- Email: [email protected]
Probing peptide substrate specificities of N-glycosyltranferase isoforms from different bacterial species
Carbohydrate Research 473 (2019) 82–87
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Probing peptide substrate specificities of N-glycosyltranferase isoforms from different bacterial species
T
Qingyun Meng, Kun Li, Yongheng Rong, Qizheng Wu, Xunlian Zhang, Yun Kong∗, Min Chen∗∗ The State Key Laboratory of Microbial Technology, National Glycoengineering Research Center, Shandong University, Qingdao, Shandong, 266237, China
ARTICLE INFO
ABSTRACT
Keywords: N-glycosyltransferase Peptide Substrate specificities RP-HPLC Mass spectrometry
N-glycosyltransferase (NGT) is responsible for transferring hexose monosaccharides to the asparagine side chain of proteins and polypeptides in the consensus sequon (N-(X≠P)-T/S) with nucleotide-activated sugars as donor substrates. Here, we expressed and purified four different N-glycosyltransferases derived from diverse bacteria, including Actinobacillus pleuropneumoniae, Aggregatibacter aphrophilus, Kingella kingae and Bibersteinia trehalosi, and measured their catalytic activities of four synthesized peptides via in vitro glycosylation assays. RP-HPLC and mass spectrometry were used to identify and quantify the glycopeptide formation by distinct NGT isoforms. We then analyzed and compared the glycosylation efficiencies of different peptides for these four NGT isoforms, which showed distinct substrate selectivities. We sought to probe peptide specificities among various NGT isoforms, which could broaden the application of NGT-catalyzed N-glycosylation of a variety of therapeutic proteins.
1. Introduction N-linked protein glycosylation is a universal post-translational protein modification in all eukaryotes [1], some bacteria and many archaea [2–6]. More than half of the proteins are N-glycosylated, including structural proteins, carrier proteins, enzymes, immunoglobulins, hormones and lectins [7,8]. N-linked glycosylation plays an important physiological role in organisms, such as cell-cell recognition [9], signal transduction, fertilization, development and differentiation, regulating hormone levels and modulating the nervous and immune system [10,11]. In the secretory pathway of eukaryotic organisms, N-linked protein glycosylation directs correct folding of proteins, including recognizes and degrades misfolded proteins [12,13]. In classical N-linked glycosylation, the assembled oligosaccharide is transferred from the lipid carrier oligosaccharide donor onto the asparagine residue of the acceptor proteins. The essential enzyme of this pathway is the oligosaccharyltransferase (OST/PglB) locating in endoplasmic reticulum (in eukaryotes) or periplasm (in bacteria) [14–16]. Recently, a cytoplasmic soluble N-glycosyltransferase (NGT) has been described in some bacteria, which uses nucleotide-activated hexose monosaccharides as donors to glycosylate the same consensus sequon N-X-(T/S) (where X≠ Pro) as eukaryotic OST [17–19]. The N-glycosyltransferase from Actinobacillus pleuropneumoniae
∗
(ApNGT) is the best characterized NGT in enzymatic properties [17,18]. ApNGT is capable to modify polypeptides containing consensus sequons (N-X(X≠P)-T/S), and could also glycosylate the peptide with other amino acids (Ala, Gly, Val and Asp) at the +2 position in low efficiency [18,20,21]. Compared to the -NXS-sequence context, ApNGT also has a 10-fold advantage over the -NXT-sequence [17]. In vitro, the substrate of ApNGT is preferably a peptide or a long polypeptide other than a folded protein substrate [22]. Compared with the complex OST-mediated N-glycosyaltion pathway, NGT is more likely to be utilized to add N-linked glycans onto recombinant proteins in vitro. A novel approach using NGT coupled with the transglycosylation activities of endoglycosidases has been utilized to produce peptides bearing different types of N-glycans [20,23,24]. Thus, more NGT isoforms with distinct donor or substrate specificities are needed to be identified and probed to promote the application of NGTs in the future. In this study, the NGT isoforms from Kingella kingae (KkNGT), and Bibersteinia trehalosi (BtNGT) were characterized and compared with ApNGT and AaNGT from Aggregatibacter aphrophilus [24]. We reported and demonstrated that two novel Nglycosyltransferases, KkNGT and BtNGT, were capable of transferring glucose onto synthetic peptides in vitro. Furthermore, we analyzed the substrate specificities of these four enzymes by testing several peptides using RP-HPLC and mass spectrometry, and explored the substrate
Corresponding author. Corresponding author. E-mail addresses: [email protected] (Y. Kong), [email protected] (M. Chen).
∗∗
https://doi.org/10.1016/j.carres.2018.12.016 Received 31 October 2018; Received in revised form 27 December 2018; Accepted 28 December 2018 Available online 30 December 2018 0008-6215/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Sequence alignment of four NGT sequences. Complete alignment of four NGT sequences (from A. pleuropneumoniae, A. aphrophilus, B. trehalosi and K. kingae). The alignment was performed using BioXM. Coloring indicates conservation of residues (from white, not conserved, to black, fully conserved).
selectivities for each enzyme for the application in future.
