Identification of an active-site residue in invertase SUC2 by mass spectrometry-based proteomics and site-directed mutagenesis

International Journal of Mass Spectrometry 409 (2016) 9–15

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Identification of an active-site residue in invertase SUC2 by mass spectrometry-based proteomics and site-directed mutagenesis Zhen Sun, Yuanyuan Du, Fan Yang, Xue Wang, Yafang Wang, He Li, Rong Li, Wenzhu Tang, Xianzhen Li ∗ School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, PR China

a r t i c l e

i n f o

Article history: Received 18 July 2016 Received in revised form 7 September 2016 Accepted 15 September 2016 Available online 16 September 2016 Keywords: Invertase Ubiquitination Mass spectrometry Site-directed mutagenesis Active-site residue

a b s t r a c t Invertase SUC2 is well-studied as a model hydrolase for carbohydrate catabolism in yeast strains. However, little is known about the ubiquitination of SUC2 and its contribution on the enzyme. In this paper, we demonstrated for the first time, that invertase SUC2 was ubiquitinated at three lysine residue sites using mass spectrometry-based proteomics. The role of ubiquitination was investigated through sitedirected mutagenesis. The K185, K312 and K430 were changed to arginine, respectively. The expression level, activity and stability of all the SUC2 isoforms together with the wild-type one were measured and compared. Results showed that ubiquitination of SUC2 could not lead to the degradation of the protein. Interestingly, ubiquitination of SUC2 in the site of 185 contributes to the high enzyme activity, and ubiquitin-tagged residue K185 in SUC2 is probably a novel active-site residue in invertase SUC2. This study provides an insight into the role of post-translational modifications in regulating the stability and activity of enzymes. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Invertase SUC2 (␤-d-fructofuranoside fructohydrolase, EC 3.2.1.26) is critical for inulin hydrolysis by yeast in industrial sections [13,29,32]. Its physiological role, crystallographic structure, key domains/residues, and historical relevance have been well studied [10,20–22,31]. As for post-translational modifications in SUC2, most researches focused on glycosylation. Two forms of invertase were found: an extracellular highly-glycosylated form and an intracellular non-glycosylated form [5,25]. Previous reports showed that the active form of invertase SUC2 was normally secreted as a heavily glycosylated octamer consisting of 513 amino acid residues deduced from the nucleotide sequence of the SUC2 gene (Fig. 1) [25,31]. However, as another critical post-translational modification, the ubiquitination of invertase has not yet been reported. Ubiquitin is a highly conserved polypeptide with 76 amino residues and fulfills a host of critical cellular processes in eukaryotes via post-translational modification [18]. At present, most studies just focus on the ubiquitin/proteasome-dependent proteolysis, a major role of ubiquitination [9,12,30]. When ubiquitin molecules

∗ Corresponding author. E-mail address: [email protected] (X. Li). http://dx.doi.org/10.1016/j.ijms.2016.09.008 1387-3806/© 2016 Elsevier B.V. All rights reserved.

are covalently linked via its C-terminal carboxyl group to the ␧-amino groups of lysine residues on target proteins, ubiquitinconjugated proteins will be instantly recognized by 26S proteasome or lysosomes/vacuole, resulting in rapid degradation of target proteins. Such proteolysis regulates many processes, such as the progression of the cell cycle, the induction of the inflammatory response, and antigen presentation [18]. However, as for invertase SUC2, there is no report on both ubiquitination and biological function caused by ubiquitination. With the technological advances of two “soft” ionization methods, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), biological mass spectrometry (MS) is evolving rapidly. For instance, a new type of mass spectrometer that combines an ESI ion source with a highly efficient tandem mass spectrometer that can fragment the individual peptides has been widely applied to analyzing biological samples, such as nucleotide acid, protein, etc [23]. Moreover, MS has evolved into an indispensable tool for proteomics research that has contributed greatly to our understanding of gene function in the post-genomic era [3]. Currently, identification of ubiquitin-modified proteins by MS-based proteomics has been achieved using a number of approaches, including His6 -tagged ubiquitin-conjugated proteins, ubiquitin-specific antibodies, multiple reaction monitoring-initiated detection and sequencing, etc [2,7,14,17,18].

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Fig. 1. Amino acid sequence analysis of S. cerevisiae invertase SUC2. “*” refers to N-glycosylation sites. Substrate binding/catalysis sites were marked by arrows. High conserved regions were wave lined.

