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The effect of N-glycosylation on the expression of the tetanus toxin fragment C in Pichia pastoris
Protein Expression and Purification 166 (2020) 105503
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The effect of N-glycosylation on the expression of the tetanus toxin fragment C in Pichia pastoris
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Nan Wanga, Kevin Yueju Wangb, Fangfang Xua, GangQiang Lia, DeHu Liua,∗ a b
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China Department of Natural Sciences, Northeastern State University, Broken Arrow, OK, 74014, USA
A B S T R A C T
The N-glycosylation process that occurs in the Pichia pastoris protein expression system can have a significant effect on the yield of heterologous glycoproteins secreted from the yeast. The basis of the effect of N-glycosylation on yield, however, has not been elucidated. In order to investigate the effect of N-glycosylation on heterologous protein production, site-directed mutation was performed on five potential N-glycosylation sites of the tetanus toxin fragment C (TetC). Unaltered TetC (wild-TetC) and eight mutants, in which different numbers and locations of N-glycosylation sites were altered, were expressed in P. pastoris GS115. The recombinant target proteins presented different levels of N-glycosylation. The wild Tet-C and 4 mutations sites of putative N-glycosylation (4Gly mutant: N280Q) had the highest level of secreted protein, while 1 mutation of putative N-glycosylation sites (1Gly mutant: N39/64/85/205Q) had the highest level of intracellular, non-secreted heterologous protein. Reducing the number of native N-glycosylation sites decreased the level of glycosylation, as well as the level of secretion. Introduction of a Nglycosylation site at position 320, however, also reduced the level of expression and secretion of recombinant protein. These results indicate that the number and location of N-glycosylation sites jointly have an effect on the expression and secretion of heterologous glycoproteins in P. pastoris.
1. Introduction Pichia pastoris is one of the most common eukaryotic organisms used for the expression of heterologous proteins [1,2]. In most cases, the recombinant proteins need to undergo post-translational modifications prior to secretion. Some heterologous proteins, however, do not migrate into the transport and secretion pathway, and are thus eventually degraded. This drastically limits the production of the target protein and reduces or eliminates the potential for industrial application. This is often a major problem when using P. pastoris as an expression system. A recent study found that the process of N-glycosylation has a significant effect on the level of heterologous glycoproteins that are secreted [3]. N-glycosylation is one of the most important post-translational modifications that occurs in yeast expression systems and plays a variety of functions related to protein secretion, protein folding, stability, antigenicity, enzyme activity, etc [4–7]. A similar secretory pathway for glycoproteins is shared in eukaryotic cells from a variety of species [8]. The nascent proteins are introduced in to ER lumen via the use of a signal peptide, where a glycan (Glc3Man9GlcNAc2) is transferred to an Asn residue wherever an N-glycosylation consensus sequence Asn-Xxx-Ser/Thr is present in the nascent protein sequence. The modified glycoprotein then folds with the aid of ER-resident enzymes and chaperones (e.g. Glucosidase, UDP-glucose:glycoprotein glucosyltransferase (UGGT), calreticulin,
calnexin, etc). Properly modified and folded glycoproteins subsequently exit the ER and enter the Golgi apparatus where glycosylation continues. The correctly modified and folded proteins are then successfully secreted outside of the cell. In contrast, the presence of misfolded glycoproteins activates the “unfolded protein response (UPR) and enter ERAD or ER-phagy and are finally degraded in the cytoplasm by proteasomes. This degradation process is referred to as the N-glycan-dependent ER quality control system [8–11]. Most of the information on the N-glycan-dependent ER quality control system has been derived from studies on Saccharomyces cerevisiae or mammalian cells, and many aspects of this system have not been studied in the yeast, P. pastoris. For example, the P. pastoris genome contains the homologous gene of UGGT1, which acts as a central component of the glycoprotein quality control system in mammalian cells [12], but UGGT1 has not yet been identified in S. cerevisiae [13]. Additionally, the self-splicing of HAC1 mRNA, which is the marker transcription factor of UPR, is induced by ER stress in S. cerevisiae and mammalian cells, but that event is constitutive and independent of ER stress in P. pastoris [14]. The two yeast species also have other differences related to glycoprotein quality control, e.g. N-glycosylation type [15,16], Golgi morphology [17], etc. Based on the knowledge of the effect of N-glycosylation on levels of protein secretion, some researchers have explored improving the yield of secreted heterologous glycoproteins in P. pastoris by modifying N-
∗
Corresponding author. E-mail addresses: [email protected] (N. Wang), [email protected] (K.Y. Wang), xffxff[email protected] (F. Xu), [email protected] (G. Li), [email protected] (D. Liu). https://doi.org/10.1016/j.pep.2019.105503 Received 21 March 2019; Received in revised form 22 May 2019; Accepted 20 September 2019 Available online 21 September 2019 1046-5928/ © 2019 Elsevier Inc. All rights reserved.
