TY - JOUR
T1 - An inhibitory antibody blocks the first step in the dithiol/disulfide relay mechanism of the enzyme QSOX1
AU - Grossman, Iris
AU - Alon, Assaf
AU - Ilani, Tal
AU - Fass, Deborah
N1 - Funding Information:
The authors are grateful to Y. Yarden for advice and support. The authors acknowledge O. Leitner, Z. Landau, and H. Hamawi from the Weizmann Institute Antibody Unit for performing all technical aspects of antibody elicitation and production. E. Wong, Y. Udi, and A. Trahtenhercts provided advice on antibody sequencing. N. Sela assisted with antibody isotyping. This research was supported by the Israel Science Foundation , the Israel Cancer Association , and the European Research Council under the European Union's Seventh Framework Programme (ERC Grant agreement number 310649).
PY - 2013/11/15
Y1 - 2013/11/15
N2 - 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.
AB - 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.
KW - anti-metastatic drugs
KW - disulfide bonds
KW - extracellular matrix
KW - laminin
KW - monoclonal antibody
UR - http://www.scopus.com/inward/record.url?scp=84886729300&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2013.07.011
DO - 10.1016/j.jmb.2013.07.011
M3 - Article
C2 - 23867277
AN - SCOPUS:84886729300
SN - 0022-2836
VL - 425
SP - 4366
EP - 4378
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 22
ER -