Arylsulfatase K,a Novel Lysosomal Sulfatase

The human sulfatase family has 17 members, 13 of which have been characterized biochemically. These enzymes specifically hydrolyze sulfate esters in glycosaminoglycans, sulfolipids, or steroid sulfates, thereby playing key roles in cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked to severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase K (ARSK), was identified bioinformatically through its conserved sulfatase signature sequence directing posttranslational generation of the catalytic formylglycine residue in sulfatases. However, overall sequence identity of ARSK with other human sulfatases is low (18–22%). Here we demonstrate that ARSK indeed shows desulfation activity toward arylsulfate pseudosubstrates. When expressed in human cells, ARSK was detected as a 68-kDa glycoprotein carrying at least four N-glycans of both the complex and high-mannose type. Purified ARSK turned over p-nitrocatechol and p-nitrophenyl sulfate. This activity was dependent on cysteine 80, which was verified to undergo conversion to formylglycine. Kinetic parameters were similar to those of several lysosomal sulfatases involved in degradation of sulfated glycosaminoglycans. An acidic pH optimum (∼4.6) and colocalization with LAMP1 verified lysosomal functioning of ARSK. Further, it carries mannose 6-phosphate, indicating lysosomal sorting via mannose 6-phosphate receptors. ARSK mRNA expression was found in all tissues tested, suggesting a ubiquitous physiological substrate and a so far non-classified lysosomal storage disorder in the case of ARSK deficiency, as shown before for all other lysosomal sulfatases.     

The Structure of Human GALNS Reveals the Molecular Basis for Mucopolysaccharidosis IV A

Lysosomal enzymes catalyze the breakdown of macromolecules in the cell. In humans, loss of activity of a lysosomal enzyme leads to an inherited metabolic defect known as a lysosomal storage disorder. The human lysosomal enzyme galactosamine-6-sulfatase (GALNS, also known as N-acetylgalactosamine-6-sulfatase and GalN6S; E.C. 3.1.6.4) is deficient in patients with the lysosomal storage disease mucopolysaccharidosis IV A (also known as MPS IV A and Morquio A). Here, we report the three-dimensional structure of human GALNS, determined by X-ray crystallography at 2.2 Å resolution. The structure reveals a catalytic gem diol nucleophile derived from modification of a cysteine side chain. The active site of GALNS is a large, positively charged trench suitable for binding polyanionic substrates such as keratan sulfate and chondroitin-6-sulfate. Enzymatic assays on the insect‐cell-expressed human GALNS indicate activity against synthetic substrates and inhibition by both substrate and product. Mapping 120 MPS IV A missense mutations onto the structure reveals that a majority of mutations affect the hydrophobic core of the structure, indicating that most MPS IV A cases result from misfolding of GALNS. Comparison of the structure of GALNS to paralogous sulfatases shows a wide variety of active‐site geometries in the family but strict conservation of the catalytic machinery. Overall, the structure and the known mutations establish the molecular basis for MPS IV A and for the larger MPS family of diseases.     

Recognition of arylsulfatase A and B by the UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-phosphotransferase

The critical step for sorting of lysosomal enzymes is the recognition by a Golgi-located phosphotransferase. The topogenic structure common to all lysosomal enzymes essential for this recognition is still not well defined, except that lysine residues seem to play a critical role. Here we have substituted surface-located lysine residues of lysosomal arylsulfatases A and B. In lysosomal arylsulfatase A only substitution of lysine residue 457 caused a reduction of phosphorylation to 33% and increased secretion of the mutant enzyme. In contrast to critical lysines in various other lysosomal enzymes, lysine 457 is not located in an unstructured loop region but in a helix. It is not strictly conserved among six homologous lysosomal sulfatases. Based on three-dimensional structure comparison, lysines 497 and 507 in arylsulfatase B are in a similar position as lysine 457 of arylsulfatase A. Also, the position of oligosaccharide side chains phosphorylated in arylsulfatase A is similar in arylsulfatase B. Despite the high degree of structural homology between these two sulfatases substitution of lysines 497 and 507 in arylsulfatase B has no effect on the sorting and phosphorylation of this sulfatase. Thus, highly homologous lysosomal arylsulfatases A and B did not develop a single conserved phosphotransferase recognition signal, demonstrating the high variability of this signal even in evolutionary closely related enzymes.     

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

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

Detection of arylsulfatase A activity after electrophoresis in polyacrylamide gels: problems and solutions.