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glycosylation sites. The number of reports where the modification was successful in improving the yield of secreted protein have been very few, and reports have often been contradictory. The secreted level of heterologous, hydrophobic cutinase was significantly increased in both P. pastoris and S. cerevisiae when an N-glycosylation site was introduced into the N-terminal before the appearance of exposed hydrophobic stretches [18]. An ovalbumin (N292Q) that was mutated by deleting an N-glycosylation site had decreased levels of secretion, but the secretion of this protein was not increased by the addition of a N-glycosylation site close to the N-terminal of the mutant N292Q at position 168 or 236 [15]. The secretion level of a mutated elastase, D11 N, possessing an additional N-glycosylation site at D11, was lower in P. pastoris than of the secretion level of K53S or A95S mutated elastases; even though the N-glycosylation site in the D11 N mutant was closer to the N-terminal end of the protein [19]. Most studies have indicated that reducing the number of native Nglycosylation sites in a glycoprotein results in decreased levels of secretion [20–22], but the opposite results have also been reported in some studies [23–25]. Since the studies on the effect of N-glycosylation on secretion of heterologous glycoproteins in P. pichia or S. cerevisiae have been contradictory or protein-specific, it is obvious that additional studies need to be conducted in order to better understand the relationship between N-glycosylation and the level of protein secretion. The tetanus toxin Fragment C (TetC) from Clostridium tetani is a nontoxic fragment composed of 451 amino acids. TetC was expressed and secreted in S. cerevisiae and was present in two forms; a hyper-glycosylated protein (75–200 kDa) and a core-glycosylated protein (65 kDa) [26]. TetC was also expressed intracellularly in P. pastoris, with an approximate yield of 12 g/L of culture [27], but its secretion outside of the P. pastoris yeast cells has not been reported. In the present study, potential N-glycosylation sites in TeC were predicted in silico and mutated prior to use in the P. pastoris protein expression system. Unaltered TetC (wild-TetC) and eight mutant gene fragments were inserted into P. pastoris. The level of intracellular expressed proteins and secreted proteins were measured and compared among the control (wild-TetC) and eight mutants. The results of this study provide further insight into the relationship between N-glycosylation and the expression and secretion level of heterologous glycoprotein in P. pastoris.
Fig. 1. Map of the expression vector used for wild-TetC-9kG or muta-TetC9kG. AOX1 indicates the alcohol oxidase 1 promoter from P. pastoris; α-factor indicates the α-factor secretion signal from S. cerevisiae; Kex2 indicates the signal cleavage site; wild (muta)-TetC indicates the gene coding for wild (native) or mutant TetC protein; TT indicates the alcohol oxidase transcriptional terminator sequence; HIS4 indicates the histidinol dehydrogenase gene; Kan indicates the kanamycin resistance gene.
medium, and cultured for 2 days at 30 °C and 200 rpm. 2.3. Anti-TetC antibody production The TetC gene was amplified by PCR utilizing the primers: TetC-F: 5′-ATGAAAAATCTGGATTGTTGGGTTG-3′ and TetC-R: 5′-TTAATCATT TGTCCATCCTTCATC- 3′, with the wild-TetC-9kG vector serving as a template. The PCR product was cloned into the E. coli expression vector, pEASY-E2, and transformed into cloning competent cells. The TetC gene was confirmed by DNA sequencing. The expression vector was then transformed into E. coli expression competent cells, Transetta (DE3). Synthesis of the recombinant protein was induced by 1 mM IPTG and the production of the recombinant protein was confirmed SDSPAGE. Inclusion bodies were collected from the E. coli cells by breaking the bacterial cell walls ultrasonically and centrifugation of the culture at 12000 rpm at room temperature for 5 min. The inclusion bodies were dissolved in 8 M urea and utilized as an antigen. Polyclonal Anti-TetC antibody was produced in mice by Beijing Protein Innovation (Beijing, China).