In this paper, yeast invertase SUC2 was heterogenous expressed in Pichia pastoris and Escherichia coli cells, respectively. Great difference in invertase activity was observed between the purified SUC2 proteins from different expression hosts. To further investigate the role of ubiquitinated-site residue on protein acitivity, MS-based proteomics was applied to identify ubiquitin modified sites of yeast invertase SUC2 expressed in different expressing hosts, and an active ubiquitinated-site residue was found by comparing the activities of yeast invertase SUC2 expressed in different versions using site-directed mutagenesis [14]. This result is significant to provide an interesting insight into biological functions of ubiquitination. 2. Materials and methods 2.1. Plasmids, bacterial and yeast strains Escherichia coli DH5␣ was used in all cloning experiments. The plasmid pPICZ␣A and wild-type Pichia pastoris strain X-33 were used for the eukaryotic expression of SUC2, whereas the plasmid pET28a and E. coli BL21 (DE3) were ready for prokaryotic expression of SUC2. All above plasmids and strains were kind gifts from Prof. Zhao (Dalian Institute of Chemical Physics, CAS). Plasmid pYC230-B-SUC2 containing the SUC2 gene of S. cerevisiae BY4741 was constructed in our previous work. 2.2. Construction of the SUC2 expression vectors To obtain the eukaryotic expression vector, SUC2 gene was amplified by PCR using PrimeSTAR HS DNA Polymerase (TaKaRa Bio Inc., Dalian, China) with the template pYC230-B-SUC2 and the forward primer 5 -TCCTCGAGAAAAGATCAATGACAAACGAAACTAGC3 (XhoI restriction site underlined) and the reverse primer 5 -CTCGCGGCCGCCTATTTTACTTCCCTTACTTG-3 (NotI restriction site underlined). The PCR products were digested with XhoI and NotI and ligated into the downstream of the alcohol oxidase 1 promoter (AOX1) in the vector pPICZ␣A. The construct that had the full length SUC2 gene sequence was termed as pPICZ␣A-suc2. After digested by the restriction enzyme SacI, the linearized form of the plasmid pPICZ␣A-suc2 was transformed into P. pastoris X-33 cells by electroporation at 1.5 kV, 25 ␮F, and 200  in a 0.2 cm gap electroporation cuvette, using Bio-Rad Gene Pulser. The recombinants were selected on YPDS (1% yeast extract, 2% peptone, 2% dextrose, and 1 M D-sorbitol) plates containing 100 ␮g/ml of Zeocin.

For the construction of prokaryotic expression vector, the same XhoI and NotI digested SUC2 fragment was ligated into the XhoINotI-cut pET28a vector, the resulted plasmid pET-suc2 was then transformed into electrocompetent E. coli BL21 (DE3). 2.3. Site-directed mutagenesis of SUC2 gene Site-directed mutagenesis of SUC2 was performed by use of a restriction-free (RF) cloning method described before [26]. The introduction of specific mutations into SUC2 gene was achieved by the use of the following pairs of mutagenic primers: for isoform K185R, K185Rfwd (5 TCCTCTGATGACTTGAGATCCTGGAAGCTAGAATC-3 ) and K185Rrev (5 -CTATTTTACTTCCCTTACTTGGAAC-3 ); for K312R, K312Rfwd (5 -TCAATGACAAACGAAACTAGCG-3 ) and K312Rrev (5 CAGTGTTCAAAGAAAATCTGCGGACCAAAGACAT-3 ); for K430R, K430Rfwd (5 -TCAATGACAAACGAAACTAGCG-3 ) and K430Rrev GTGAAATATGGGTTCTCTCTGACAAACTTGACCTT-3 ) (the (5 nucleotide changes giving the appropriate mutations are underlined). The initial template for isoform K185R was pPICZ␣A-suc2, the resulted plasmid pPICZ␣A-185suc2 was used as the template for introducing K312R, giving birth to the plasmid pPICZ␣A185-312suc2, which became the template for K430R, and pPICZ␣A-185-312-430suc2 was obtained. The reaction mixtures were placed on ice for 5 min, and then the parental, supercoiled plasmid was digested with 0.5 ␮L of DpnI at 37 ◦ C for 1 h before being transformed into competent E. coli DH5␣ cells. Mutations were verified by DNA sequencing. Three resulting plasmids (pPICZ␣A-185suc2, pPICZ␣A-185312suc2, and pPICZ␣A-185-312-430suc2) will express three SUC2 isoforms (K185R, K185, 312R and K185, 312, 430R). Of the SUC2 mutants, K185R was used for studying the biological role of ubiquitination in amino acid site K185. K185R was also set as the control for K185, 312R to study the site K312. And K185, 312R was subsequently set as the control for K185, 312, 430R to study the site K430. Meanwhile, K185, 312R and K185, 312, 430R were used to investigate the coupling biological effect of two and three ubiquitination sites on SUC2. 2.4. Expression and purification of different SUC2 versions To express SUC2 and SUC2 isoforms in P. pastoris X-33, midscale cultures were performed. Briefly, the recombinant was grown