phase high performance liquid chromatography (RP-HPLC) analysis was carried out using a C18 column. Compared with the negative control without any enzyme added, the NGT-glycosylated peptide was observed as a new peak on the chromatogram with a shifted retention time (Fig. 2A). The eluted product was collected and subsequently confirmed by LC/MS-IT-TOF analysis based on molecular weight (Fig. 2B). The reaction of the four enzymes with the short peptide DANYTK showed a product peak at 14.5 min and the substrate peak at 15 min via RP-HPLC analysis. Additionally, from the MS spectrum, the [M+2H]2+ product peak demonstrated a m/z of 643.27, which is in good agreement with the calculated molecular weight of TAMRA-DANYTK-Glc of 1284.55 Da. Thus, all four NGTs showed glycosylation activities through RP-HPLC and MS analyses (Fig. 2). We next used HPLC-based analyses to optimize reaction conditions of BtNGT and KkNGT, such as pH, temperature and ionic strength of the buffer. We found that the metal chelating agent, EDTA (20 mM), had little influence on reaction rate of both BtNGT and KkNGT (Figs. S2A and S3A), which indicated that neither BtNGT nor KkNGT was a metalloenzyme. BtNGT exhibited better tolerance for Na+ and Mg2+, but was strongly inhibited by Ca2+, Mn2+, Cu2+ and Zn2+ (Fig. S2A), while KkNGT exhibited better tolerance for Ca2+, Mn2+, Mg2+, Zn2+ and Cu2+ (Fig. S3A). The optimum temperature of BtNGT was found to be 20 °C (Fig. S2B) and the optimum pH for the enzyme was 8.0 in the PBS buffer (Fig. S2C), which was similar to ApNGT. The optimum temperature of KkNGT was 40 °C and the three buffers, PBS, HEPES and
2. Results 2.1. Identification and purification of NGTs According to the sequence alignment results, KkNGT, and BtNGT are shown to be close homologs of ApNGT and AaNGT (Fig. 1), and KkNGT has been reported as a functional glycosyltransferase in vivo [21]. In our previous work, we have expressed and characterized AaNGT as a novel N-glycosyltransferase with different catalytic activities from ApNGT [24]. Among all four NGTs, the sugar donor binding site (Q469 in ApNGT, Q468 in AaNGT, Q471 in BtNGT and Q474 in KkNGT) is conserved, but some differences exist in amino acid residues around this site, which might affect their donor specificities. In this study, we expressed and purified BtNGT and KkNGT from Escherichia coli and obtained yields of about 30–40 mg from 1 L of cell culture (Fig. S1). 2.2. Recombinant NGTs showed glycosyltransferase activity in vitro To test the activity of these glycosyltransferases, we incubated the purified enzymes with UDP-Glc and the hexapeptide DANYTK, which was labeled at the N-terminus with the fluorescent dye TAMRA. In vitro glycosylation reactions were performed in PBS buffer, pH 8.0. Reversed 83
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Fig. 2. HPLC and Mass spectrometry analysis of NGT peptide receptor selectivities. A. The purified recombinant NGTs and UDP-Glc were incubated with different peptide receptors in 20 μL PBS buffer, pH 8.0, under optimum temperatures for 30 min, and the reaction mixture was boiled for 5 min, of which 5 μL was added to 45 μL of 0.1% TFA. After centrifugation, the naked peptide and glycosylated peptide were separated by RP-HPLC and quantified by the intensity of TAMRA. B. The purified four recombinant NGTs were incubated with UDP-Glc and different synthetic peptides at optimum temperatures in a 20 μL system. The mixture was boiled for 5 min, then centrifuged at 12000 rpm for 10 min 1 μL sample was taken at 30 min and diluted with 99 μL of 0.1% FA. 2 μL of the supernatant was analyzed by MS. 2 + represents [M+2H] 2 +.
which is two amino acid residues longer showed a slightly lower efficiency in the BtNGT reaction (Fig. 3C and D). But, AaNGT preferred the longer peptide (13mer) than the shorter one (11mer). Our results showed that the residues beside the N-glycosylation sequon, even in −5 or +7 position also affected the catalytic efficiencies of NGTs.
Tris, showed relatively higher enzyme activity when pH was 8.0 (Figs. S3B and C). Therefore, PBS buffer, pH 8.0, without salt was used for all further experiments. 2.3. The comparison of in vitro catalytic efficiencies with different NGT isoforms
2.4. Effects of C-terminal truncation of BtNGT on utilizing different sugar donors
In the assay, we found that the all four NGTs showed great differences in catalytic efficiencies in vitro. Under optimum conditions, AaNGT was capable of glycosylating most of the DANYTK peptide after 30 min of incubation, while few glycopeptide (approximately 16% of peptide) could be detected in the ApNGT reaction. In addition, the glycosylation efficiency of KkNGT was lower (approximately 5%), while the activity of BtNGT is slightly higher (about 29%) than ApNGT (Fig. 3A). The residues within and surrounding the N-glycosylation sequon were reported to affect the catalytic efficiency of NGTs [20]. Therefore, we analyzed more peptides to examine their potentials as NGT substrates. After 30 min incubation, the glycosylation efficiencies of the same peptide were compared for different enzymes (Fig. S4). The GGNWTT peptide has been reported to be the best preferred short peptide substrate for several NGTs via screening the peptide substrate microarray [25], thus we have also compared its glycosylation with our four NGTs. We found that KkNGT and AaNGT has a relatively low catalytic efficiency in vitro, about 50%, and the catalytic efficiency of ApNGT and BtNGT is similar, about 80% (Fig. 3B). We have reported the WPAVGNCSSALRW peptide derived from human Hemopexin was catalyzed more efficiently than the usually used DANYTK peptide by both ApNGT and AaNGT [24]. Thus, in this work we also tested two long peptides, WPAVGNCTSALRW (13mer) and PAVGNCTSALR (11mer), which have a T189S mutation to optimize the substrate utilization. The 11mer peptide can be almost completely glycosylated in 30 min by ApNGT, BtNGT and KkNGT, whereas the 13mer peptide
In BtNGT and KkNGT, there are about 70 extra amino acids in the Cterminal, which were not conserved in all NGTs. To identify the function of this domain, we further expressed and purified the protein fragment corresponding to the N-terminus amino acids 1–620 of BtNGT (referred to as BtNGTct) as a soluble protein with a His tag at C-terminal. The specificities of sugar donors for BtNGT and BtNGTct were examined using the TAMRA-DANYTK peptide and three different nucleotide-activated monosaccharides, including UDP-Glc, UDP-Gal and GDP-Glc. The reaction products were detected by RP-HPLC. The reactions of BtNGT and BtNGTct using nucleotide-activated hexose showed a product peak at about 14 min and a substrate peak at about 15 min (Fig. 4). Both BtNGT and BtNGTct were able to utilize the UDP-Glc (Fig. 4A), UDP-Gal (Fig. 4B) and GDP-Glc (Fig. 4C) in vitro glycosylation assays. Furthermore, the glycosylation rate of BtNGTct (Fig. 4) using UDP-Glc and GDP-Glc as sugar donors was even higher than that of BtNGT. The removal of C-terminal amino acids did not affect the glycosyltransferase activity and the recognition of sugar donors, which indicated these residues might not be necessary for their catalytic activities. 3. Discussion Cytoplasmic N-glycosyltransferase (NGT) transferring a single monosaccharide to the N-X-T/S acceptor sequence motif provides a 84
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Fig. 3. For the same peptide receptor, the conversion rate of each enzyme. After centrifugation of the reaction mixture, the supernatant was analyzed by RP-HPLC, and the peptide was detected by carboxymethyltetramethylrhodamine (TAMRA) (excitation wavelength: 542 nm, emission wavelength: 568 nm). Percent conversion was calculated from the peak areas of the naked and glycosylated peptides. All trials were conducted in triplicate.