2. Material and methods 2.1. Strains, vectors, and reagents E. coli cloning vector, pEASY-T, the expression vector, pEASY-E2, as well as the cloning competent cells and Transetta (DE3) competent cells were purchased from TransGen Biotech Co., Ltd. (Beijing, China). The yeast expression vector, p9K-G, and the yeast, P. pastoris (strain GS115) have been previously described [28]. All of the utilized restriction enzymes, ligase, DNA polymerase, and N-glycosidase F (PNGase F) were obtained from New England Biolabs (Ipswich, MA, USA). A yeast total Protein Extraction Kit was purchased from Sangon Biotech. All other chemicals and reagents were acquired from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China).
2.4. Prediction of glycosylation sites and site-directed mutation Glycosylation sites present in wild-TetC were predicted using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/). Based on the site predictions, the Asn in wild-TetC was mutated into Gln, as Gln has similar structural and chemical properties as Asn. An additional N-glucosylation site was added by replacing the Ile at position 320 with Thr. Various mutated TetC genes were obtained from TransGen Biotech (Beijing, China). The mutated TetC genes were inserted into the expression vector, p9K-G, and subsequently processed and inserted into P. pastoris as described above.
2.2. Construction of expression vectors and yeast transformation The TetC gene (1356bp, GenBank accession no. AF154828.1) was synthesized by Inovogen (Beijing, China). The synthesized gene was inserted into the p9K-G vector between the Xho I and Not I restriction sites to produce the wild-TetC-9kG expression vector (Fig. 1). The wildTetC-9kG expression vector was linearized with Sal I and transformed into P. pastoris GS115 by electroporation according to the manufacturer's instructions (ThermoFisher, Waltham, MA, USA). Positive transformants were identified using a selective YPD agar plates (1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar, and 0.5 mg/mL G418). The positive colonies were inoculated in 5 mL of YPD broth
2.5. Analysis of glycosylation and the expression level of the recombinant proteins The supernatants from cultures of positive colonies were collected by centrifugation at 12000 rpm for 2 min at room temperature. Glycosylation of the recombinant proteins was determined using PNGase F according to the manufacturer's protocol (NEB, CITY, USA). 2
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Both crude and PNGase F-treated supernatants were analyzed by SDS–PAGE and Western blotting [29]. The relative level of secreted recombinant proteins was determined by Enzyme Linked Immunosorbent Assay (ELISA). Accordingly, 50 μL of supernatant from the culture medium containing transformant cell lines was mixed with 50 μL sodium carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, 8 M urea, 0.1 M DTT, pH9.6), and added to 100 μL of substrate in a 96 well plate at 4 °C and incubated overnight. The cells of the 96-well plate were then washed with PBST (137 mM NaCl, 1.8 mM KH2PO4, 8 Mm Na2HPO4·12H2O, 2.7 Mm KCl, 0.05% Tween-20, pH7.4) and then incubated with a blocking solution (PBST with 1% BSA) at 37 °C with shaking for 1 h. The plates were then washed and the anti-TetC antibody solution (the antiserum was diluted 1:5000 by PBST) was added, and the plates were incubated at 37 °C s for 1 h with shaking. The plates were then washed again with buffer and a solution containing the alkaline phosphatase conjugate anti-mouse IgG (diluted 1:5000 by PBST) was added, and the plates were incubated at 37 °C for 1 h with shaking. The plates were then washed with buffer and a detection solution (1 mg/mL pNPP, 10% diethanol amine, 0.5 mM MgCl, pH9.8) was added and the plates were incubated in the dark for 15 min at room temperature. Absorbance of the reaction mixture in each well was measured at 405 nm. Relative intracellular and total expression levels were determined using cell lysate and cell suspensions treating by Yeast total Protein Extraction Kit as the substrates. ELISA was then conducted as described above.