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Fig. 2. Comparison of purified SUC2 samples from different expression strains. (A) SDS-PAGE analysis of purified SUC2 samples. Lane1: Ec-SUC2; lane2: Pp-SUC2; (B) Inulinase activity (diagonal) and invertase activity (blank) of Ec-SUC2 and Pp-SUC2. The lowercase letters represent the multiple comparison results obtained by LSD method (p < 0.05). The different letters mean significant difference, while the same letter means no significant difference.

in 50 mL of BMGY medium (100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10−5 % d-biotin, and 1% glycerol) at 30 ◦ C for 10 h. The cultures had an optical density at 600 nm (OD600nm ) around 4.0. Cells were collected by centrifugation at 4 ◦ C, resuspended in 200 mL of BMMY medium (100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10−5 % d-biotin, and 0.5% methanol) in 2-l shake flasks, and then incubated at 30 ◦ C for 96 h. Methanol was added to a final concentration of 0.5% every 24 h to maintain induction of SUC2. The effect of ubiquitination on the proteolysis of SUC2 was assessed by SDS-PAGE and the total protein concentration of the fermentation supernatant was measured using the Bio-Rad protein assay kit (Bio-Rad, USA). The fermentation supernatant was filtered through a 0.22 ␮m durapore membrane (Millipore Corporation, USA), concentrated using a stirred ultrafiltration cell (Millipore Corporation, USA) over a 10-KDa molecular weight cut-off membrane. The concentrated crude enzyme solutions were ready for purification. For the expression of SUC2 in E. coli BL21 (DE3), transformants were grown at 37 ◦ C in Luria–Bertani (LB) medium (10 g/L tryptone, 5.0 g/L yeast extract, and 5 g/L sodium chloride, pH 7.0) supplemented with 30 mg/L kanamycin until OD600nm reached 0.5. For induction, IPTG was added at a final concentration of 1 mM and the cells were further cultured at 16 ◦ C for 12 h. Cells were harvested by centrifugation, and the cell pellet was suspended and sonicated 30 times on ice for 10 s with 30 s intervals in between with an ultrasonic processor (Branson Digital Sonifier; Branson Inc., USA). Following removal of cell debris by centrifugation, the supernatant was ready for subsequently purification. His-tagged SUC2 both from P. pastoris and E. coli were purified using a Ni-NTA purification system as described before [4]. Chromatograms were recorded by absorbance detection at 280 nm. The purity of the fractions was assessed by SDS-PAGE. Protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad, USA).

[24]. Approximately 100 ␮g protein for each sample was digested with trypsin for the following experiments. The lyophilized peptides were dissolved in 0.1% FA, directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific) with a linear gradient of 7–35% solvent B for 12 min, 35–80% for 4 min then holding at 80% for last 4 min at a constant flow rate of 280 nl/min on an EASY-nLC 1000 UPLC system, The resulting peptides were analyzed by Q Exactive hybrid quadrupoleOrbitrap Plus mass spectrometer (ThermoFisher Scientific). The peptides were subjected to nanoESI source followed by tandem mass spectrometry (MS/MS) in Q Exactive (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using 30% normalized collision energy; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 1E4 in the MS survey scan with 10.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350–1800. The resulting MS/MS data were processed using Mascot search engine (version 3.2). Tandem mass spectra were searched against UniProt Saccharomyces cerevisiae database (6629 sequences) concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 4 missing cleavages. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as fixed modification, oxidation on Met, GlyGly on Lys, acetylation on Lys, and acetylation on protein N-terminal was specified as variable modification. Peptide ion score was set to >20.