Fig. 4. HPLC analysis of the specificities of sugar donors for BtNGT and BtNGTct. The reaction in a total volume of 10 μL containing different nucleotideactivated monosaccharides (A: UDP-Glc; B: UDP-Gal; C: GDP-Glc), the TAMRA-DANYTK peptide and BtNGT or BtNGTct was conducted in PBS buffer (pH 8.0, 100 mM) at 20 °C for 16 h. The mixture was quenched by boiling for 10 min and then added with 40 μL 0.1%TFA. After centrifugation, the naked peptide and glycosylated peptide were separated by RP-HPLC and quantified by the intensity of TAMRA. The naked peptide was used as control.
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powerful tool to produce novel N-glycosylation modification in therapeutic proteins. Our previous work on the N-glycosyltransferase from A. aphrophilus (named AaNGT) has provided important information of the enzyme properties of NGTs. In this work, we investigated two more ApNGT and AaNGT homologues derived from other species, including Kingella kingae (KkNGT) and Bibersteinia trehalosi (BtNGT), for further studies of the NGT family. The recombinant BtNGT and KkNGT have the activity to transfer a single hexose moiety to peptides in vitro and shares similar enzymatic properties with ApNGT and AaNGT, such as buffer and pH [26]. But there are also some differences of enzymatic properties among four NGT isoforms. BtNGT showed lower optimum temperature (20 °C), while KkNGT exhibited higher optimum temperature (40 °C) than AaNGT (30 °C) and ApNGT (37 °C). BtNGT was sensitive to metal ions and just tolerant of Na+ and Mg2+, while KkNGT exhibited better tolerance for tested metal ions. The diversity of the enzymatic properties of different NGTs provide more application in some diverse conditions. The peptide substrate specificities and nucleotide-activated monosaccharide selectivities of AaNGT have been analyzed in our previous work [24]. The KkNGT and BtNGT were found to be functional glycosyltransferases in vitro, but the turnover rates of several peptides were widely different. For example, the long peptide derived from human Hemopexin, PAVGNCTSALR, could be almost completely glycosylated in 30 min by BtNGT, whereas the WPAVGNCTSALRW peptide showed only a little glycosylated product. These results indicate that NGT enzymes have quite different preferences for amino acids residues surrounding the N-glycosylation site, even these residues located at −5 or +7 position. It is demonstrated that the receptor selectivities of these four NGT enzymes are similar but not identical, which broadens the peptide selection patterns of the NGT family. The diverse NGT isoforms identified and characterized might contribute to the application of NGT in the glycosylation of distinct therapeutic glycoproteins. In classical N-glycosylation pathway, GlcNAc is the first monosacharide residue linked to the asparagines residues of proteins [8]. But the direct transfer of a single GlcNAc moiety to produce asparagineslinked GlcNAc (N-GlcNAc) glycan has not been found. AaNGT and ApNGTQ469A mutant have been reported to transfer GlcN and produce N-GlcNAc glycan by coupling with GlmA [20,24]. Without the mutation in the conserved Gln (Gln468 in AaNGT), AaNGT can utilize the GlcN and the AaNGTQ468A mutant exhibited no increased utilization of UDPGlcN [24]. We hypothesized that the amino acids around the conserved Gln also affected the molecular mechanism of NGT recognizing sugar donors. Thus, further work is necessary to explore the sugar donor specificities of these two novel NGT isoforms to broaden the nucleotideactivated monosaccharide patterns utilized by the NGT family.