Fig. 3. Schematic representation of N-glycosylation sites in the wild and mutant TetC proteins that were synthesized in P. pastoris.
3. Results
of the highly glycosylated form of the recombinant wild-TetC protein decreased from 55–100kDa to 50 kDa when the recombinant protein was treated with PNGase F. This result indicates that the wild-TetC glycoprotein possessed all N-linked glycosylation sites.
3.1. Prediction of N-glycosylation sites N-glycosylation sites predominate over predicted O-glycosylation sites in eukaryotic glycoproteins, so the NetNGlyc 1.0 Server was used to predict N-glycosylation sites present in the native TetC protein. A total of five N-glycosylation sites (39NSS, 64NES, 85NFT, 205NIT, and 280NIT) were predicted (Fig. 2). Four of the five N-glycosylation sites were located in the N-terminal section of the TetC protein. In order to reduce the interference of N-glycans in the secretion process, an additional N-glycosylation site was introduced in the C-terminal section at position 320 (Fig. 2). A series of mutants possessing different numbers and locations of N-glycosylation sites were generated (Fig. 3). The results of the immunoblotting (Fig. 4) indicated that the molecular mass
3.2. Effect of N-glycosylation (number and location) on the degree of Nglycosylation in the expressed TetC The degree of N-glycosylation in the mature protein produced by each of the mutant strains was assessed by examining their molecular mass in immunoblots (Fig. 5). Two forms of the wild-TetC protein were detected in the immunoblots, a broad major band of approximately 55–70 kDa and a diffuse fainter band of approximately 75–100 kDa. The broader, major band of the 4Gly (N280Q) mutant was 50–65 kDa,
Fig. 2. Three-dimensional structure of a TetC. Molecule obtained from the Protein Data Bank (http://www.pdb.org PDB ID: 1a8d) and illustrated using PyMOL. The five Asn residues (putative native N-glycosylation sites) are displayed in blue whilean introduced N-glycosylation site is indicated in red. 3
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ratio of hyper-glycosylated form in total secreted target protein increased when the extra N-glycosylation site was introduced at site 320. The sharp band that was evident in the 2Gly/2Gly protein was still present, even though this mutant possessed four N-glycosylation sites just as the 4Gly mutant, which did not produce a sharp band. The shape of the band that was observed in the immunblots of the 1Gly/2Gly mutant was similar to the band produced by the 2Gly/2Gly mutant. The secreted target protein synthesized by the 0Gly/2Gly mutant, however, could not be detected. The collective results described above indicate that the degree of N-glycosylation in the mature TetC protein is related with both the number and the location of the glycosylation sites. 3.3. Effect of N-glycosylation sites on the intercellular residual levels of TetC protein The immunoblots of cell lysates indicated that N-glycosylated target proteins were also present within cells (Fig. 6). Additionally, a slight band appeared at ~25 kDa in immunoblots made from lysates of the 2Gly, 1Gly, and 0Gly mutants, as well as the 2Gly/1Gly, 1Gly/1Gly, and 0Gly/1Gly mutants These slight bands were assumed to represent degradation products of the intracellular taget proteins. The Wild-TetC, 4Gly, and wild/1Gly strains did not exhibit this band and thus suggests that the hyper-glycosylated protein is more resistant to degradation.
Fig. 4. Immunoblot analysis of glycosylated TetC protein and TetC protein that was deglycosylated by treatment with PNGase F. Lane M, protein marker. Lane 1, secreted wild-type TetC protein. Lane 2, supernatant from the control. Lane 3, 500 units of PNGase F. Lane 4, supernatant from the control treated with PNGase F. Lane 5, wild-TetC protein treated with PNGase F.
