Engineering and structural characterization of a linear polyubiquitin-specific antibody.
Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking. We therefore engineered a linear-linkage-specific antibody that is functional in Western blot, immunoprecipitation, and immunofluorescence applications. A crystal structure of the linear-linkage-specific antibody Fab fragment in complex with linear diubiquitin provides molecular insight into the nature of linear chain specificity. We use the antibody to demonstrate that linear polyUb is up-regulated upon tumor necrosis factor α stimulation of cells, consistent with a critical role in nuclear factor κB signaling. This antibody provides an essential tool for further investigation of the function of linear chains.
Department of Antibody Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Marissa L
ML
Matsumoto
Ken C
KC
Dong
Christine
C
Yu
Lilian
L
Phu
Xinxin
X
Gao
Rami N
RN
Hannoush
Sarah G
SG
Hymowitz
Donald S
DS
Kirkpatrick
Vishva M
VM
Dixit
Robert F
RF
Kelley
D000906:Antibodies / Q000737:chemistry* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism; D047468:Immunoprecipitation; D008958:Models, Molecular; D025821:Polyubiquitin / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D014409:Tumor Necrosis Factor-alpha; D025821:Polyubiquitin