Residues Critical for Formylglycine Formation and/or Catalytic Activity of Arylsulfatase A

Abstract

Sulfatases contain a unique posttranslational modification in their active site, a formylglycine residue generated from a cysteine or a serine residue. The formylglycine residue is part of a sequence that is highly conserved among sulfatases, suggesting that it might direct the generation of this unique amino acid derivative. In the present study residues 68-86 flanking formylglycine 69 in arylsulfatase A were subjected to an alanine/glycine scanning mutagenesis. The mutants were analyzed for the conversion of cysteine 69 to formylglycine and their kinetic properties. Only cysteine 69 turned out to be essential for formation of the formylglycine residue, while substitution of leucine 68, proline 71, and alanine 74 within the heptapeptide LCTPSRA reduced the formylglycine formation to about 30-50%. Several residues that are part of or directly adjacent to an alpha-helix presenting the formylglycine 69 at the bottom of the active site pocket were found to be critical for catalysis. A surprising outcome of this study was that a number of residues fully or highly conserved between all known eukaryotic and prokaryotic sulfatases turned out to be essential neither for generation of formylglycine nor for catalysis.

Sulf1A and HGF regulate satellite-cell growth

“…HSPG sulfation is also extracellularly regulated by Sulf1, a member of the sulfatase gene family, related to the lysosomal Nacetyl glucosamine sulfatases (Lukatela et al, 1998;Knaust et al, 1998;Robertson et al, 1992) that has been shown to modulate the activities of many growth factors and signalling molecules by regulating the sulfation status of specific HSPGs as it specifically reduces the 6-O-sulfation of HS, a component of the HSPGs. For example, FGF activity has been shown to be inhibited by Sulf1 during chick angiogenesis and in ovarian cells by removing 6-O sulfates from glucosamine residues in HS of HSPGs, required as secondary receptors for FGF2 and FGF4 function (MorimotoTomita et al, 2002;Ai et al, 2003;Lai et al, 2004;Wang et al, 2004).…”

Human sulfatase transiently and functionally active expressed in E. coli K12

“…In Linfoblastoid and COS cells it has been found that the mature forms presents a molecular weight between 45-55 kDa. As other members of the sulfatases family, the hIDS undergo a common posttranslational modification in which a Cys or Ser residue at the active enzyme core is converted into FGly (Schmidt et al 1995;Dierks et al 1998a;Dierks et al 1998b;Knaust et al 1998;Dierks et al 1999;Waldow et al 1999). The Cys at position 84 in hIDS is co-translationally modified to formylglycine (FGly) within the endoplasmic reticulum (ER) to produce an active enzyme (von ).…”

Amino Acid Residues Forming the Active Site of Arylsulfatase A

“…3 , and Asp 281 were replaced by codons for asparagine, and the codon for Asn 282 was replaced by that of aspartate. Mutagenesis was performed using the pAlter cloning kit (Promega Corp.), the cDNA for human ASA (11) cloned into the pAlter mutagenesis vector (12), and appropriate primers. Following mutagenesis mutant cDNAs were cloned as EcoRI fragments into the pMPSVEH vector downstream of the myeloproliferative sarcoma virus promoter.…”

“…After selection and expansion of the clones stably expressing the mutants, sulfatases were purified from cell secretions by affinity chromatography and dialyzed against 10 mM Tris/HCl, 150 mM NaCl, pH 7.5. V max , K m , and the pH optimum of sulfatase activity of the purified sulfatases were determined using 2, 5, 10, and 20 mM pnitrocatechol sulfate as substrate as described earlier (12). All kinetic data were determined under initial rate conditions.…”

“…All kinetic data were determined under initial rate conditions. Purity and amount of ASA proteins were calculated from the integral of the ASA peak at 280 nm after reversed phase HPLC as described (12). To determine the percentage of Cys 69 converted to FGly 69 , purified mutants were digested with trypsin.…”