3.4. Effect of N-glycosylation sites on the expression level of TetC
which slightly smaller than the native wild-TetC protein. The 2Gly (N39/64/85Q) mutant, however, appeared a sharp band with a molecular mass of ~52 kDa and a faint smear at about 60 kDa. The diffuse, fainter bands with higher and broader molecular mass were referred to as hyper-glycosylated forms. The 1Gly (N39/64/85/205Q) mutant was evident as a single sharp band at~52 kDa. The 0Gly mutant was also evident as a sharp band with a molecular mass of 50 kDa which corresponded with the mass of the wild-TetC protein that had been deglycosylated by treatment with PNGase F. Based on the above results, it can be concluded that the degree of N-glycosylation of the TetC protein increased with the number of N-glycosylation sites present in the protein. Importantly, various mutants with the same number of N-glycosylation sites but at different positions were found to have different degrees of N-glycosylation. An additional N-glycosylation site at position 320 was introduced into the Wild-TetC and mutated genes using sitedirected mutation. The results of the immunoblots indicate that the
In order to determine the effect of the level of N-glycosylation on the yield of expressed target protein, the secreted, intracellular, and total protein expression level of the different target proteins were compared by ELISA (Fig. 7). The wild-TetC and the 4Gly mutant had the highest level of secreted protein (Fig. 7A). The level of secreted protein decreased as the number of N-glycosylation sites decreased in the mutated protein. The 0Gly mutant had the lowest level of secreted protein among all of the mutants. The introduction of an N-glycosylation site at position 320, further reduced the level of secreted protein but otherwise followed the same trend; with secretion levels decreasing with a reduction in the number of glycosylation sites. The additional N-glycan at position 320, therefore, appeared to have an inhibitory effect on secretion. The 2Gly and1Gly/1Gly mutants had the same number of Nglycosylation sites but exhibited different levels of secretion. These results indicate that both the number and the location of N-glycosylation sites have an effect on the level of protein that is secreted. Additionally, the 1Gly mutant, with only one N-glycosylation site at
Fig. 5. Immunoblot analysis of the degree of N-glycosylation in the wild- and mutant TetC proteins. Each recombinant strain was cultured for 48 h at 30 °C and 250 rpm. Fifteen microliters of supernatant from each culture was loaded into a lane on a 12% acrylamide gel. 4
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Fig. 6. Immunoblot analysis of the intracellular target proteins of each recombinant strain. Each strain was cultured for 48 h at 30 °C and 250 rpm. The cells were then harvested by centrifugation at 12000 rpm for 2min. Each sample of cells was boiled in 6 × loading buffer for 10min to lyse the cells. After subsequent centrifugation at 12000 rpm for 2min, 15 μL of the supernatant were loaded into a lane of a 12% acrylamide gel.
demonstrated effect of introducing an N-glycosylation site on decreasing the level of secretion confirmed earlier studies indicating that specific locations of N-glycosylation sites have a significant impact on secretion. The present study provides additional insight into the function of N-glycans on secretion in P. pastoris. We also found that the effect of the introduction of a single N-glycan on the secretion of recombinant protein was not independent but rather was dependent on the number and presumably the location of other Nglycans. When a single N-glycosylation site at position 280 was present in the target protein sequence, the intracellular, residual level of protein increased but secretion levels decreased. When this single site was deleted by site-directed mutagenesis, however, the residual and secreted levels of recombinant protein both increased rather than decreased. This result was probably due to the glycan at position 280 being concealed by other glycans. As a result, it can be assumed that the N-glycans in target proteins jointly affect the secretion of recombinant protein. The level and location of N-glycosylation in the translated protein affected the total expression level and the ratio between inter- and extracellular levels of the recombinant protein. The 2Gly, 1Gly, and wild/1Gly mutants all had similar levels of total expressed protein but different ratios of inter-vs. extracellular levels of protein. The total expression levels of protein in the various mutants was also different. Whether the difference in total expression was due to differences in the rate of protein synthesis of the recombinant protein or the degradation rate, however, is not known. More specifically, it is not known if the rate of protein synthesis was different, or whether a feedback mechanism existed in the ER glycosylation quality control system that affected the rate of degradation. If the rate of degradation was different, the question arises of how do N-glycans affect the rate of degradation? Future studies are warranted and necessary in order to address this question. In summary, this paper complements the existing knowledge on the relationship between N-glycosylation and the expression and secretion of heterologous glycoproteins in P. pastoris. Additional research will be needed to determine the optimum combination of the number and location of N-glycosylation sites needed for maximum production of heterologous proteins in P. pastoris.