5.2. Protein sequence alignments Sequence comparisons and database searches were accomplished with BLAST programs (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The amino acid sequence of BtNGT, which is one of the closest homologs of ApNGT, was exported from the NCBI database. Protein sequences of ApNGT (accession number ALC78880.1), AaNGT (accession number ALC78880.1), KkNGT (accession number WP_015433617.1) and BtNGT (accession number CRZ19328.1) were retrieved from the NCBI database, and a structure-aided multiple sequence alignment was performed using BioXM. 5.3. Cloning, expression, and purification Four NGT genes were synthesized and cloned into pET45b plasmid by Genscript Bio-Technologies. All open reading frames were in frame with the C-terminal hexahistidine tag and the plasmid was transformed into E. coli BL21 (DE3) chemically competent cells (TransGen Biotech) for protein expression. The plasmid-harboring E. coli cells were grown at 37 °C in LB medium (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl) containing 100 μg/mL ampicillin and induced with IPTG when OD600 reached 0.8 at 16 °C. After 16 h of incubation, cells were harvested by centrifugation at 12,000 rpm for 10 min at 4 °C, and disrupted using sonication in lysis buffer (10 mM Phosphate Buffer pH 7.4, 137 mM NaCl and 2.7 mM KCl). Cell supernatants were obtained by centrifugation at 12,000 rpm for 30 min at 4 °C. Subsequently the recombinant proteins were purified with Ni-NTA affinity column, which was pre-equilibrated with binding buffer (10 mM Phosphate Buffer pH 7.4, 137 mM NaCl and 2.7 mM KCl). The column was washed with 30 mL of binding buffer containing 20 mM imidazole, and the purified proteins were eluted with the elution buffer (10 mM Phosphate Buffer pH 7.4, 137 mM NaCl, 2.7 mM KCl and 200 mM imidazole). The fractions containing the desired protein were pooled and concentrated using an Amicon Ultracel 10-kDa filter unit (Merck Millipore Ltd.). The purified protein was detected by SDS-PAGE and quantified by a BCA protein detection kit (Thermo Scientific). 30–40 mg of each purified NGT was obtained from 1 L of cell culture. 5.4. In vitro enzyme assays of synthetic peptides The enzymatic activity of NGT was evaluated using different peptide receptors with 0.5 μg of purified enzyme in a 20 μL system containing 1 mM receptor peptide labeled with carboxymethylrhodamine (TAMRA) and 10 mM sugar donor. Glycosylation reactions were performed in PBS buffer, pH 8.0. The glycosylation reactions were incubated at the optimum temperature of each enzyme for the same time, then boiled for 10 min and centrifuged. Then the supernatants were subjected onto reversed-phase high performance liquid chromatography (RP-HPLC, Phenomenex Aeris PEPTIDE 3.6 μm XB-C18, 250 × 4.6 mm column) and eluted with a gradient of 5–65% ACN and detected by TAMRA (excitation wavelength: 542 nm, emission wavelength: 568 nm). The glycosylated peptide was collected and further characterized by mass spectrometry (MS) using Shimadzu LC/MS-ITTOF (ion trap and time-of-flight mass spectrometer).
4. Conclusions In summary, we have purified and characterized NGT isoforms from various bacterial species. Through in vitro glycosylation experiments, we found that each NGT preferred different peptide receptors for glycosylation, together enhancing the receptor selections of the NGT family. NGT should provide wide application prospects to generate a variety of therapeutic glycopeptides or glycoproteins with natural or unnatural N-glycans.
5.5. Mass spectrometry analyses All sample analyses were performed by mass spectrometry (MS) using Shimadzu LC/MS-IT-TOF (ion trap and time-of-flight mass spectrometer). The instrument was calibrated in the range of 100–2000 m/z with a sodium trifluoroacetate (TFA) solution as a standard sample. The data scanned was processed by LC-MS solution software (Shimadzu, Tokyo, Japan). The parameters used were as follows: negative mode EFI voltage 3.5 kV, positive mode EFI voltage 4.5 kV, CDL temperature 200 °C, nebulizer gas flow rate 1.5 L/min, 20AB pump with a flow rate of 0.2 mL/min. The samples of in vitro enzymatic assays were diluted
5. Material and methods 5.1. Material UDP-Glc and UDP-Gal were purchased from Sigma-Aldrich (St. Louis, MO, USA). GDP-Glc was kindly provided by Dr. Guofeng Gu. All the synthetic peptides were purchased from Genscript BioTechnologies. 86
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100 times, and 5 μL were injected and analyzed in positive ion mode. The mass spectrum was scanned in the m/z range from 500 to 2000.
[10] Y. Kaneko, F. Nimmerjahn, J.V. Ravetch, Science 313 (2006) 670. [11] P.M. Rudd, T. Elliott, P. Cresswell, I.A. Wilson, R.A. Dwek, Science 291 (2001) 2370–2376. [12] M. Aebi, R. Bernasconi, S. Clerc, M. Molinari, Trends Biochem. Sci. 35 (2010) 74–82. [13] A. Helenius, M. Aebi, Annu. Rev. Biochem. 73 (2004) 1019–1049. [14] A. Dell, A. Galadari, F. Sastre, P. Hitchen, Int. J. Microbiol. 2010 (2010) (2010) 148178 2010-11-24. [15] M. Abuqarn, J. Eichler, N. Sharon, Curr. Opin. Struct. Biol. 18 (2008) 544–550. [16] P. Burda, M. Aebi, Biochim. Biophys. Acta 1426 (1999) 239–257. [17] A. Naegeli, G. Michaud, M. Schubert, C.W. Lin, C. Lizak, T. Darbre, J.L. Reymond, M. Aebi, J. Biol. Chem. 289 (2014) 24521. [18] A. Naegeli, C. Neupert, Y.Y. Fan, C.W. Lin, K. Poljak, A.M. Papini, F. Schwarz, M. Aebi, J. Biol. Chem. 289 (2014) 2170. [19] Y. Xu, Z. Wu, P. Zhang, H. Zhu, H. Zhu, Q. Song, L. Wang, F. Wang, P.G. Wang, J. Cheng, Chem. Commun. 53 (2017). [20] Q. Song, Z. Wu, Y. Fan, W. Song, P. Zhang, L. Wang, F. Wang, Y. Xu, P.G. Wang, J. Cheng, J. Biol. Chem. 292 (2017) 8856. [21] K.A. Rempe, L.A. Spruce, E.A. Porsch, S.H. Seeholzer, N. Nørskov-Lauritsen, J.W.S. Geme, 6 (2015) e01206-01215. [22] F. Schwarz, Y.Y. Fan, M. Schubert, M. Aebi, J. Biol. Chem. 286 (2011) 35267–35274. [23] J.V. Lomino, A. Naegeli, J. Orwenyo, M.N. Amin, M. Aebi, L.X. Wang, Bioorg. Med. Chem. 21 (2013) 2262–2270. [24] Y. Kong, J. Li, X. Hu, Y. Wang, Q. Meng, G. Gu, P.G. Wang, M. Chen, Carbohydr. Res. 462 (2018) 7–12. [25] W. Kightlinger, L. Lin, M. Rosztoczy, W. Li, M.P. Delisa, M. Mrksich, M.C. Jewett, Nat. Chem. Biol. 14 (2018). [26] A. Naegeli, G. Michaud, M. Schubert, C.W. Lin, C. Lizak, T. Darbre, J.L. Reymond, M. Aebi, J. Biol. Chem. 289 (2014) 24521–24532.
