Proteome-wide Mapping of Endogenous SUMOylation Sites in Mouse Testis

Abstract

SUMOylation is a reversible post-translational modification involved in various critical biological processes. To date, there is limited approach for endogenous wild-type SUMO-modified peptides enrichment and SUMOylation sites identification. In this study, we generated a high-affinity SUMO1 antibody to facilitate the enrichment of endogenous SUMO1-modified peptides from Trypsin/Lys-C protease digestion. Following secondary Glu-C protease digestion, we identified 53 high-confidence SUMO1-modified sites from mouse testis by using high-resolution mass spectrometry. Bioinformatics analyses showed that SUMO1-modified proteins were enriched in transcription regulation and DNA repair. Nab1 was validated to be an authentic SUMOylated protein and Lys⁴⁷⁹ was identified to be the major SUMOylation site. The SUMOylation of Nab1 enhanced its interaction with HDAC2 and maintained its inhibitory effect on EGR1 transcriptional activity. Therefore, we provided a novel approach to investigating endogenous SUMOylation sites in tissue samples.

Fig. 1.

Strategy for mapping endogenous SUMO1-modified sites. A, Sequence alignment of SUMO1 orthologs and strategy of antibody preparation. The tryptic remnant peptide of SUMO1 C-terminal attached to εNH₂ of lysine was synthesized as an antigen to immunize rabbits and obtain a polyclonal antibody specifically identifying SUMO1-modified peptide. B, Verification of the efficiency of TC-Ab antibody. HEK293T cells were transfected with Flag-SUMO1 and Ubc9-Myc plasmids to enhance the overall SUMOylation level. Whole cell lysates were divided into groups for IP assay. The same amount of antiserum, TC-Ab antibody, and commercial SUMO1 antibody were used to enrich SUMOylated proteins. IgG was used as the negative control. Com refers to a commercial SUMO1 antibody. C, Schematic overview of endogenous SUMO1-modified peptides identification. Tissue samples were subjected to trypsin/Lys-C protease digestion, followed by peptide IP to enrich SUMO1-modified peptides. After Glu-C protease digestion, QTGG featured peptides were successfully exposed for further MS identification. High pH RP pre-separation is optional before LC-MS/MS.

Fig. 2.

Mouse testis is highly SUMOylated in vivo. SUMO1-modified proteins are highly enriched in mouse testis. The excised tissues from C57BL/6 mice were lysed and treated for immunoblot using SUMO1 antibody (Abcam) to detect the SUMOylation status of different tissues (up). Coomassie brilliant blue staining indicates the same amount of loading of each lane (middle). The bottom figure shows the comparison of the gray-scale value of SUMOylated bands (bottom). Results are displayed as mean ± S.E. from quadruplicate experiments of four mice.

Fig. 3.

Identification and bioinformatics analysis of endogenous SUMO1-modified proteins and sites. A and B, Bar chart shows endogenous SUMO1-modified sites identified in one experiment (sample EndoSUMO1–45) (A) and all experiments in this study (B). The red bar represents the number of total putative SUMO1-modified sites and the green bar represents the number of high-confidence sites which were assigned at least by two spectra. C, SUMO1-modified lysines are enriched in the KxE consensus motif. The pie chart shows distribution of identified endogenous SUMO1 modification sites based on sequence motif (upper). SUMO1 sites located within consensus KxE sequence motif are represented in red, sites located at a reverse consensus motif are represented in green. The bottom panel shows the graphical representation of SUMO site motifs identified in this study. D, GO enrichment analysis of potential endogenous SUMO1-modified proteins. The bar plot shows the top 5 terms of significantly enriched biological process, cellular component and molecular function for potential SUMO targets. All the analysis results were listed in supplemental Table S7. E, Network analysis of identified endogenous SUMO1-modified proteins. Network enrichment representation of SUMOylated proteins associated with Post-Translational Modification, DNA Replication, and Repair.

Fig. 4.

Validation of endogenous SUMO1-modified targets of mouse testis. A and B, HEK293T cells were cotransfected with HA/GFP-tagged SUMO1 and Flag-tagged wild-type or K-to-R mutant expression plasmids for selected candidates, that is, Nab1 (A) and Eid3 (B). Cell lysates were subjected to IP with anti-Flag M2 beads, and SUMOylated bands were detected by Western blot analysis using anti-Flag and anti-HA (or anti-GFP) antibodies. Black arrows represent unmodified candidate proteins, and the white represent the SUMO1-modified forms.

Fig. 5.

SUMOylation maintained the transcriptional repressing activity of NAB1. A, Interaction of wild-type and K480R NAB1 with HDAC2. Flag-tagged NAB1 (WT or K480R) was immunoprecipitated with anti-Flag M2 beads. NAB1 and HDAC2 were detected by Western blot analysis using anti-Flag or anti-HA antibodies, respectively. B, SUMOylation site mutation impaired the inhibitory effect of NAB1 on EGR1 transcriptional activity. Luciferase reporter assay used EP300 promoter as reporter construct and co-transfected it with different combination of wild-type or mutant NAB1 plasmids in HepG2 cells with EGR1 and SUMO1 plasmids. Data were corrected for Renilla activity. * p < 0.05, ** p < 0.01, unpaired Student's t test.