Engineering and structural characterization of a linear polyubiquitin-specific antibody |
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Authors: | Matsumoto Marissa L Dong Ken C Yu Christine Phu Lilian Gao Xinxin Hannoush Rami N Hymowitz Sarah G Kirkpatrick Donald S Dixit Vishva M Kelley Robert F |
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Affiliation: | Department of Antibody Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. |
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Abstract: | 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. |
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