Acknowledgments This work was supported by the National Natural Science Foundation of China (grant numbers 31770997 and 31500648). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.carres.2018.12.016. References [1] A. Varki, R.D. Cummings, J.D. Esko, H.H. Freeze, P. Stanley, C.R. Bertozzi, G.W. Hart, M.E. Etzler, Cold Spring Harbor Laboratory Press, (2009). [2] A. Varki, Essentials of Glycobiology, second ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2009. [3] G.W. Hart, R.J. Copeland, Cell 143 (2010) 672–676. [4] D. C, L. K, J. E, Glycobiology 20 (2010) 1065–1076. [5] J.D. Marth, P.K. Grewal, Nat. Rev. Immunol. 8 (2008) 874. [6] R. Jefferis, Nat. Rev. Drug Discov. 8 (2009) 226–234. [7] R. A, H. H, N. S, Biochim. Biophys. Acta 1473 (1999) 4–8. [8] C. Li, L.-X. Wang, Chem. Rev. 118 (2018) 8359–8413. [9] N. Sharon, H. Lis, Essays Biochem. 30 (1995) 59–75.
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Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Probing peptide substrate specificities of N-glycosyltranferase isoforms from different bacterial species
T
Qingyun Meng, Kun Li, Yongheng Rong, Qizheng Wu, Xunlian Zhang, Yun Kong∗, Min Chen∗∗ The State Key Laboratory of Microbial Technology, National Glycoengineering Research Center, Shandong University, Qingdao, Shandong, 266237, China
ARTICLE INFO
ABSTRACT
Keywords: N-glycosyltransferase Peptide Substrate specificities RP-HPLC Mass spectrometry
N-glycosyltransferase (NGT) is responsible for transferring hexose monosaccharides to the asparagine side chain of proteins and polypeptides in the consensus sequon (N-(X≠P)-T/S) with nucleotide-activated sugars as donor substrates. Here, we expressed and purified four different N-glycosyltransferases derived from diverse bacteria, including Actinobacillus pleuropneumoniae, Aggregatibacter aphrophilus, Kingella kingae and Bibersteinia trehalosi, and measured their catalytic activities of four synthesized peptides via in vitro glycosylation assays. RP-HPLC and mass spectrometry were used to identify and quantify the glycopeptide formation by distinct NGT isoforms. We then analyzed and compared the glycosylation efficiencies of different peptides for these four NGT isoforms, which showed distinct substrate selectivities. We sought to probe peptide specificities among various NGT isoforms, which could broaden the application of NGT-catalyzed N-glycosylation of a variety of therapeutic proteins.
1. Introduction N-linked protein glycosylation is a universal post-translational protein modification in all eukaryotes [1], some bacteria and many archaea [2–6]. More than half of the proteins are N-glycosylated, including structural proteins, carrier proteins, enzymes, immunoglobulins, hormones and lectins [7,8]. N-linked glycosylation plays an important physiological role in organisms, such as cell-cell recognition [9], signal transduction, fertilization, development and differentiation, regulating hormone levels and modulating the nervous and immune system [10,11]. In the secretory pathway of eukaryotic organisms, N-linked protein glycosylation directs correct folding of proteins, including recognizes and degrades misfolded proteins [12,13]. In classical N-linked glycosylation, the assembled oligosaccharide is transferred from the lipid carrier oligosaccharide donor onto the asparagine residue of the acceptor proteins. The essential enzyme of this pathway is the oligosaccharyltransferase (OST/PglB) locating in endoplasmic reticulum (in eukaryotes) or periplasm (in bacteria) [14–16]. Recently, a cytoplasmic soluble N-glycosyltransferase (NGT) has been described in some bacteria, which uses nucleotide-activated hexose monosaccharides as donors to glycosylate the same consensus sequon N-X-(T/S) (where X≠ Pro) as eukaryotic OST [17–19]. The N-glycosyltransferase from Actinobacillus pleuropneumoniae
∗
(ApNGT) is the best characterized NGT in enzymatic properties [17,18]. ApNGT is capable to modify polypeptides containing consensus sequons (N-X(X≠P)-T/S), and could also glycosylate the peptide with other amino acids (Ala, Gly, Val and Asp) at the +2 position in low efficiency [18,20,21]. Compared to the -NXS-sequence context, ApNGT also has a 10-fold advantage over the -NXT-sequence [17]. In vitro, the substrate of ApNGT is preferably a peptide or a long polypeptide other than a folded protein substrate [22]. Compared with the complex OST-mediated N-glycosyaltion pathway, NGT is more likely to be utilized to add N-linked glycans onto recombinant proteins in vitro. A novel approach using NGT coupled with the transglycosylation activities of endoglycosidases has been utilized to produce peptides bearing different types of N-glycans [20,23,24]. Thus, more NGT isoforms with distinct donor or substrate specificities are needed to be identified and probed to promote the application of NGTs in the future. In this study, the NGT isoforms from Kingella kingae (KkNGT), and Bibersteinia trehalosi (BtNGT) were characterized and compared with ApNGT and AaNGT from Aggregatibacter aphrophilus [24]. We reported and demonstrated that two novel Nglycosyltransferases, KkNGT and BtNGT, were capable of transferring glucose onto synthetic peptides in vitro. Furthermore, we analyzed the substrate specificities of these four enzymes by testing several peptides using RP-HPLC and mass spectrometry, and explored the substrate
Corresponding author. Corresponding author. E-mail addresses: [email protected] (Y. Kong), [email protected] (M. Chen).