When arylsulfatase extracted from normal human skin fibroblasts was electrophoresed in polyacrylamide gels with a Tris-glycine buffer at pH 8.4–8.6, two problems occurred. First, no arylsulfatase A activity was detected. Second, an artifactual fluorescent spot was generated when the gels were stained for arylsulfatase activity with 4-methylumbelliferyl sulfate as substrate. The artifact simulated arylsulfatase A activity in mobility but also appeared when 4-methylumbelliferyl substrates for other enzymes were used. It can be eliminated by prerunning or prolonged storage of the gets before use. The arylsulfatase A activity, however, could be recovered only when a low pH buffer system (pH 58–68) was used for electrophoresis. The highest percentage recovery (70%) of activity was obtained in acrylamide gels polymerized with ammonium persulfate, prerun for 0.5 h before use and electrophoresed with an anode buffer of acetic acid-triethanolamine at pH 5.8.     

Cloning and expression of human steroid-sulfatase. Membrane topology, glycosylation, and subcellular distribution in BHK-21 cells

A 2.4-kilobase cDNA clone for human steroid-sulfatase (STS) was isolated and sequenced, which encoded an enzymatically active protein. The deduced amino acid sequence comprises 583 amino acids with an N-terminal signal peptide of 21 or 23 residues and four potential N-glycosylation sites. Two of the N-glycosylation sites are utilized and were localized to the asparagine residues 47 and 259. STS has the solubility properties of an integral membrane protein. The resistance of STS toward proteinase K after translocation into microsomes suggests that most, if not all, sequences of STS are exposed at the luminal side of microsomes. The deduced amino acid sequence predicts two membrane-spanning domains (amino acids 185-211 and 213-237) separated by a helix-breaking proline residue. We propose for STS a three-domain model. Two glycosylated luminally oriented domains of 161 and 346 residues are separated by a hydrophobic domain spanning the membrane twice in opposite directions. STS expressed in BHK-21 cells is located predominantly in the endoplasmic reticulum; smaller fractions are found in the Golgi, at the cell surface, multivesicular endosomes, as well as in lysosomes. The stability of STS in lysosomes may be related to the high homology of the two luminal domains of STS with the lysosomal sulfatases, arylsulfatase A, and arylsulfatase B. In spite of its similarity with these two lysosomal sulfatases, STS does not contain mannose 6-phosphate residues and is transported to lysosomes by a mannose 6-phosphate receptor-independent mechanism.     

Histochemistry of estrogen sulfatases in human breast diseases

Two estrogen sulfatases, arylsulfatase C-estrone sulfatase (ASC-ES) and d-equilenin sulfatase (EqS) were demonstrated histochemically in the normal human female breast, in benign breast diseases and in infiltrating mammary ductal carcinomas to study their significance in the pathogenesis of epithelial proliferations. By hydrolyzing estrone sulfate, the amount of which in female blood is about ten times greater than that of estradiol or estrone, estrogen sulfatases can produce a high local concentration of estrogens. A simultaneous azo-coupling method for histochemical demonstration of ASC-ES is described in the present study; EqS was demonstrated by a previously described method. Estrogen sulfatases were not found in the normal female breast. Both estrogen sulfatases were found in epithelial cells in some examples of mastopathic disease and in fibroadenomas, while ASC-ES was found in periductal fibroblasts. In some cases of infiltrating ductal carcinomas, estrogen sulfatases were present in carcinoma cells. In most of these tumors ASC-ES activity was observed in fibroblasts around infiltrative cell cords. There was no correlation between the presence of estrogen sulfatases and of hormone receptors in carcinomas. It is concluded that estrogen sulfatases play no role in the early stages of benign or malignant epithelial proliferations. However, the induction of estrogen sulfatases may promote epithelial proliferation in some cases if estrogen receptors are present in epithelial cells.     

A specific ultrastructural stain for arylsulfatase A activity in human cultured fibroblasts

A staining reaction was developed to specifically detect arylsulfatase A activity in the presence of arylsulfatases B and C. Nitrocatechol, generated by all arylsulfatases from the substrate p-nitrocatechol sulfate, can be coupled to produce Hatchett 's brown which reacts with 3,3'-diaminobenzidine to yield an osmiophilic polymer visible under the electron microscope. The reaction was made specific for arylsulfatase A by inhibiting arylsulfatase C activity with low pH and arylsulfatase B activity with pyrophosphate. The specificity was confirmed both by electrophoretic analysis and by patient fibroblasts deficient only in arylsulfatase A activity. Under optimal conditions for preserving structural integrity and enzyme activity, enzyme reaction deposits were found mainly around vesicles. Some of these vesicles were large and heterogeneous (48-330 nm in diameter), distributed randomly within the cytoplasm, but most of the positive-reacting vesicles were uniform in size (86 +/- 18 nm in diameter) and distributed in a peripheral zone about 0.1-0.5 micron wide. These periplasmic vesicles might be partly fused with each other or with the plasma membrane. In conclusion, a specific stain for arylsulfatase A activity suitable for light and electron microscopy and the optimal conditions for structural and enzymatic preservations were developed. Although this enzyme has been considered to be lysosomal in origin, most of the activity was detected in periplasmic vesicles near the cell surface.     

