Full-length SENP1 was poorly expressed in E. coli BL21 and only several 100 μg of SENP1 could be partially purified ( Figure 2 A). The in vitro hydrolysis activity of SENP1 in SUMO maturation was studied and detected by SDS/PAGE and immunoblotting ( Figures 2 B and and2C). 2 C). To distinguish the SUMO precursors and their mature forms on gels, a GST module was inserted at the C-terminus of the precursors. Proteolytic cleavage at the ‘GG’ region by the protease will release a 16 kDa mature form and a 27 kDa GST module. When 2 μg of partially purified SENP1 was added to the assay, over 90% of SUMO-1 and -2 were hydrolysed; however, surprisingly, only 50% of SUMO-3 was hydrolysed. To examine the substrate specificity of SENP1 in SUMO maturation, different concentrations of SENP1 were tested ( Figure 3 ). When the SENP1 dosage reduced from 2 to 0.4 μg, substrate preferences were clearly illustrated; the maturation efficiency is in the order of SUMO-1 (90%), SUMO-2 (50%) and SUMO-3 (10%). Furthermore, cleavage of SUMO-3 could not be detected when 0.08 μg of SENP1 was added. These results imply that SENP1 is capable of processing all SUMO-1, -2 and -3 in vitro but with different efficiencies. Since the maturation reaction is the first committed step for subsequent sumoylation, the different maturation efficiencies catalysed by SENP1 may regulate the availability of different SUMO proteins for conjugation.
To identify the residues involved in maturation efficiency, three further mutants were constructed on the basis of SUMO-2 as it has the shortest tail of the three precursors. We noticed that the two residues after the ‘GG’ site are HS, VY and VP in SUMO-1, -2 and -3 respectively. Therefore the rationale of this analysis is to create three mutants that allow us to examine the effect of the amino acid sequence after the ‘GG’ region in the maturation process. In comparing the maturation efficiencies of mutants SUMO-2/V94H, SUMO-2/Y95P and SUMO-2/V94H/Y95S with those of the wild-type precursors, we were able to distinguish the role of the amino acid sequence C-terminal to the cleavage site. The three mutants were assayed using the same condition as described above. The results show that the SENP1C can hydrolyse SUMO-2/V94H and SUMO-2/V94H/Y95S as efficiently as hydrolysing SUMO-1. Substitution of Tyr-95 to proline in SUMO-2 reduced the maturation rate to that of SUMO-3 ( Figure 6 ). These results demonstrated that although the sequence of SUMO-1, -2 and -3 are not identical, the differences in the N-terminal of the cleavage site do not contribute towards proteolytic efficiency ( Figure 5 ). Instead, His-98 in SUMO-1 confers the highest maturation efficiency observed as described in Figures 3 and and4, 4 , whereas Pro-94 in SUMO-3 is responsible for the lowest maturation efficiency. In summary, this experiment implies that the first two residues of the tail play a crucial role in controlling the maturation efficiency.
Recombinant SENP1C was expressed in Escherichia coli BL21 by induction with 0.1 mM isopropyl β- D -thiogalactoside at 25 °C for 12 h. Cell pellets were resuspended in buffer I (500 mM NaCl, 10 mM Tris/HCl, pH 8.0, 0.2 mM benzamidine, 0.2 mM PMSF, 0.5 mM EDTA and 2 mM dithiothreitol) and lysed by sonication on ice. Lysate was clarified by centrifugation at 40000 g for 1 h and the protein was purified by affinity chromatography on glutathione–Sepharose beads (Amersham Biosciences). The GST tag was cleaved from the fusion protein by PreScission protease and SENP1C was further purified by Superdex 75 chromatography column (Amersham Biosciences). Expression of His-SUMO-1–GST fusion protein (SUMO-1 fusion protein) in E. coli BL21 was induced by 0.1 mM isopropyl β- D -thiogalactoside at 37 °C for 4.5 h. Cell pellets were resuspended in buffer II (500 mM NaCl, 10 mM Tris/HCl, pH 8.0, 0.2 mM benzamidine and 0.2 mM PMSF). Lysate was centrifuged for 40000 g for 1 h. The soluble fraction was loaded on to a nickel agarose column (Qiagen) under standard conditions followed by size exclusion chromatography (Superdex 75; Amersham Biosciences). Expression and purification of SENP1 and other SUMO fusion proteins are the same as described above, with the exception that SENP1 was only partially purified by nickel affinity chromatography as the yield was too low for further purification. SENP1C and the SUMO fusion proteins were purified to beyond 95% homogeneity.
