Determination of the mechanism of action of anti-FasL antibody by epitope mapping and homology modeling.
Fas ligand (FasL) is a 40-kDa type II transmembrane protein belonging to the tumor necrosis factor (TNF) family of proteins and binds to its specific receptor, Fas, a member of the TNF receptor family. Membrane-bound FasL can be processed into a soluble form by a metalloprotease similar to that which cleaves TNFalpha. Elevated levels of FasL have been implicated in a wide variety of diseases ranging from cancer to inflammatory abnormalities, which could be targeted by antibody therapy. We generated a fully human high-affinity antibody against FasL that binds to and neutralizes the activity of both soluble and membrane-associated human FasL. In order to elucidate the mechanism of function of this antibody, we have mapped the region and critical residues involved in the recognition of FasL using a combination of homology modeling, immunoprecipitation, hydrogen-deuterium exchange mass spectrometry (H/DXMS), and alanine scanning site-directed mutagenesis. These studies have revealed the antibody binding site on human FasL. Furthermore, through molecular homology modeling, we have proposed a mechanism for the neutralizing activity of this antibody that involves interference with the docking of the ligand to its receptor by the antibody.
Obungu
Victor H
VH
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, USA. Obunguvi@lilly.com
Gelfanova
Valentina
V
Rathnachalam
Radhakrishnan
R
Bailey
Anna
A
Sloan-Lancaster
Joanne
J
Huang
Lihua
L
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D017209:Apoptosis / Q000276:immunology; D001665:Binding Sites; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D053222:Fas Ligand Protein / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D000906:Antibodies; D000939:Epitopes; D053222:Fas Ligand Protein; D011993:Recombinant Fusion Proteins; D014357:Trypsin