∗∗
https://doi.org/10.1016/j.carres.2018.12.016 Received 31 October 2018; Received in revised form 27 December 2018; Accepted 28 December 2018 Available online 30 December 2018 0008-6215/ © 2018 Elsevier Ltd. All rights reserved.
Carbohydrate Research 473 (2019) 82–87
Q. Meng et al.
Fig. 1. Sequence alignment of four NGT sequences. Complete alignment of four NGT sequences (from A. pleuropneumoniae, A. aphrophilus, B. trehalosi and K. kingae). The alignment was performed using BioXM. Coloring indicates conservation of residues (from white, not conserved, to black, fully conserved).
selectivities for each enzyme for the application in future.
phase high performance liquid chromatography (RP-HPLC) analysis was carried out using a C18 column. Compared with the negative control without any enzyme added, the NGT-glycosylated peptide was observed as a new peak on the chromatogram with a shifted retention time (Fig. 2A). The eluted product was collected and subsequently confirmed by LC/MS-IT-TOF analysis based on molecular weight (Fig. 2B). The reaction of the four enzymes with the short peptide DANYTK showed a product peak at 14.5 min and the substrate peak at 15 min via RP-HPLC analysis. Additionally, from the MS spectrum, the [M+2H]2+ product peak demonstrated a m/z of 643.27, which is in good agreement with the calculated molecular weight of TAMRA-DANYTK-Glc of 1284.55 Da. Thus, all four NGTs showed glycosylation activities through RP-HPLC and MS analyses (Fig. 2). We next used HPLC-based analyses to optimize reaction conditions of BtNGT and KkNGT, such as pH, temperature and ionic strength of the buffer. We found that the metal chelating agent, EDTA (20 mM), had little influence on reaction rate of both BtNGT and KkNGT (Figs. S2A and S3A), which indicated that neither BtNGT nor KkNGT was a metalloenzyme. BtNGT exhibited better tolerance for Na+ and Mg2+, but was strongly inhibited by Ca2+, Mn2+, Cu2+ and Zn2+ (Fig. S2A), while KkNGT exhibited better tolerance for Ca2+, Mn2+, Mg2+, Zn2+ and Cu2+ (Fig. S3A). The optimum temperature of BtNGT was found to be 20 °C (Fig. S2B) and the optimum pH for the enzyme was 8.0 in the PBS buffer (Fig. S2C), which was similar to ApNGT. The optimum temperature of KkNGT was 40 °C and the three buffers, PBS, HEPES and
2. Results 2.1. Identification and purification of NGTs According to the sequence alignment results, KkNGT, and BtNGT are shown to be close homologs of ApNGT and AaNGT (Fig. 1), and KkNGT has been reported as a functional glycosyltransferase in vivo [21]. In our previous work, we have expressed and characterized AaNGT as a novel N-glycosyltransferase with different catalytic activities from ApNGT [24]. Among all four NGTs, the sugar donor binding site (Q469 in ApNGT, Q468 in AaNGT, Q471 in BtNGT and Q474 in KkNGT) is conserved, but some differences exist in amino acid residues around this site, which might affect their donor specificities. In this study, we expressed and purified BtNGT and KkNGT from Escherichia coli and obtained yields of about 30–40 mg from 1 L of cell culture (Fig. S1). 2.2. Recombinant NGTs showed glycosyltransferase activity in vitro To test the activity of these glycosyltransferases, we incubated the purified enzymes with UDP-Glc and the hexapeptide DANYTK, which was labeled at the N-terminus with the fluorescent dye TAMRA. In vitro glycosylation reactions were performed in PBS buffer, pH 8.0. Reversed 83
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Fig. 2. HPLC and Mass spectrometry analysis of NGT peptide receptor selectivities. A. The purified recombinant NGTs and UDP-Glc were incubated with different peptide receptors in 20 μL PBS buffer, pH 8.0, under optimum temperatures for 30 min, and the reaction mixture was boiled for 5 min, of which 5 μL was added to 45 μL of 0.1% TFA. After centrifugation, the naked peptide and glycosylated peptide were separated by RP-HPLC and quantified by the intensity of TAMRA. B. The purified four recombinant NGTs were incubated with UDP-Glc and different synthetic peptides at optimum temperatures in a 20 μL system. The mixture was boiled for 5 min, then centrifuged at 12000 rpm for 10 min 1 μL sample was taken at 30 min and diluted with 99 μL of 0.1% FA. 2 μL of the supernatant was analyzed by MS. 2 + represents [M+2H] 2 +.
which is two amino acid residues longer showed a slightly lower efficiency in the BtNGT reaction (Fig. 3C and D). But, AaNGT preferred the longer peptide (13mer) than the shorter one (11mer). Our results showed that the residues beside the N-glycosylation sequon, even in −5 or +7 position also affected the catalytic efficiencies of NGTs.