2.6. Enzyme assay 2.5. Detection of ubiquitination sites of SUC2 by MS-based proteomics For digestion, the protein solution was reduced with 10 mM DTT for 1 h at 37 ◦ C and alkylated with 20 mM IAA for 45 min at room temperature in darkness. Then, trypsin was added with trypsinto-protein mass ratio of 1:50 for the first digestion overnight and trypsin-to-protein mass ratio of 1:100 for a second 4 h-digestion

Inulinase or invertase activity was measured as described previously [6,29]. The reaction mixture consists of 0.0002 mg purified enzyme samples and 450 ␮L of 2% inulin or sucrose in 100 mM acetate buffer (pH 5.0), and distilled H2 O was added to make a total volume of 500 ␮L. The reaction was performed at 50 ◦ C for 15 min and terminated by boiling. The reducing sugar was assayed by the 3,5-dinitrosalicylic acid method [16]. One unit of enzyme activ-

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Fig. 3. LC–MS/MS fragment ion spectra of ubiquitination on the peptides of SUC2. (A) The ubiquitinated site located on K185 in the peptide IEIYSSDDLKSWK was unambiguously assigned from a mass increment of 242.1400 Da (K + G1 G2 ) between the y-ion series of y3 + and y4 + . (B) The ubiquitinated site located on K312 in the peptide KFSLNTEYQANPETELINLK was identified from the a2 + (K + F + G1 G2 + H+ − 28 Da) and b3 + ions (K + F + S + G1 G2 + H+ ). (C) The ubiquitinated site located on K430 in the peptide FVKENPYFTNR was distinguished identified from the ions of a2 + and b3 + , the increment of which is assigned to be (K + G1 G2 + 28 Da).

ity was defined as the amount of enzyme that produced 1 ␮mol fructose per min under the assay condition used in this study. All assays were done in triplicates, and the mean values were presented.

All experiments were done in triplicates.

3. Results

2.7. Determination of the thermal and pH stability of SUC2 isoforms

3.1. Purified invertase SUC2 from different expression hosts showed different properties

The thermostability of SUC2 isoforms was determined by assaying the remaining specific invertase activity of enzyme samples that were exposed to 100 mM acetate buffer (pH 5.0) at various temperatures (20–60 ◦ C) for 30 min. To measure the pH stability, activity assays were done using enzyme samples that were pre-incubated at different pH values (3.0–10.0) for 2 h.

Invertase SUC2 was heterologously expressed in P. pastoris strain X-33 (Pp-SUC2) and E. coli BL21 (DE3) (Ec-SUC2) respectively and purified by affinity chromatography. As shown in Fig. 2A, Ec-SUC2 migrated as a single band with apparent molecular weight of 60 kDa, whereas the molecular weight of Pp-SUC2 was much higher, achieved ∼85 kDa. It indicated that when expressed

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in P. pastoris strain X-33, Pp-SUC2 probably underwent posttranslational modifications to form the mature protein product. Moreover, great difference in invertase activity was observed between Pp-SUC2 and Ec-SUC2 (Fig. 2B). The specific activities of Pp-SUC2 were 18721.0 U/mg and 1247.4 U/mg, when using sucrose and inulin as substrates, respectively. However, Ec-SUC2 showed a much lower specific activity of 6336.5 U/mg and 267.5 U/mg respectively towards sucrose and inulin. All the results suggested that SUC2 expressed by P. pastoris strain was effectively functioned presumably due to post-translational modifications [15]. It was presumed that besides glycosylation, ubiquitination of invertase could affect its expression, conformation or activity [7]. Therefore, the discrepancy in ubiquitination between different SUC2 samples was investigated. 3.2. MS-based proteomics demonstrated several ubiquitination sites in P. pastoris expressed SUC2 As described before, in eukaryotic cells, the ubiquitin is covalently attached to the side chain of lysine residue in proteins, in a post-translational manner [27]. Whereas in prokaryotes (e.g. E. coli), a prokaryotic ubiquitin-like protein, Pup, can be investigated. An ubiquitin-conjugated protein produces a signature peptide Gly–Gly at the ubiquitination site after the tryptic digestion, which causes a mass shift of 114.1 Da. The ubiquitination site will then be identified by database-searching algorithms with the variable modification of Gly–Gly on the lysine [17]. Although amino acid sequence varies between ubiquitin and Pup, a two-residue remnant (Gly-Gly) was detected on K430 in the peptide FVKENPYFTNR of both Pp-SUC2 and Ec-SUC2 after trypsin proteolysis. The ubiquitinated site located on K430 was distinguished identified from the ions of a2 + and b3 + , the increment of which is assigned to be (K + G1 G2 + 28 Da) (Fig. 3C). Therefore, Pup attached to Ec-SUC2 probably shared the same Gly–Gly in the C-terminus with the ubiquitin conjugated to Pp-SUC2. Besides the shared ubiquitinated-site residue K430, K185 and K312 in Pp-SUC2 were also detected to be tagged with the ubiquitin. As shown in Fig. 3A, the ubiquitination site located on K185 was unambiguously assigned from a mass increment of 242.1400 Da (K + G1 G2 ) between the y-ion series of y3 + and y4 + . By analyzing the information from the LC–MS/MS fragment ion spectra of KFSLNTEYQANPETELINLK, a2 + ion was assigned to be (K + F + G1 G2 + H+ − 28 Da) and b3 + ion to be (K + F + S + G1 G2 + H+ ) (Fig. 3B). Moreover, K312 was also a missed proteolytic cleavage which was in accordance with the fact that trypsin proteolysis cannot occur at the modified lysine. Thus, K312 was obviously ubiquitinated. 3.3. Ubiquitination of SUC2 could not lead to its proteolysis in P. pastoris X-33 It is well established that, in most cases, proteins marked with ubiquitin are selectively targeted to the 26 proteasome or the lysosome, which ultimately resulting in proteolysis [18]. In order to investigate whether the ubiquitination of SUC2 will induce its degradation, the deubiquitination of protein was carried out. The lysines in position K185, K312 and K430 were substituted with arginines, respectively, by site-directed mutagenesis. The nucleotide substitutions in all the gene mutants were confirmed by DNA sequence analysis. The mutant enzymes were expressed in P. pastoris X-33 and purified, resulting three SUC2 isoforms including K185R, K185, 312R and K185, 312, 430 R. All the SUC2 mutants lost the ability to conjugate ubiquitin at the mutant site, which will decrease the proteolysis, if the ubiquitination really causes such a consequence.

