Strategy for Development of Site-Specific Ubiquitin Antibodies

Abstract

Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin-76 amino acids-complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments.

Keywords: H2B-K123ub; PCNA; histone H2B; monoclonal antibody; ubiquitin.

Figure 1

Outline of site-specific Ub antibody development. (i) design and synthesis of non-hydrolyzable Ub-peptide conjugates for immunization; (ii) design and synthesis of extended native iso-peptide linked Ub-peptide conjugates for screening; (iii) immunization, and generation and selection of hybridomas; (iv) selection of clones and antibody validation.

Figure 2

Synthesis of ubiquitinated polypeptides. (A) Design of the proteolytically-stable and native isopeptide-linked ubiquitinated polypeptides; (B) Schematic representation of the key CuAAC reaction to generate non-hydrolyzable Ub-polypeptides; (C) Schematic representation of the key steps to generate isopeptide linked Ub-polypeptides in which the sequence used in panel B is extended (indicated by -X- and explained further in the text).

Figure 3

LC-MS analysis of ubiquitinated polypeptides. Example of LC-MS analysis based on PCNA-K164 (UniProtKB P12004). Diode Array chromatogram (top), MS spectrum of peak (bottom) and Deconvoluted mass of product peak (insert) of (A) the non-hydrolyzable Ub-conjugate and (B) the isopeptide linked Ub-conjugate.

Figure 4

Expected types of antibodies upon immunization with peptide-Ub conjugates. Polyclonal serum after immunization with a non-hydrolyzable end-modified Ub-peptide conjugate will typically contain a mix of antibodies. (A) Antibodies recognizing the site-specific ubiquitin on the native protein can be identified using an extended version of the antigen used for immunization. Other antibodies might recognize (B) an epitope that includes the terminus of the immunization antigen and hence these antibodies will not recognize ubiquitin in the native extended protein, (C) the peptide independent of Ub, (D) Ub independent of the peptide, and (E) a K-Ub conjugate independent of the peptide context.

Figure 5

Validation of the site-specific antibody against yeast histone H2B-K123ub1 using engineered cell lines. (A) Example of ELISA screening of supernatants of hybridoma cultures that identified clones specific for the H2B-K123ub1 conjugate (#a,b) and clones non-specifically recognizing the H2B peptide (#c,d) or ubiquitin (#e,f). (B–F) Following ELISA screens, antibodies produced from selected hybridomas against yH2B-K123ub were validated by immunoblot analysis using whole-cell extracts of a panel of engineered yeast strains. The panel includes (1) WT (normal H2B-K123ub1), (2) bre1Δ (no H2B-K123ub1), (3) DOT1-OE (more H2B-K123ub1), (4) ubp8Δ; FLAG-H2B (more H2B-K123ub1 and size shift due to FLAG-tag), (5) ubp8Δ; FLAG-H2B-K123R (no H2B-K123ub1 and H2B size shift due to FLAG-tag). The strains (BY4741, NKI4558, NKI4553, NKI2563, NKI2564) and protocols have been described previously (Vlaming et al., ; van Welsem et al., 2018). The antibodies and polyclonal serum indicated are described in the main text and section 2.2.2.

Figure 6

Characterization of candidate site-specific antibody against human PCNA-K164ub1 using recombinant proteins and cell lysates. (A) Following ELISA screens, antibodies produced from selected hybridomas against PCNA-K164ub were examined by immunoblot analysis using recombinant proteins that were enzymatically ubiquitinated. The recombinant proteins used are shown on a Coomassie-stained gel. (B–F) Immunoblots of recombinant proteins with the indicated antibodies and recombinant proteins. Asterisk indicates degradation product; double asterisk indicates aggregation product. The selected PCNA-K164ub clone produced non-specific antibodies since ubiquitinated H2A was also detected. (G) Immunoblot analysis of whole-cell lysates of HEK-293T cells treated or not with 20 J/m² UV for 5 h, confirmed the lack of specificity of the selected clone (non-specific), while a UV-induced PCNA-K164ub1 increase was detected with a commercially available site-specific antibody (CST). Histone H4 was used as a loading control.