harbors two highly conserved cysteines (50 and 52) (see above).

Subdomain I (residues 91-154) has a sequence identity of 39% and a similarity of 85% within the six known full-length eukaryotic FGE orthologs. This domain

6 Chapter1. Introduction

Figure 1.6: FGE domain architecture. FGE protein sequence shows three highly conserved subdomains (I, II and III) determined by phylogenetic sequence conservation analysis. Binding between residue 33 and 34 represents signal peptide (SP) cleavage site. FGE possess a single glycosylation site at residue Asn141 and eight cysteine residues represented as (C) show specific disulfide bridging.

carries the glycosylation site at Asn141, which is conserved in all orthologs. The N-glycosylation of FGE in the intracellular and secreted FGE is different. Intracellular FGE contains a high mannose-type while the extracellular FGE contains a complex-type oligosaccharide side chain [Preusser-Kunze et al., 2005]. The middle part of FGE (residues 179-308) determined by the tryptophan-rich subdomain II contains two cysteines which form two disulfide bridges to subdomain III. The identity of this subdomain in eukaryotic orthologs is 48%, the similarity 72%. Subdomain III (residues 327-366) is the most highly conserved sequence within the FGE family.

The sequence identity in eukaryotic orthologs is 85%, the similarity 100%. This subdomain carries four cysteine residues of which 336, 341 and 365 are fully conserved.

Limited proteolysis with thermolysin and elastase revealed that FGE is made up of two protease-resistant domains [Preusser-Kunze et al.,2005]. The first protease-resistant domain coincides with subdomain I and the cleavage site between the protease-resistant domains is located in the linker region separating subdomain I and II. Moreover, subdomains II and III are in fact part of one protease-resistant domain that is stabilized by the two intersubdomain disulfide bonds (see above).

However, from the crystal structure data [Dierks et al., 2005](see Section 1.2.4) FGE is found to be a compact molecule without obvious domain boundaries that has been suggested from sequence comparisons [Landgrebe et al.,2003] and proteolytic experiments [Preusser-Kunze et al.,2005].

1.2.3 Mutations in SUMF1 are the cause of MSD

Deficiency to generate FGly residues in sulfatases by FGE is the cause for MSD.

The FGE encoding SUMF1 gene is therefore a candidate gene for MSD. Mutations

1.2. FGE and its salient features 7 of SUMF1 gene were identified in all patients, that including missense, nonsense, microdeletion and splicing mutations. A total of 18 missense mutations over 17 different residues of mature FGE have been found to date, of which 12 have been published [Cosma et al.,2003,2004;Dierks et al.,2003]. These missense mutations found in MSD patients is explained by the FGE structure, providing a molecular basis of MSD [Dierks et al.,2005]. Most of the mutations appear to cluster in subdomain III of FGE, suggesting that this domain is crucial for FGE activity.

1.2.4 Substrate binding site of FGE

Photoaffinity labeling experiments with an ASA-derived peptide carrying a photore-active p-benzoyl-phenylalanine (Bpa) showed a crosslink between the substrate and proline 182, which is located close to the N-terminal end of subdomain II of FGE.

In accordance with that, a yeast two-hybrid assay showed that subdomain II is sufficient for binding a sulfatase fragment harboring the FGly modification motif [Preusser-Kunze et al., 2005]. However, resolving the crystal structure of FGE revealed a single domain monomer without further subdivisions in subdomains [Dierks et al., 2005]. According to the crystal structure FGE shows a surprising paucity of secondary structure and adopts a unique fold, called FGE fold [Dierks et al., 2005] (Fig. 1.7A). Main features of FGE structure include two stabilizing calcium ions, N-glycosylation at Asn 141, two cis-peptide bonds at Pro115 and Pro266, and three disulfide bonds. The N-terminal residues up to Gln85 including cysteine 50 and 52 are missing in the crystal structure. Two disulfide bonds, Cys218-Cys365 and Cys235-Cys346, are permanently present in FGE and contribute to its stability. Cysteines 336 and 341 have varying redox states depending on reducing or oxidizing conditions during crystallization and have therefore been considered to be candidates for catalytically active residues. Indeed, a surface representation of FGE shows an oval-shaped groove that is bordered by these cysteines at one end and Pro182 at the other end constituting a functionally bipartite binding/active site for the unfolded sulfatase substrates (Fig. 1.7B).

Later, by co-crystalization of FGE Cys336Ser and the ASA-derived peptide LCTPSRA containing the FGly motif it could be shown that this groove indeed constitutes the binding site for the CTPXR motif of sulfatases (Fig. 1.7C). Based on the structural and biochemical data a mechanism has been proposed for FGE, ascribing a key role to Cys336 and Cys341 during catalysis of FGly formation in sulfatases (Fig. 1.8).

8 Chapter1. Introduction

A B C

Figure 1.7: Crystal structure of FGE with and without bound substrate peptide. A) Diagram of FGE with the secondary structure elements and calcium ions as spheres. B) Surface representation of FGE showing the groove with the redox-active cysteine pair Cys336/Cys341 at one end. Pro 182 marks the site of a cross-link with a photoreactive substrate peptide and hence this site to be close to the substrate-binding site. C) FGE-peptide complex. The peptide LCTPSRA binds to Cys341 via an intermolecular disulfide bond (not seen in the figure [Dierks et al.,2005;Roeser et al.,2006].

H₂S

Figure 1.8: Possible catalytic mechanism of FGE. Color code: Black FGE; Green -Sulfatase; Oxygen(O) in red - from molecular oxygen and in blue - from water molecule.

1. Binding of substrate and formation of a mixed disulfide intermediate between the substrate and Cys341 of FGE.

2. Binding of molecular oxygen and formation of a hydroperoxide at Cys336.

3. Generation of a sulfenic acid at Cys336 and the oxidation of a reducing factor (XH2).

4. Transfer of the hydroxyl group from Cys336 to the substrate cysteine residue, regeneration of the Cys336-SS-Cys341 disulfide of FGE and release of the substrate as sulfenic acid.

5. Elimination of water (β-elimination) from the sulfenic acid forming a thioaldehyde intermediate and spontaneous hydrolysis to FGly [Dierks et al.,2005].