position 280, had the highest level of intracellular, residual protein among all of the mutant strains; even higher than the 0Gly mutant (Fig. 7B). When the N-glycosylation site at 280 was deleted from the 4Gly mutant, however, the intracellular residual level of protein in the 4Gly mutant was still higher than in the wild-TetC strain. This indicates that the native N-glycan at position 280 had an inhibitory effect on protein secretion and this effect was not independent but rather associated with the impact of other N-glycan sites. Lastly, the level of total expressed protein (combined intracellular, residual and secreted protein) in the different strains is presented in Fig. 7C. The trend of the total expression level was similar to the secreted protein levels. Total levels of expressed protein differed among the different strains. Interestingly, some strains had similar levels of secreted protein but different levels of total expressed protein, as evidenced in the 0Gly and 1Gly/1Gly mutants. These results indicate that N-glycosylation affects not only the secreted level but also the total level of expressed proteins. 4. Discussion Over the past few decades, many researchers have studied the function of N-glycans in the quality control of glycoproteins as they are processed in the ER [4,9]. Quality control in the ER is closely related to the secretion of heterologous proteins and it is known that the specific location of N-glycan has a significant effect on the secretion of recombinant protein in P. pastoris [3,15,18,19,22], and other eukaryotic cells [5,20,23]. The details of how N-glycan affects secretion, however, have not been clearly elucidated. Our current study mainly focused on the influence of the number of N-glycosylation sites on protein expression and secretion levels rather than the specific location of N-glycosylation sites. Native wild-TetC and eight other mutated genes in which the number of glycosylation sites that would be present in the translated protein were either decreased or increased, were inserted into P. pastoris and the resulting levels of expression and secretion of recombinant protein were monitored. Results indicated that increases in the number of N-glycosylation sites increased the level of N-glycosylation in the translated protein which also enhanced the yield of secreted protein. Although the level of secreted protein decreased when a N-glycosylation site was added at position 320, the overall trend still existed. At the same time, however, the 5
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Conflicts of interest The authors declare that they have no competing interests. Authors’ contributions NW- performed experiments, analyzed data, and wrote manuscript. KYW- Analyzed data, designed experiments and prepare manuscript. GL- Performed experiments, ran bioreactor, FX- Analyzed data, performed study. DL- Conceived of study, analyzed data, and wrote manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by the National Biotechnology Development Plan (2016ZX08005-004), and the Researcher Foundation of the Chinese Academy of Agricultural Sciences. References [1] M. Ahmad, M. Hirz, H. Pichler, H. Schwab, Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production, Appl. Microbiol. Biotechnol. 98 (12) (2014) 5301–5317. [2] L.M. Damasceno, H. Chung Jr., C.A. Batt, Protein secretion in Pichia pastoris and advances in protein production, Appl. Microbiol. Biotechnol. 93 (1) (2012) 31–39. [3] M. Han, X. Yu, Enhanced expression of heterologous proteins in yeast cells via the modification of N-glycosylation sites, Bioengineered 6 (2) (2015) 115–118. [4] A. Tannous, G.B. Pisoni, D.N. Hebert, M. Molinari, N-linked sugar-regulated protein folding and quality control in the ER, Semin. Cell Dev. Biol. 41 (2015) 79–89. [5] J.H. Li, W. Huang, P. Lin, B. Wu, Z.G. Fu, H.M. Shen, et al., N-linked glycosylation at Asn 152 on CD147 affects protein folding and stability: promoting tumour metastasis in hepatocellular carcinoma, Sci. Rep. 6 (2016) 35210, https://doi.org/10. 