Chondroitin sulfate/dermatan sulfate sulfatases from mammals and bacteria

Sulfatases that specifically catalyze the hydrolysis of the sulfate groups on chondroitin sulfate (CS)/dermatan sulfate (DS) poly- and oligosaccharides belong to the formylglycine-dependent family of sulfatases and have been widely found in various mammalian and bacterial organisms. However, only a few types of CS/DS sulfatase have been identified so far. Recently, several novel CS/DS sulfatases have been cloned and characterized. Advanced studies have provided significant insight into the biological function and mechanism of action of CS/DS sulfatases. Moreover, further studies will provide powerful tools for structural and functional studies of CS/DS as well as related applications. This article reviews the recent progress in CS/DS sulfatase research and is expected to initiate further research in this field.     

Examining the promiscuous phosphatase activity of Pseudomonas aeruginosa arylsulfatase: a comparison to analogous phosphatases

Pseudomonas aeruginosa arylsulfatase (PAS) is a bacterial sulfatase capable of hydrolyzing a range of sulfate esters. Recently, it has been demonstrated to also show very high proficiency for phosphate ester hydrolysis. Such proficient catalytic promiscuity is significant, as promiscuity has been suggested to play an important role in enzyme evolution. Additionally, a comparative study of the hydrolyses of the p-nitrophenyl phosphate and sulfate monoesters in aqueous solution has demonstrated that despite superficial similarities, the two reactions proceed through markedly different transition states with very different solvation effects, indicating that the requirements for the efficient catalysis of the two reactions by an enzyme will also be very different (and yet they are both catalyzed by the same active site). This work explores the promiscuous phosphomonoesterase activity of PAS. Specifically, we have investigated the identity of the most likely base for the initial activation of the unusual formylglycine hydrate nucleophile (which is common to many sulfatases), and demonstrate that a concerted substrate-as-base mechanism is fully consistent with the experimentally observed data. This is very similar to other related systems, and suggests that, as far as the phosphomonoesterase activity of PAS is concerned, the sulfatase behaves like a "classical" phosphatase, despite the fact that such a mechanism is unlikely to be available to the native substrate (based on pK(a) considerations and studies of model systems). Understanding such catalytic versatility can be used to design novel artificial enzymes that are far more proficient than the current generation of designer enzymes.     

Complementation of multiple sulfatase deficiency in somatic cell hybrids

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

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

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

SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies

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

Mycobacterium tuberculosis Rv3406 Is a Type II Alkyl Sulfatase Capable of Sulfate Scavenging

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb’s single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase’s role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases.     

Synthesis and evaluation of general mechanism-based inhibitors of sulfatases based on (difluoro)methyl phenyl sulfate and cyclic phenyl sulfamate motifs

Several model mechanism-based inhibitors (MbIs) were designed and evaluated for their ability to inhibit sulfatases. The MbI motifs were based on simple aromatic sulfates, which are known to be commonly accepted substrates across this highly conserved enzyme class, so that they might be generally useful for sulfatase labeling studies. (Difluoro)methyl phenol sulfate analogs, constructed to release a reactive quinone methide trap, were not capable of irreversibly inactivating the sulfatase active site. On the other hand, the cyclic sulfamates (CySAs) demonstrated inhibition profiles consistent with an active site-directed mode of action. These molecules represent a novel scaffold for labeling sulfatases and for probing their catalytic mechanism.     

Biochemical characterization of arylsulfatase E and functional analysis of mutations found in patients with X-linked chondrodysplasia punctata.

