title: Molecular mechanism for the action of the anti-CD44 monoclonal antibody MEM-85.
abstract: The hyaluronate receptor CD44 plays role in cell adhesion and migration and is involved in tumor metastasis. The extracellular domain of CD44 comprises the hyaluronate-binding domain (HABD) and the membrane-proximal stem region; the short intracellular portion interacts with adaptor proteins and triggers signaling pathways. Binding of hyaluronate to CD44 HABD induces an allosteric conformational change, which results in CD44 shedding. A poorly characterized epitope in human CD44 HABD is recognized by the murine monoclonal antibody MEM-85, which cross-blocks hyaluronate binding to CD44 and also induces CD44 shedding. MEM-85 is of therapeutic interest, as it inhibits growth of lung cancer cells in murine models. In this work, we employed a combination of biophysical methods to determine the MEM-85 binding epitope in CD44 HABD and to provide detailed insight into the mechanism of MEM-85 action. In particular, we constructed a single-chain variable fragment (scFv) of MEM-85 as a tool for detailed characterization of the CD44 HABD-antibody complex and identified residues within CD44 HABD involved in the interaction with scFv MEM-85 by NMR spectroscopy and mutational analysis. In addition, we built a rigid body model of the CD44 HABD-scFv MEM-85 complex using a low-resolution structure obtained by small-angle X-ray scattering. The MEM-85 epitope is situated in the C-terminal part of CD44 HABD, rather than the hyaluronate-binding groove, and the binding of MEM-85 induces a structural reorganization similar to that induced by hyaluronate. Therefore, the mechanism of MEM-85 cross-blocking of hyaluronate binding is likely of an allosteric, relay-like nature.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, AS CR, v.v.i., Flemingovo nam. 2, Prague 6, 166 10, Czech Republic; Institute of Molecular Genetics, AS CR, v.v.i., Videnska 1083, Prague 4, 142 20, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Albertov 6, Prague 2, 128 40, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Škerlová
authors.affiliation: Institute of Molecular Genetics, AS CR, v.v.i., Videnska 1083, Prague 4, 142 20, Czech Republic.
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Král
authors.affiliation: European Molecular Biology Laboratory, Hamburg Outstation, c/o Deutsches Elektronen-Synchrotron (DESY), Notkestrasse 85, D-22603 Hamburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Kachala
authors.affiliation: Institute of Molecular Genetics, AS CR, v.v.i., Videnska 1083, Prague 4, 142 20, Czech Republic.
authors.forename: Milan
authors.initials: M
authors.lastname: Fábry
authors.affiliation: Institute of Microbiology, AS CR, v.v.i., Videnska 1083, Prague 4, 142 20, Czech Republic.
authors.forename: Ladislav
authors.initials: L
authors.lastname: Bumba
authors.affiliation: European Molecular Biology Laboratory, Hamburg Outstation, c/o Deutsches Elektronen-Synchrotron (DESY), Notkestrasse 85, D-22603 Hamburg, Germany.
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.affiliation: Faculty of Science, Charles University in Prague, Albertov 6, Prague 2, 128 40, Czech Republic.
authors.forename: Zdeněk
authors.initials: Z
authors.lastname: Tošner
authors.affiliation: Institute of Organic Chemistry and Biochemistry, AS CR, v.v.i., Flemingovo nam. 2, Prague 6, 166 10, Czech Republic. Electronic address: veverka@uochb.cas.cz.
authors.forename: Václav
authors.initials: V
authors.lastname: Veverka
authors.affiliation: Institute of Organic Chemistry and Biochemistry, AS CR, v.v.i., Flemingovo nam. 2, Prague 6, 166 10, Czech Republic; Institute of Molecular Genetics, AS CR, v.v.i., Videnska 1083, Prague 4, 142 20, Czech Republic. Electronic address: rezacova@uochb.cas.cz.
authors.forename: Pavlína
authors.initials: P
authors.lastname: Řezáčová
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001665:Binding Sites; D018604:Epitope Mapping; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry*; D006820:Hyaluronic Acid / Q000737:chemistry; D019169:Jurkat Cells; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary
chemical_list: D000911:Antibodies, Monoclonal; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid
keywords: CD44; Epitope mapping; MEM-85; Monoclonal antibody; NMR; SAXS