1038/srep35210. [6] D. Skropeta, The effect of individual N-glycans on enzyme activity, Bioorg. Med. Chem. 17 (7) (2009) 2645–2653. [7] N. Mitra, S. Sinha, T.N. Ramya, A. Surolia, N-linked oligosaccharides as outfitters for glycoprotein folding, form and function, Trends Biochem. Sci. 31 (3) (2006) 156–163. [8] J.J. Caramelo, A.J. Parodi, A sweet code for glycoprotein folding, FEBS Lett. 589 (22) (2015) 3379–3387. [9] Q. Wang, J. Groenendyk, M. Michalak, Glycoprotein quality control and endoplasmic reticulum stress, Molecules 20 (8) (2015) 13689–13704. [10] Y. Chen, D. Hu, R. Yabe, H. Tateno, S.Y. Qin, N. Matsumoto, et al., Role of malectin in Glc(2)Man(9)GlcNAc(2)-dependent quality control of alpha1-antitrypsin, Mol. Biol. Cell 22 (19) (2011) 3559–3570. [11] C. Galli, R. Bernasconi, T. Solda, V. Calanca, M. Molinari, Malectin participates in a backup glycoprotein quality control pathway in the mammalian ER, PLoS One 6 (1) (2011) e16304. [12] S.P. Ferris, N.S. Jaber, M. Molinari, P. Arvan, R.J. Kaufman, UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum, Mol. Biol. Cell 24 (17) (2013) 2597–2608. [13] F.S. Fernández, S.E. Trombetta, U. Hellman, A.J. Parodi, Purification to homogeneity of UDP-glucose:glycoprotein glucosyltransferase from Schizosaccharomyces pombe and apparent absence of the enzyme fro Saccharomyces cerevisiae, J. Biol. Chem. 269 (48) (1994) 30701–30706. [14] M. Guerfal, S. Ryckaert, P.P. Jacobs, P. Ameloot, K. Van Craenenbroeck, R. Derycke, et al., The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins, Microb. Cell Factories 9 (2010) 49, https://doi.org/10.1186/1475-2859-9-49. [15] K. Ito, T. Ishimaru, F. Kimura, N. Matsudomi, Importance of N-glycosylation positioning for secretion and folding of ovalbumin, Biochem. Biophys. Res. Commun. 361 (3) (2007) 725–731. [16] M. Delic, M. Valli, A.B. Graf, M. Pfeffer, D. Mattanovich, B. Gasser, The secretory pathway: exploring yeast diversity, FEMS Microbiol. Rev. 37 (6) (2013) 872–914. [17] S. Mogelsvang, N. Gomez-Ospina, J. Soderholm, B.S. Glick, L.A. Staehelin, Tomographic evidence for continuous turnover of Golgi cisternae in Pichia pastoris, Mol. Biol. Cell 14 (6) (2003) 2277–2291. [18] C.M.J. Sagt, B. Kleizen, R. Verwaal, M.D.M.D. Jong, W.H. Müller, A. Smits, et al., Introduction of an N-glycosylation site increases secretion of heterologous proteins in yeasts, Appl. Environ. Microbiol. 66 (11) (2000) 4940–4944. [19] M. Han, W. Wang, G. Jiang, X. Wang, X. Liu, H. Cao, et al., Enhanced expression of recombinant elastase in Pichia pastoris through addition of N-glycosylation sites to the propeptide, Biotechnol. Lett. 36 (12) (2014) 2467–2471. [20] K.M. Turner, L.C. Wright, T.C. Sorrell, J.T. Djordjevic, N-linked glycosylation sites affect secretion of cryptococcal phospholipase B1, irrespective of glycosylphosphatidylinositol anchoring, Biochim. Biophys. Acta 1760 (10) (2006) 1569–1579. [21] Q. Yan, X.P. Li, N.E. Tumer, N-glycosylation does not affect the catalytic activity of ricin a chain but stimulates cytotoxicity by promoting its transport out of the endoplasmic reticulum, Traffic 13 (11) (2012) 1508–1521. [22] M. Han, X. Wang, H. Ding, M. Jin, L. Yu, J. Wang, et al., The role of N-glycosylation
Fig. 7. Relative expression of recombinant protein in the Wild and various TetC mutants of P. pastoris. The secreted (A), intracellular (B) and total (C) protein expression levels produced by the various yeast strains were determined by measuring the OD405 value in an ELISA assay system. The left and right sides of the dotted line indicate the protein expression level of the target protein containing native and introduced N-glycosylation sites.