Tris, showed relatively higher enzyme activity when pH was 8.0 (Figs. S3B and C). Therefore, PBS buffer, pH 8.0, without salt was used for all further experiments. 2.3. The comparison of in vitro catalytic efficiencies with different NGT isoforms
2.4. Effects of C-terminal truncation of BtNGT on utilizing different sugar donors
In the assay, we found that the all four NGTs showed great differences in catalytic efficiencies in vitro. Under optimum conditions, AaNGT was capable of glycosylating most of the DANYTK peptide after 30 min of incubation, while few glycopeptide (approximately 16% of peptide) could be detected in the ApNGT reaction. In addition, the glycosylation efficiency of KkNGT was lower (approximately 5%), while the activity of BtNGT is slightly higher (about 29%) than ApNGT (Fig. 3A). The residues within and surrounding the N-glycosylation sequon were reported to affect the catalytic efficiency of NGTs [20]. Therefore, we analyzed more peptides to examine their potentials as NGT substrates. After 30 min incubation, the glycosylation efficiencies of the same peptide were compared for different enzymes (Fig. S4). The GGNWTT peptide has been reported to be the best preferred short peptide substrate for several NGTs via screening the peptide substrate microarray [25], thus we have also compared its glycosylation with our four NGTs. We found that KkNGT and AaNGT has a relatively low catalytic efficiency in vitro, about 50%, and the catalytic efficiency of ApNGT and BtNGT is similar, about 80% (Fig. 3B). We have reported the WPAVGNCSSALRW peptide derived from human Hemopexin was catalyzed more efficiently than the usually used DANYTK peptide by both ApNGT and AaNGT [24]. Thus, in this work we also tested two long peptides, WPAVGNCTSALRW (13mer) and PAVGNCTSALR (11mer), which have a T189S mutation to optimize the substrate utilization. The 11mer peptide can be almost completely glycosylated in 30 min by ApNGT, BtNGT and KkNGT, whereas the 13mer peptide
In BtNGT and KkNGT, there are about 70 extra amino acids in the Cterminal, which were not conserved in all NGTs. To identify the function of this domain, we further expressed and purified the protein fragment corresponding to the N-terminus amino acids 1–620 of BtNGT (referred to as BtNGTct) as a soluble protein with a His tag at C-terminal. The specificities of sugar donors for BtNGT and BtNGTct were examined using the TAMRA-DANYTK peptide and three different nucleotide-activated monosaccharides, including UDP-Glc, UDP-Gal and GDP-Glc. The reaction products were detected by RP-HPLC. The reactions of BtNGT and BtNGTct using nucleotide-activated hexose showed a product peak at about 14 min and a substrate peak at about 15 min (Fig. 4). Both BtNGT and BtNGTct were able to utilize the UDP-Glc (Fig. 4A), UDP-Gal (Fig. 4B) and GDP-Glc (Fig. 4C) in vitro glycosylation assays. Furthermore, the glycosylation rate of BtNGTct (Fig. 4) using UDP-Glc and GDP-Glc as sugar donors was even higher than that of BtNGT. The removal of C-terminal amino acids did not affect the glycosyltransferase activity and the recognition of sugar donors, which indicated these residues might not be necessary for their catalytic activities. 3. Discussion Cytoplasmic N-glycosyltransferase (NGT) transferring a single monosaccharide to the N-X-T/S acceptor sequence motif provides a 84
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Fig. 3. For the same peptide receptor, the conversion rate of each enzyme. After centrifugation of the reaction mixture, the supernatant was analyzed by RP-HPLC, and the peptide was detected by carboxymethyltetramethylrhodamine (TAMRA) (excitation wavelength: 542 nm, emission wavelength: 568 nm). Percent conversion was calculated from the peak areas of the naked and glycosylated peptides. All trials were conducted in triplicate.
Fig. 4. HPLC analysis of the specificities of sugar donors for BtNGT and BtNGTct. The reaction in a total volume of 10 μL containing different nucleotideactivated monosaccharides (A: UDP-Glc; B: UDP-Gal; C: GDP-Glc), the TAMRA-DANYTK peptide and BtNGT or BtNGTct was conducted in PBS buffer (pH 8.0, 100 mM) at 20 °C for 16 h. The mixture was quenched by boiling for 10 min and then added with 40 μL 0.1%TFA. After centrifugation, the naked peptide and glycosylated peptide were separated by RP-HPLC and quantified by the intensity of TAMRA. The naked peptide was used as control.
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powerful tool to produce novel N-glycosylation modification in therapeutic proteins. Our previous work on the N-glycosyltransferase from A. aphrophilus (named AaNGT) has provided important information of the enzyme properties of NGTs. In this work, we investigated two more ApNGT and AaNGT homologues derived from other species, including Kingella kingae (KkNGT) and Bibersteinia trehalosi (BtNGT), for further studies of the NGT family. The recombinant BtNGT and KkNGT have the activity to transfer a single hexose moiety to peptides in vitro and shares similar enzymatic properties with ApNGT and AaNGT, such as buffer and pH [26]. But there are also some differences of enzymatic properties among four NGT isoforms. BtNGT showed lower optimum temperature (20 °C), while KkNGT exhibited higher optimum temperature (40 °C) than AaNGT (30 °C) and ApNGT (37 °C). BtNGT was sensitive to metal ions and just tolerant of Na+ and Mg2+, while KkNGT exhibited better tolerance for tested metal ions. The diversity of the enzymatic properties of different NGTs provide more application in some diverse conditions. The peptide substrate specificities and nucleotide-activated monosaccharide selectivities of AaNGT have been analyzed in our previous work [24]. The KkNGT and BtNGT were found to be functional glycosyltransferases in vitro, but the turnover rates of several peptides were widely different. For example, the long peptide derived from human Hemopexin, PAVGNCTSALR, could be almost completely glycosylated in 30 min by BtNGT, whereas the WPAVGNCTSALRW peptide showed only a little glycosylated product. These results indicate that NGT enzymes have quite different preferences for amino acids residues surrounding the N-glycosylation site, even these residues located at −5 or +7 position. It is demonstrated that the receptor selectivities of these four NGT enzymes are similar but not identical, which broadens the peptide selection patterns of the NGT family. The diverse NGT isoforms identified and characterized might contribute to the application of NGT in the glycosylation of distinct therapeutic glycoproteins. In classical N-glycosylation pathway, GlcNAc is the first monosacharide residue linked to the asparagines residues of proteins [8]. But the direct transfer of a single GlcNAc moiety to produce asparagineslinked GlcNAc (N-GlcNAc) glycan has not been found. AaNGT and ApNGTQ469A mutant have been reported to transfer GlcN and produce N-GlcNAc glycan by coupling with GlmA [20,24]. Without the mutation in the conserved Gln (Gln468 in AaNGT), AaNGT can utilize the GlcN and the AaNGTQ468A mutant exhibited no increased utilization of UDPGlcN [24]. We hypothesized that the amino acids around the conserved Gln also affected the molecular mechanism of NGT recognizing sugar donors. Thus, further work is necessary to explore the sugar donor specificities of these two novel NGT isoforms to broaden the nucleotideactivated monosaccharide patterns utilized by the NGT family.