Fig. 4. Analysis of secreted expression level of different SUC2 isoforms. SDS-PAGE analysis of the supernatants of the P. pastoris recombinants expressing wild-type SUC2 (lane1), K185R (lane2), K185,312R (lane3), K185,312,430R (lane4), respectively, after cultured in BMMY medium at 30 ◦ C for 72 h. Total protein concentrations were showed below.

Secreted expression levels of SUC2 mutants were then observed. After cultured in BMMY medium at 30 ◦ C for 96 h, equal-OD600nm fermentation broths of P. pastoris recombinants were treated by centrifugation, the supernatants containing different SUC2 isoforms were separated by SDS-PAGE. As shown in Fig. 4, compared with P. pastoris strain expressing wild-type SUC2, no significant changes in the secretion level of K185R, K185, 312R and K185, 312, 430 R was detected. Total protein concentrations of the fermentation supernatants containing wild-type SUC2 or SUC2 isoforms also showed no significant difference (Fig. 4). All the results indicating that deubiquitination of any lysine residue could not increase the expression level of SUC2 and on the other hand, ubiquitination could not lead to the degradation of SUC2 in P. pastoris X-33. 3.4. Deubiquitination of SUC2 revealed an active-site residue in invertase SUC2 To confirm the biological role of ubiquitination in amino acid site K185, K312 and K430, specific activity of mutant K185R, K185,312R and K185,312,430 R together with wild-type SUC2 were determined and compared. All the three purified invertase mutants revealed no difference in their specific activity values towards sucrose and inulin. However, when compared with the wildtype enzyme, the average specific activities of the pure mutant enzymes decreased to ∼11,000 U/mg (for sucrose) and ∼600 U/mg (for inulin), respectively, which were about 70% and 68% those of wild-type SUC2 (Fig. 5). All the results suggested that the deubiquitination of K312 and K430 had no effects on the activity of invertase SUC2, but deubiquitination of K185 through amino acid mutagenesis significantly decreased the enzyme activity. In order to investigate the effect of ubiquitinated K185 on invertase stability, thermal and pH stabilities for wild-type SUC2 and deubiquitinated mutant enzyme K185R were determined. As shown in Fig. 6A, a temperature-dependent assay was performed by incubating the enzyme samples at a range of temperatures for 30 min, and measuring their specific activities. Both wild-type SUC2 and K185R were relatively stable when temperature ranged from 25 ◦ C to 55 ◦ C. Interestingly, the K185R showed higher stability than SUC2 at 40 ◦ C–50 ◦ C. These results suggested that the deubiquitination of K185 slightly affect the thermal stability of SUC2. To evaluate if deubiquitination of K185 can induce a difference in protein stability against pH, SUC2 and K185R were incubated at a range of pH, and their activities after 2 h of incubation at 50 ◦ C was measured. The experiments revealed both SUC2 and K185R to be

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Fig. 5. Specific activities of SUC2 isoforms. Inulinase activity (diagonal) and invertase activity (blank) of purified enzymes including SUC2, K185R, K185,312R and K185,312,430R. The lowercase letters represent the multiple comparison results obtained by LSD method (p <0.05). The different letters mean significant difference, while the same letter means no significant difference.

relatively stable up to pH 6.0, but SUC2 showed higher stability than K185R at pH 6.5 (Fig. 6B). These results suggest that ubiquitination of K185 stabilized the invertase protein at a higher pH value.

