title: An inhibitory antibody blocks the first step in the dithiol/disulfide relay mechanism of the enzyme QSOX1.
abstract: Quiescin sulfhydryl oxidase 1 (QSOX1) is a catalyst of disulfide bond formation that undergoes regulated secretion from fibroblasts and is over-produced in adenocarcinomas and other cancers. We have recently shown that QSOX1 is required for incorporation of particular laminin isoforms into the extracellular matrix (ECM) of cultured fibroblasts and, as a consequence, for tumor cell adhesion to and penetration of the ECM. The known role of laminins in integrin-mediated cell survival and motility suggests that controlling QSOX1 activity may provide a novel means of combating metastatic disease. With this motivation, we developed a monoclonal antibody that inhibits the activity of human QSOX1. Here, we present the biochemical and structural characterization of this antibody and demonstrate that it is a tight-binding inhibitor that blocks one of the redox-active sites in the enzyme, but not the site at which de novo disulfides are generated catalytically. Sulfhydryl oxidase activity is thus prevented without direct binding of the sulfhydryl oxidase domain, confirming the model for the interdomain QSOX1 electron transfer mechanism originally surmised based on mutagenesis and protein dissection. In addition, we developed a single-chain variant of the antibody and show that it is a potent QSOX1 inhibitor. The QSOX1 inhibitory antibody will be a valuable tool in studying the role of ECM composition and architecture in cell migration, and the recombinant version may be further developed for potential therapeutic applications based on manipulation of the tumor microenvironment.
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Iris
authors.initials: I
authors.lastname: Grossman
authors.forename: Assaf
authors.initials: A
authors.lastname: Alon
authors.forename: Tal
authors.initials: T
authors.lastname: Ilani
authors.forename: Deborah
authors.initials: D
authors.lastname: Fass
mesh_terms: D000595:Amino Acid Sequence; D019138:Antibodies, Blocking / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism; D014050:Toluene / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D004220:Disulfides; D046912:Multiprotein Complexes; D057127:Single-Chain Antibodies; D014050:Toluene; D050862:Oxidoreductases Acting on Sulfur Group Donors; C117183:QSOX1 protein, human; C004848:dithiol
keywords: 5,5′-dithiobis-(2-nitrobenzoic acid); BM; BSA; CDR; DTNB; ECM; EDTA; ER; Erv; Erv-fold; FAD; HRP; HsQSOX1; LC–MS/MS; LOX; PBS; PBS with 0.1% Tween; PBS-T; PDB; PEG; Protein Data Bank; QSOX1; RT; Trx; anti-metastatic drugs; basement membrane; bovine serum albumin; complementarity-determining region; disulfide bonds; endoplasmic reticulum; ethylenediaminetetraacetic acid; extracellular matrix; flavin adenine dinucleotide; horseradish peroxidase; laminin; liquid chromatography–tandem mass spectrometry; lysyl oxidase; monoclonal antibody; phosphate-buffered saline; polyethylene glycol; quiescin sulfhydryl oxidase 1; rdRNase; recombinant human QSOX1; reduced and denatured RNase A; room temperature; scFv; single-chain variable fragment; thioredoxin-fold