X-linked chondrodysplasia punctata (CDPX) is a congenital disorder characterized by abnormalities in cartilage and bone development. Mutations leading to amino acid substitutions were identified recently in CDPX patients, in the coding region of the arylsulfatase E (ARSE) gene, a novel member of the sulfatase gene family. Transfection of the ARSE full-length cDNA, in Cos7 cells, allowed us to establish that its protein product is a 60-kD precursor, which is subject to N-glycosylation, to give a mature 68-kD form that, unique among sulfatases, is localized to the Golgi apparatus. Five missense mutations found in CDPX patients were introduced into wild-type ARSE cDNA by site-directed mutagenesis. These mutants were transfected into Cos7 cells, and the arylsulfatase activity and biochemical properties were determined, to study the effect of these substitutions on the ARSE protein. One of the mutants behaves as the wild-type protein. All four of the other mutations resulted in a complete lack of arylsulfatase activity, although the substitutions do not appear to affect the stability and subcellular localization of the protein. The loss of activity due to these mutations confirms their involvement in the clinical phenotype and points to the importance of these residues in the correct folding of a catalytically active ARSE enzyme.     

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

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

Targeting of phosphomannosyl-deficient arylsulfatase A to lysosomes of I-cell fibroblasts

Fibroblasts from I-cell disease, a genetically-determined lysosomal storage disease, are shown to contain large amounts of phase-dense lysosomes. These lysosomes accumulated acridine orange and were specifically labeled with antibodies to arylsulfatase A. In normal skin fibroblasts the number of arylsulfatase-containing lysosomes was considerably lower. By immunocytochemistry, metabolic labeling and enzyme assay, the arylsulfatase A in I-cell fibroblasts was shown to be synthesized, stored and secreted at a level that was several-fold higher than that present in heterozygous I-cell or normal fibroblasts. Arylsulfatase A in I-cell fibroblasts differed from arylsulfatase in normal fibroblasts by the absence of endoglycosidase H-sensitive phosphorylated oligosaccharides. These findings indicate that arylsulfatase A in I-cells is targeted to lysosomes by a mechanism that does not appear to involve the phosphorylated mannose marker.     

Crystal structure of human pFGE, the paralog of the Calpha-formylglycine-generating enzyme

In eukaryotes, sulfate esters are degraded by sulfatases, which possess a unique Calpha-formylglycine residue in their active site. The defect in post-translational formation of the Calpha-formylglycine residue causes a severe lysosomal storage disorder in humans. Recently, FGE (formylglycine-generating enzyme) has been identified as the protein required for this specific modification. Using sequence comparisons, a protein homologous to FGE was found and denoted pFGE (paralog of FGE). pFGE binds a sulfatase-derived peptide bearing the FGE recognition motif, but it lacks formylglycine-generating activity. Both proteins belong to a large family of pro- and eukaryotic proteins containing the DUF323 domain, a formylglycine-generating enzyme domain of unknown three-dimensional structure. We have crystallized the glycosylated human pFGE and determined its crystal structure at a resolution of 1.86 A. The structure reveals a novel fold, which we denote the FGE fold and which therefore serves as a paradigm for the DUF323 domain. It is characterized by an asymmetric partitioning of secondary structure elements and is stabilized by two calcium cations. A deep cleft on the surface of pFGE most likely represents the sulfatase polypeptide binding site. The asymmetric unit of the pFGE crystal contains a homodimer. The putative peptide binding site is buried between the monomers, indicating a biological significance of the dimer. The structure suggests the capability of pFGE to form a heterodimer with FGE.     

HpSumf1 is involved in the activation of sulfatases responsible for regulation of skeletogenesis during sea urchin development

Sulfatases such as arylsulfatase and heparan sulfate 6-O-endosulfatase play important roles in morphogenesis during sea urchin development. For the activation of these sulfatases, Cα-formylglycine formation by sulfatase modifying factor (Sumf) is required. In this study, to clarify the regulatory mechanisms for the activation of sulfatases during sea urchin development, we examined the expression and function of the Hemicentrotus pulcherrimus homologs of Sumf1 and Sumf2 (HpSumf1 and HpSumf2, respectively). Expression of HpSumf1 but not HpSumf2 mRNA was dynamically changed during early development. Functional analyses of recombinant HpSumf1 and HpSumf2 using HEK293T cells expressing mouse arylsulfatase A (ArsA) indicated that HpSumf1 and HpSumf2 were both able to activate mammalian ArsA. Knockdown of HpSumf1 using morpholino antisense oligonucleotides caused abnormal spicule formation in the sea urchin embryo. Injection of HpSumf2 mRNA had no effect on skeletogenesis, while injection of HpSumf1 mRNA induced severe supernumerary spicule formation. Taken together, these findings suggest that HpSumf1 is involved in the activation of sulfatases required for control of skeletogenesis.