The sequences of SUMO-1, -2 and -3 are shown. Residues that are completely conserved are in boldface and the ‘GG’ cleavage site is boxed.
Department of Biochemistry, Faculty of Science, The Chinese University of Hong Kong, Shatin, Hong Kong
Tissue distributions of SENP1, SUMO-1, -2 and -3 were analysed by PCR using normalized Human MTC™ panel I and II (BD Biosciences) as templates. PCRs were preformed as described in the Experimental section. The PCR products of SENP1, SUMO-1, -2 and -3 were separated on 1% agarose gel by the same sequence: lane 1, thymus; lane 2, kidney; lane 3, lung; lane 4, prostate; lane 5, pancreas; lane 6, spleen; lane 7, liver; lane 8, leukocyte; lane 9, brain; lane 10, placenta; lane 11, testis; lane 12, colon; lane 13, ovary; lane 14, small intestine; lane 15, heart; lane 16, muscle. The results were detected by staining the agarose gel with ethidium bromide.
Analysis of the effect of the tail in maturation efficiency. (A) Schematic diagram showing the construction of chimaeras SUMO-1M, -2M and -3M by swapping the tails after the ‘GG’ cleavage site. Only the protein sequence at the tail is shown. (B) SENP1 (2 and 0.4 μg) and (C) SENP1C (0.2 and 0.01 μg) were added to the assay mixture containing 0.1 nM of SUMO-1M, -2M or -3M. Reactions were incubated for 20 min at 37 °C in 50 ml mixture. After incubation, 12 μl of the reaction mixture was analysed by SDS/PAGE. The control (–) reactions do not contain SENP1 or SENP1C.
Human SENP1, SENP1C, wild-type and various SUMO mutants were amplified from a human control cDNA library of Human MTC™ panel I (BD Biosciences, Franklin Lakes, NJ, U.S.A.) by PCR. SENP1 and SENP1C were cloned into an expression vector pTWO-E encoding an N-terminal His6 tag (gift from Dr A. Oliver, The Instititute of Cancer Research, London, U.K.) between NheI and EcoRI, whereas SENP1C was cloned into expression vector pGEX-6P-1 (Amersham Biosciences, Uppsala, Sweden) between EcoRI and NotI for the expression of GST (glutathione S-transferase) fusion proteins. To assay for the SUMO C-terminal hydrolysis activity of a protease, SUMO precursors were engineered to have C-terminal extensions of a GST (amino acids 1–220, GenBank® accession no. <"type":"entrez-protein","attrs":<"text":"AAB37346","term_id":"1699061","term_text":"AAB37346">> AAB37346). Therefore, the amplified SUMO DNA was cloned into pTWO-E between NdeI and BamHI, followed by insertion of the fragment of GST between BamHI and XhoI. All clones were sequenced and shown to be identical with those previously reported for SENP1, SUMO-1, -2 and -3 (GenBank® accession nos. <"type":"entrez-protein","attrs":<"text":"Q9P0U3","term_id":"215273882","term_text":"Q9P0U3">> Q9P0U3, <"type":"entrez-protein","attrs":<"text":"AAH53528","term_id":"31565512","term_text":"AAH53528">> AAH53528, <"type":"entrez-protein","attrs":<"text":"AAH68465","term_id":"46250410","term_text":"AAH68465">> AAH68465 and <"type":"entrez-protein","attrs":<"text":"NP_008867","term_id":"48928058","term_text":"NP_008867">> NP_008867 respectively).
For SUMO-1, -2 and -3, the cDNAs were amplified for 30 cycles in a programme of 30 s at 94 °C, 36 s at 58 °C and 90 s at 72 °C. For SENP1, the cDNAs were amplified by the same programme with the exception that the annealing temperature was 55 °C. The DNA products were detected by 1% agarose gel staining with ethidium bromide.
Mapping residues of SUMO precursors essential in differential maturation by SUMO-specific protease, SENP1 Zheng Xu Department of Biochemistry, Faculty of Science, The Chinese University of