5.2. Protein sequence alignments Sequence comparisons and database searches were accomplished with BLAST programs (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The amino acid sequence of BtNGT, which is one of the closest homologs of ApNGT, was exported from the NCBI database. Protein sequences of ApNGT (accession number ALC78880.1), AaNGT (accession number ALC78880.1), KkNGT (accession number WP_015433617.1) and BtNGT (accession number CRZ19328.1) were retrieved from the NCBI database, and a structure-aided multiple sequence alignment was performed using BioXM. 5.3. Cloning, expression, and purification Four NGT genes were synthesized and cloned into pET45b plasmid by Genscript Bio-Technologies. All open reading frames were in frame with the C-terminal hexahistidine tag and the plasmid was transformed into E. coli BL21 (DE3) chemically competent cells (TransGen Biotech) for protein expression. The plasmid-harboring E. coli cells were grown at 37 °C in LB medium (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl) containing 100 μg/mL ampicillin and induced with IPTG when OD600 reached 0.8 at 16 °C. After 16 h of incubation, cells were harvested by centrifugation at 12,000 rpm for 10 min at 4 °C, and disrupted using sonication in lysis buffer (10 mM Phosphate Buffer pH 7.4, 137 mM NaCl and 2.7 mM KCl). Cell supernatants were obtained by centrifugation at 12,000 rpm for 30 min at 4 °C. Subsequently the recombinant proteins were purified with Ni-NTA affinity column, which was pre-equilibrated with binding buffer (10 mM Phosphate Buffer pH 7.4, 137 mM NaCl and 2.7 mM KCl). The column was washed with 30 mL of binding buffer containing 20 mM imidazole, and the purified proteins were eluted with the elution buffer (10 mM Phosphate Buffer pH 7.4, 137 mM NaCl, 2.7 mM KCl and 200 mM imidazole). The fractions containing the desired protein were pooled and concentrated using an Amicon Ultracel 10-kDa filter unit (Merck Millipore Ltd.). The purified protein was detected by SDS-PAGE and quantified by a BCA protein detection kit (Thermo Scientific). 30–40 mg of each purified NGT was obtained from 1 L of cell culture. 5.4. In vitro enzyme assays of synthetic peptides The enzymatic activity of NGT was evaluated using different peptide receptors with 0.5 μg of purified enzyme in a 20 μL system containing 1 mM receptor peptide labeled with carboxymethylrhodamine (TAMRA) and 10 mM sugar donor. Glycosylation reactions were performed in PBS buffer, pH 8.0. The glycosylation reactions were incubated at the optimum temperature of each enzyme for the same time, then boiled for 10 min and centrifuged. Then the supernatants were subjected onto reversed-phase high performance liquid chromatography (RP-HPLC, Phenomenex Aeris PEPTIDE 3.6 μm XB-C18, 250 × 4.6 mm column) and eluted with a gradient of 5–65% ACN and detected by TAMRA (excitation wavelength: 542 nm, emission wavelength: 568 nm). The glycosylated peptide was collected and further characterized by mass spectrometry (MS) using Shimadzu LC/MS-ITTOF (ion trap and time-of-flight mass spectrometer).
4. Conclusions In summary, we have purified and characterized NGT isoforms from various bacterial species. Through in vitro glycosylation experiments, we found that each NGT preferred different peptide receptors for glycosylation, together enhancing the receptor selections of the NGT family. NGT should provide wide application prospects to generate a variety of therapeutic glycopeptides or glycoproteins with natural or unnatural N-glycans.
5.5. Mass spectrometry analyses All sample analyses were performed by mass spectrometry (MS) using Shimadzu LC/MS-IT-TOF (ion trap and time-of-flight mass spectrometer). The instrument was calibrated in the range of 100–2000 m/z with a sodium trifluoroacetate (TFA) solution as a standard sample. The data scanned was processed by LC-MS solution software (Shimadzu, Tokyo, Japan). The parameters used were as follows: negative mode EFI voltage 3.5 kV, positive mode EFI voltage 4.5 kV, CDL temperature 200 °C, nebulizer gas flow rate 1.5 L/min, 20AB pump with a flow rate of 0.2 mL/min. The samples of in vitro enzymatic assays were diluted
5. Material and methods 5.1. Material UDP-Glc and UDP-Gal were purchased from Sigma-Aldrich (St. Louis, MO, USA). GDP-Glc was kindly provided by Dr. Guofeng Gu. All the synthetic peptides were purchased from Genscript BioTechnologies. 86
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100 times, and 5 μL were injected and analyzed in positive ion mode. The mass spectrum was scanned in the m/z range from 500 to 2000.
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