Defective Oligomerization of Arylsulfatase A as a Cause of Its Instability in Lysosomes and Metachromatic Leukodystrophy

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The sulfatases constitute a conserved family of enzymes that specifically hydrolyze sulfate esters in a wide variety of substrates such as glycosaminoglycans, steroid sulfates, or sulfolipids. By modifying the sulfation state of their substrates, sulfatases play a key role in the control of physiological processes, including cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked with various severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase G (ASG), was initially described as an enzyme lacking in vitro arylsulfatase activity and localizing to the endoplasmic reticulum. Contrary to these results, we demonstrate here that ASG does indeed have arylsulfatase activity toward different pseudosubstrates like p-nitrocatechol sulfate and 4-methylumbelliferyl sulfate. The activity of ASG depends on the Cys-84 residue that is predicted to be post-translationally converted to the critical active site C ␣ -formylglycine. Phosphate acts as a strong, competitive ASG inhibitor. ASG is active as an unprocessed 63-kDa monomer and shows an acidic pH optimum as typically seen for lysosomal sulfatases. In transfected cells, ASG accumulates within lysosomes as indicated by indirect immunofluorescence microscopy. Furthermore, ASG is a glycoprotein that binds specifically to mannose 6-phosphate receptors, corroborating its lysosomal localization. ARSG mRNA expression was found to be tissue-specific with highest expression in liver, kidney, and pancreas, suggesting a metabolic role of ASG that might be associated with a so far non-classified lysosomal storage disorder.

Section: Asg Is Active In the Monomericmentioning

confidence: 72%

“…Arylsulfatase A, the sulfatase with highest homology to ASG, is a lysosomal sulfatase that acts as an octamer at acidic pH (30). Deficiency of arylsulfatase A causes metachromatic leukodystrophy, a lysosomal storage disorder associated with progressive demyelination of the central and peripheral nervous system.…”

Section: Discussionmentioning

confidence: 99%

Arylsulfatase G, a Novel Lysosomal Sulfatase

Frese

Schulz²,

Dierks

2008

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“…Structural and enzymatic studies of mutants of arylsulfatase A (ASA), in which the FGly was replaced by a serine or alanine, and, namely, the recent solution of the x-ray structure of a bacterial sulfatase at 1.3 Å has shown that the FGly side chain is present as an aldehyde hydrate in the resting state of the enzyme (4, 10 -14). During catalysis one of the two hydroxyls of this geminal diol performs a nucleophilic attack on the sulfur of the substrate's sulfate group leading to a covalently sulfated enzyme intermediate (4,11,12). The second hydroxyl is required for desulfation of the intermediate and for concomitant aldehyde regeneration.…”

mentioning

confidence: 99%

Characterization of Posttranslational Formylglycine Formation by Luminal Components of the Endoplasmic Reticulum

Fey¹,

Balleininger²,

Borissenko³

et al. 2001

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C ␣ -formylglycine is the key catalytic residue in the active site of sulfatases. In eukaryotes formylglycine is generated during or immediately after sulfatase translocation into the endoplasmic reticulum by oxidation of a specific cysteine residue. We established an in vitro assay that allowed us to measure formylglycine modification independent of protein translocation. The modifying enzyme was recovered in a microsomal detergent extract. As a substrate we used ribosome-associated nascent chain complexes comprising in vitro synthesized sulfatase fragments that were released from the ribosomes by puromycin. Formylglycine modification was highly efficient and did not require a signal sequence in the substrate polypeptide. Ribosome association helped to maintain the modification competence of nascent chains but only after their release efficient modification occurred. The modifying machinery consists of soluble components of the endoplasmic reticulum lumen, as shown by differential extraction of microsomes. The in vitro assay can be performed under kinetically controlled conditions. The activation energy for formylglycine formation is 61 kJ/mol, and the pH optimum is Ϸ10. The activity is sensitive to the SH/SS equilibrium and is stimulated by Ca 2؉ . Formylglycine formation is efficiently inhibited by a synthetic sulfatase peptide representing the sequence directing formylglycine modification. The established assay system should make possible the biochemical identification of the modifying enzyme.