4. Discussion Yeast invertase catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose and is one of the classical model enzymes in early biochemical studies [5,20,25,31]. Besides glycosylation, which is no doubt a major modification that exists in invertase, other modifications, especially ubiquitination, might also play a key role in the stability and activity of this protein [18]. In most cases, ubiquitinated proteins are targeted for proteolysis by 26S proteasomes or undergo endocytosis in the lysosome [9,11]. However, degradation is not the only fate for ubiquitinated proteins, some researches showed that ubiquitination could be involved in the regulation of enzymatic activities or other certain processes through some obscure mechanisms [7,8,18,28]. As mentioned above, many approaches based on proteomics such as His6 -tagged ubiquitin-conjugated proteins, ubiquitin-specific antibodies, multiple reaction monitoring-initiated detection and sequencing [2,7,14,17,18] have been applied to characterize ubiquitin-conjugated proteins. For the two former methods, ubiquitin-conjugated proteins were firstly tagged then affinity purified using nickel chromatography or directly captured by ubiquitin-specific antibodies. These two methods are suitable for large-scale analysis of ubiquitination sites. However, the technique of multiple reaction monitoring-initiated detection and sequencing needs the pre-known peptide ions knowledge of the proteins which were selected to be monitored. Therefore, to target one protein or some proteins without pre-known knowledge, the proteins should be purified before tandem mass spectrometry analysis. As Gascón et al. [5] reported that native SUC2 is trapped in the cell wall of the yeast Saccharomyces, it is therefore rather difficult for us to purify this enzyme from its natural host [31]. We carried out heterologous expression of SUC2 in P. pastoris strain X-33, which could efficiently modify and yield a sufficient amount of enzyme for studying the contribution of ubiquitination [15]. Also, expression host E. coli BL21 (DE3) was chosen for the production of unmodified SUC2 control [19]. Both the molecular weight and activity of purified SUC2 sample from P. pastoris were much higher than that from E. coli BL21, suggesting that SUC2 was sufficiently post-

Fig. 6. The thermal and pH stability of the wild-type SUC2 and its mutant K185R. Protein samples were incubated at different temperatures for 30 min and the remaining activity was measured. The specific activity at 35 ◦ C was taken as 100% (A). Protein samples were incubated at 50 ◦ C for 2 h in different pH buffer solutions and the remaining specific activity was measured. The highest invertase activity was taken into 100% (B). (Error bar-Standard deviation of the three independent experiments).

translational modified in P. pastoris, which might play an essential role for a high enzyme activity. Using MS-based proteomics, we identified that three of the 25 lysine residues in Pp-SUC2 are sites of ubiquitination. One is at the N-terminus (K185) and one is at the C-terminus (K430). According to the three-dimensional structure of SUC2 described by SainzPolo et al. [22], all the three residues are exposed on the surface of SUC2 (Fig. S1) which is in accordance with the theory that ubiquitin is preferentially added to lysines present in surface-exposed loops [1]. The discovery of discrepant SUC2 ubiquitination between Pp-SUC2 and Ec-SUC2 prompted us to investigate the importance of this modification, especially in regulating the activity and stability of the enzyme, using site-directed mutagenesis. Although this MS-based approach can distinguishably identify ubiquitination sites relying on the identification of signature peptides with an internal Gly–Gly tag on ubiquitinated lysine residues following tryptic digestion, the ubiquitinated form conjugated to each site, mono-ubiquitination or poly-ubiquitination, cannot be determined by this approach. Mutation of lysine to another residue is predicted to reduce the attachment of ubiquitin in SUC2 [9]. Of the options available, arginine was chosen as the replacement amino acid for the mutagenesis of ubiquitin in this work, because arginine is similar in size and positively charged, which would enable retention of a charged site and minimize any changes in electrostatics [14]. Three SUC2 isoform, including K185R, K185,312R and K185,312,430R were

