Abstract
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(alpha)-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.
Highlights
Sulfatases represent a family of enzymes essential for the degradation and remodeling of sulfate esters
Arylsulfatase G, a Novel Lysosomal Sulfatase consists of 11 exons, and encodes a 525-amino acid protein that shares a high degree of similarity with all sulfatases, in particular with arylsulfatase A (50% sequence similarity and 37% identity)
The enzyme was tentatively classified as an arylsulfatase, no activity toward the commonly used arylsulfate pseudosubstrates p-nitrocatechol sulfate and 4-methylumbelliferyl sulfate (4-MUS) could be detected
Summary
The ASG A501P cDNA was amplified by PCR, thereby adding a 3Ј-RGS-His6-encoding sequence followed by a stop codon and a HindIII site (reverse primer 5Ј-CCCAAGCTTAGTGATGGTGATGGTGATGCGATCCTCTTGCGGCTTGACAGCGGC-3Ј). The QuikChange mutagenesis protocol (Stratagene) was used to generate the wild-type ASG-encoding sequence with a GCA codon in position 501 (accession number NM_014960.2) For this purpose, complementary primers were used (forward 5Ј-CGACAACATCTCCAGCGCAGATTACACTCAGG-3Ј, reverse 5Ј-CCTGAGTGTAATCTGCGCTGGAGATGTTGTCG-3Ј). As analyzed by Western blotting and pNCS assays, were pooled, dialyzed against buffer A (20 mM MES, pH 6.0), and applied onto a RESOURCE S 1-ml cation exchange column (GE Healthcare). For endoglucosaminidase H treatment, samples were denatured at pH 5.0 with 0.01% SDS and 0.7% -mercaptoethanol for 5 min at 95 °C and subjected to deglycosylation In both cases, samples were incubated at 37 °C for 2 or 24 h and analyzed by Western blotting. Normalization was confirmed by primers specific for glycerol aldehyde-3-phosphate dehydrogenase
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