“…It has also been suggested that the nucleophilicity of the O-␥1 atom is enhanced by a possible proton donation to a neighboring aspartic acid residue. In our structural model of the 2-O-sulfatase, this residue would correspond to An S N 2 mechanism has been proposed to follow the above steps and eventually lead to the cleavage of the sulfate ester bond (10,12). In this mechanism, the exocyclic oxygen atom on the leaving substrate may be protonated by water or potentially by neighboring amino acids.…”

Section: Structure-based Homology Modeling Of the 2-o-sulfatasementioning

confidence: 98%

“…The crystal structures of two human lysosomal sulfatases, arylsulfatase A (cerebroside-3-sulfate 3-sulfohydrolase) (9,10) and arylsulfatase B (N-acetylgalactosamine-4-sulfate 4-sulfohydrolase) (11), and a bacterial arylsulfatase from Pseudomonas aeruginosa (12) have been solved. These three sulfatases share an identical alkaline phosphatase-like structural fold (according to the Structural Classification of Proteins Database) 2 composed of a series of mixed parallel and antiparallel ␤-strands flanked by long and short ␣-helices on either side (9 -12).…”

mentioning

confidence: 99%

The Heparin/Heparan Sulfate 2-O-Sulfatase from Flavobacterium heparinum

Raman

Myette

Shriver

et al. 2003

we described the molecular cloning, recombinant expression, and preliminary biochemical characterization of the heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. In this paper, we extend our structure-function investigation of the 2-O-sulfatase. First, we have constructed a homology-based structural model of the enzyme active site, using as a framework the available crystallographic data for three highly related arylsulfatases. In this model, we have identified important structural parameters within the enzyme active site relevant to enzyme function, especially as they relate to its substrate specificity. By docking various disaccharide substrates, we identified potential structural determinants present within these substrates that would complement this unique active site architecture. These determinants included the position and number of sulfates present on the glucosamine, oligosaccharide chain length, the presence of a ⌬4,5-unsaturated double bond, and the exolytic versus endolytic potential of the enzyme. The predictions made from our model provided a structural basis of substrate specificity originally interpreted from the biochemical and kinetic data. Our modeling approach was further complemented experimentally using peptide mapping in tandem with mass spectrometry and site-directed mutagenesis to physically demonstrate the presence of a covalently modified cysteine (formylglycine) within the active site. This combinatorial approach of structure modeling and biochemical studies provides insight into the molecular basis of enzyme function.Heparin and heparin sulfate glycosaminoglycans (HSGAGs) 1 are structurally complex linear polysaccharides (1, 2) composed of repeating disaccharides of uronic acid (␣-L-iduronic or ␤-Dglucuronic) linked 134 to ␣-D-glucosamine. This structural complexity derives principally from the variable chemical modifications made to the polysaccharide chain. Such modifications include acetylation or sulfation at the N-position of the glucosamine, epimerization of glucuronic acid to iduronic acid, and additional O-sulfation at the 2-O-position of the uronic acid in addition to the 3-O, 6-O-position of the adjoining glucosamine. It is a highly variable sulfation pattern, in particular, which ascribes to each GAG chain a unique structural signature. In turn, this signature dictates specific GAG-protein interactions underlying critical biological processes related to cell and tissue function. Given this critical structure-function relationship of GAG sulfation, enzymes that can hydrolyze these sulfates in a structurally specific manner are important tools for the determination of GAG fine structure to better ascertain these structure-function relationships. In the previous paper (21), we described the cloning, recombinant expression, and biochemical characterization of one such sulfatase, the 2-O-sulfatase from Flavobacterium heparinum.As members of a large enzyme family, the sulfatases hydrolyze a wide array of sulfate esters (for a review, see Refs. 3 and 4). Their ...