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constructed, however, deubiquitination of SUC2 at position 185, 312 or 430 made no contribution to the secretion level of SUC2 compared with wild-type SUC2, suggesting that ubiquitination of exogenously expressed SUC2 does not induce its degradation via proteolytic pathways. If ubiquitination of SUC2 is not a signal for its degradation, what is the role of this post-translational modification? To clarify this question, the activities and stabilities of different SUC2 isoforms were compared. Results showed that only the deubiquitination of the residue K185 could lead to partially inactive. Considering the previous reports that some conserved domains and residues (Trp39, His56, Phe102, Asp171, Glu223 and Cys224) were predicted to be important for the binding and hydrolyzing of sucrose (Fig. 1) [22,31]. Our investigation suggesting that ubiquitintagged K185 is probably a novel active-site residue in invertase SUC2, the chemical microenvironment surrounding the 185 lysine residue might play a role in defining the biological activity of the protein. It is worth mentioning that, the decreased activity of mutant enzyme was still much higher than that of Ec-SUC2, which suggested other important factors, e.g. glycosylation, participated in controlling the enzyme activity. Interestingly, the thermal stability of SUC2 was not changed by mutagenesis of K185, whereas the stability against pH 6.5 was significantly improved. One explanation for this might be the different mechanism between thermal denaturation and pH denaturation. When the temperature is increased, the energy transmitted to the protein induces the overall movement of the atoms in a protein, which causes a nonspecific disruption for protein folding. As a result, the deubiquitination of one residue might not so effective in changing thermal stability in the detection range. On the other hand, pH leads to the denaturation of proteins by interacting with the specific atoms involved in electrostatic interactions in a protein. Therefore, the ubiquitination of lysines, which generally involved in electrostatic interactions, might be more sensitive against the pH value. However, further researches will be still needed to more precisely understand the effect. 5. Conclusions Yeast invertase SUC2 is ubiquitinated but not degraded by the 26S proteasomes or lysosome. Ubiquitination of SUC2 contributes to the high enzyme activity, and ubiquitin-tagged residue K185 in SUC2 is probably a novel active-site residue in invertase SUC2. Acknowledgements We would like to thank Z Zhao for providing strain and plasmid. Financial supports provided by Natural Sciences Foundation of China (31201302, 31371742, 31601458), Special Fund for Agroscientific Research in the Public Interest (201303095), Science and technology Department of Dalian (2013B11NC078 and 2012J21DW009), Education Department of Liaoning (L2012197 and L2015049), Program for Liaoning Excellent Talents in University (LJQ2015009) are greatly acknowledged. References [1] A. Catic, C. Collins, G.M. Church, H.L. Ploegh, Preferred in vivo ubiquitination sites, Bioinformatics 20 (2004) 3302–3307. [2] H.J. Cooper, J.K. Heath, E. Jaffray, R.T. Hay, T.T. Lam, A.G. Marshall, Identification of sites of ubiquitination in proteins: a fourier transform ion cyclotron resonance mass spectrometry approach, Anal. Chem. 76 (2004) 6982–6988.

