title: Interaction of arylsulfatase-A (ASA) with its natural sulfoglycolipid substrates: a computational and site-directed mutagenesis study.
abstract: Arylsulfatase A (ASA) hydrolyzes sulfate esters with a pH optimum of 5. Interactions between p-nitrocatechol sulfate (NCS, artificial substrate) and active site residues of ASA are revealed from their co-crystal structure. Since equivalent ASA interactions with its natural substrates, sulfogalactosylceramide (SGC) and sulfogalactosylglycerolipid (SGG), are not known, we computationally docked SGC/SGG to the ASA crystal structure. Our dockings suggested that Cys69 was the active site residue, and Lys302 & Lys123 as residues anchoring the sulfate group of SGC/SGG to the active site, as observed for NCS. We further confirmed these results using 2 recombinant ASA mutants: C69A and CKK (Cys69, Lys302 and Lys123-all mutated to Ala). Both ASA mutants failed to desulfate SGC/SGG, and CKK showed minimal binding to [(14)C]SGC, although C69A still had affinity for this sulfoglycolipid. However, our dockings suggested additional intermolecular hydrogen bonding and hydrophobic interactions between ASA and SGC/SGG, thus contributing to the specificity of SGC/SGG as natural substrates.
authors.affiliation: Chronic Disease, Ottawa Hospital Research Institute, Ottawa, ON, Canada.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schenk
authors.forename: Chaitanya A K
authors.initials: CA
authors.lastname: Koppisetty
authors.forename: Daniela Costa
authors.initials: DC
authors.lastname: Santos
authors.forename: Euridice
authors.initials: E
authors.lastname: Carmona
authors.forename: Smita
authors.initials: S
authors.lastname: Bhatia
authors.forename: Per-Georg
authors.initials: PG
authors.lastname: Nyholm
authors.forename: Nongnuj
authors.initials: N
authors.lastname: Tanphaichitr
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D020134:Catalytic Domain; D002553:Cerebroside-Sulfatase / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D019295:Computational Biology / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D006017:Glycolipids / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D055672:Static Electricity; D013379:Substrate Specificity; D013431:Sulfates / Q000737:chemistry; D034421:Sus scrofa
chemical_list: D000596:Amino Acids; D006017:Glycolipids; D011994:Recombinant Proteins; D013431:Sulfates; C011117:sulfoglycolipids; D002553:Cerebroside-Sulfatase