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[3] J. Cox, M. Mann, Is proteomics the new genomics? Cell 130 (2007) 395–398. [4] J. Crowe, H. Dobeli, R. Gentz, E. Hochuli, D. Stuber, K. Henco, 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification, Methods Mol. Biol. 31 (1994) 371–381. [5] S. Gascón, N.P. Neumann, J.O. Lampen, Comparative study of the properties of the purified internal and external invertases of yeast, J. Biol. Chem. 243 (1968) 1573–1577. [6] N. Hu, B. Yuan, J. Sun, S.A. Wang, F.L. Li, Thermotoleran: Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from jerusalem artichoke by consolidated bioprocessing, Appl. Microbiol. Biotechnol. 95 (2012) 1359–1368. [7] H. Johnson, C.E. Eyers, Analysis of post-translational modifications by LC–MS/MS, Methods Mol. Biol. 658 (2010) 93–108. [8] E. Karousou, M. Kamiryo, S.S. Skandalis, A. Ruusala, T. Asteriou, A. Passi, H. Yamashita, U. Hellman, C.H. Heldin, P. Heldin, The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination, J. Biol. Chem. 285 (2010) 23647–23654. [9] L.A. Knodler, S. Winfree, D. Drecktrah, R. Ireland, O. Steele-Mortimer, Ubiquitination of the bacterial inositol phosphatase, sopb, regulates its biological activity at the plasma membrane, Cell. Microbiol. 11 (2009) 1652–1670. [10] Á. Lafraya, J. Sanzaparicio, J. Polaina, J. Marínnavarro, Fructo-oligosaccharide synthesis by mutant versions of Saccharomyces cerevisiae invertase, Appl. Environ. Microbiol. 77 (2011) 6148–6157. [11] W.C. Lee, M. Lee, J.W. Jung, K.P. Kim, D. Kim, Scud: Saccharomyces cerevisiae ubiquitination database, BMC Genom. 9 (2008) 440. [12] W.C. Lee, M. Lee, W.J. Jin, K.P. Kim, D. Kim, Scud: Saccharomyces cerevisiae ubiquitination database, BMC Genom. 9 (2008) 1–7. [13] Y. Li, W.J. Fu, N.N. Liu, M.J. Tan, G.L. Liu, Z.M. Chi, Role of suc2 gene and invertase of Saccharomyces sp. W0 in inulin hydrolysis, J. Mol. Catal. B—Enzym. 111 (2015) 71–78. [14] Z. Liu, S. Cheng, D.R. Gallie, R.R. Julian, Exploring the mechanism of selective noncovalent adduct protein probing mass spectrometry utilizing site-directed mutagenesis to examine ubiquitin, Anal. Chem. 80 (2008) 3846–3852. [15] S. Macauley Patrick, M.L. Fazenda, B. Mcneil, L.M. Harvey, Heterologous protein production using the pichia pastoris expression system, Yeast 22 (2005) 249–270. [16] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem. 31 (1959) 426–428. [17] J. Peng, D. Schwartz, J.E. Elias, C.C. Thoreen, D. Cheng, G. Marsischky, J. Roelofs, D. Finley, S.P. Gygi, A proteomics approach to understanding protein ubiquitination, Nat. Biotechnol. 21 (2003) 921–926. [18] C.M. Pickart, Mechanisms underlying ubiquitination, Annu. Rev. Biochem. 70 (2001) 503–533. [19] P. Rallabhandi, P.K. Yu, Production of therapeutic proteins in yeasts: a review, Australas. Biotechnol. 6 (1996) 230–237. [20] V.A. Reddy, R.S. Johnson, K. Biemann, R.S. Williams, F.D. Ziegler, R.B. Trimble, F. Maley, Characterization of the glycosylation sites in yeast external invertase: I. N-linked oligosaccharide content of the individual sequons, J. Biol. Chem. 263 (1988) 6978–6985. [21] V.A. Reddy, F. Maley, Identification of an active-site residue in yeast invertase by affinity labeling and site-directed mutagenesis, J. Biol. Chem. 265 (1990) 10817–10820. [22] M.A. Sainz-Polo, M. Ramirez-Escudero, A. Lafraya, B. Gonzalez, J. Marin-Navarro, J. Polaina, J. Sanz-Aparicio, Three-dimensional structure of Saccharomyces invertase: role of a non-catalytic domain in oligomerization and substrate specificity, J. Biol. Chem. 288 (2013) 9755–9766. [23] R.D. Smith, J.A. Loo, C.G. Edmonds, C.J. Barinaga, H.R. Udseth, New developments in biochemical mass spectrometry: electrospray ionization, Anal. Chem. 62 (1990) 882–899. [24] H. Steen, M. Mann, The abc’s (and xyz’s) of peptide sequencing, Nat. Rev. Mol. Cell Biol. 5 (2004) 699–711. [25] R.B. Trimble, F. Maley, Subunit structure of external invertase from Saccharomyces cerevisiae, J. Biol. Chem. 252 (1977) 4409–4412. [26] F. Van den Ent, J. Löwe, RF cloning: a restriction-free method for inserting target genes into plasmids, J. Biochem. Biophys. Methods. 67 (2006) 67–74. [27] A. Varshavsky, The ubiquitin system, Annu. Rev. Biochem. 67 (1998) 425–479. [28] F. Verdier, T. Valovka, A. Zhyvoloup, L.B. Drobot, V. Buchman, M. Waterfield, I. Gout, Ruk is ubiquitinated but not degraded by the proteasome, Eur. J. Biochem. 269 (2002) 3402–3408. [29] S.A. Wang, F.L. Li, Invertase suc2 is the key hydrolase for inulin degradation in Saccharomyces cerevisiae, Appl. Environ. Microbiol. 79 (2012) 403–406. [30] C. Wolberger, Mechanisms for regulating deubiquitinating enzymes, Protein Sci. 23 (2014) 344–353. [31] F. Yang, Z.C. Liu, X. Wang, L.L. Li, L. Yang, W.Z. Tang, Z.M. Yu, X. Li, Invertase suc2-mediated inulin catabolism is regulated at the transcript level in Saccharomyces cerevisiae, Microb. Cell Fact. 14 (2015) 1–10. [32] B. Yuan, S.A. Wang, F.L. Li, Improved ethanol fermentation by heterologous endoinulinase and inherent invertase from inulin by Saccharomyces cerevisiae, Bioresour. Technol. 139 (2013) 402–405.