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sites in the activity, stability, and expression of the recombinant elastase expressed by Pichia pastoris, Enzym. Microb. Technol. 54 (2014) 32–37. H. Hoshida, T. Fujita, K. Cha-aim, R. Akada, N-Glycosylation deficiency enhanced heterologous production of a Bacillus licheniformis thermostable alpha-amylase in Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 97 (12) (2013) 5473–5482. P. Wang, J. Zhang, Z. Sun, Y. Chen, J.N. Liu, Glycosylation of prourokinase produced by Pichia pastoris impairs enzymatic activity but not secretion, Protein Expr. Purif. 20 (2) (2000) 179–185. C.J. Soroka, S. Xu, A. Mennone, P. Lam, J.L. Boyer, N-Glycosylation of the alpha subunit does not influence trafficking or functional activity of the human organic solute transporter alpha/beta, BMC Cell Biol. 9 (2008) 57, https://doi.org/10. 1186/1471-2121-9-57. M.A. Romanos, A.J. Makoff, N.F. Fairweather, K.M. Beesley, D.E. Slater,
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
The effect of N-glycosylation on the expression of the tetanus toxin fragment C in Pichia pastoris
T
Nan Wanga, Kevin Yueju Wangb, Fangfang Xua, GangQiang Lia, DeHu Liua,∗ a b
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China Department of Natural Sciences, Northeastern State University, Broken Arrow, OK, 74014, USA
A B S T R A C T
The N-glycosylation process that occurs in the Pichia pastoris protein expression system can have a significant effect on the yield of heterologous glycoproteins secreted from the yeast. The basis of the effect of N-glycosylation on yield, however, has not been elucidated. In order to investigate the effect of N-glycosylation on heterologous protein production, site-directed mutation was performed on five potential N-glycosylation sites of the tetanus toxin fragment C (TetC). Unaltered TetC (wild-TetC) and eight mutants, in which different numbers and locations of N-glycosylation sites were altered, were expressed in P. pastoris GS115. The recombinant target proteins presented different levels of N-glycosylation. The wild Tet-C and 4 mutations sites of putative N-glycosylation (4Gly mutant: N280Q) had the highest level of secreted protein, while 1 mutation of putative N-glycosylation sites (1Gly mutant: N39/64/85/205Q) had the highest level of intracellular, non-secreted heterologous protein. Reducing the number of native N-glycosylation sites decreased the level of glycosylation, as well as the level of secretion. Introduction of a Nglycosylation site at position 320, however, also reduced the level of expression and secretion of recombinant protein. These results indicate that the number and location of N-glycosylation sites jointly have an effect on the expression and secretion of heterologous glycoproteins in P. pastoris.
1. Introduction Pichia pastoris is one of the most common eukaryotic organisms used for the expression of heterologous proteins [1,2]. In most cases, the recombinant proteins need to undergo post-translational modifications prior to secretion. Some heterologous proteins, however, do not migrate into the transport and secretion pathway, and are thus eventually degraded. This drastically limits the production of the target protein and reduces or eliminates the potential for industrial application. This is often a major problem when using P. pastoris as an expression system. A recent study found that the process of N-glycosylation has a significant effect on the level of heterologous glycoproteins that are secreted [3]. N-glycosylation is one of the most important post-translational modifications that occurs in yeast expression systems and plays a variety of functions related to protein secretion, protein folding, stability, antigenicity, enzyme activity, etc [4–7]. A similar secretory pathway for glycoproteins is shared in eukaryotic cells from a variety of species [8]. The nascent proteins are introduced in to ER lumen via the use of a signal peptide, where a glycan (Glc3Man9GlcNAc2) is transferred to an Asn residue wherever an N-glycosylation consensus sequence Asn-Xxx-Ser/Thr is present in the nascent protein sequence. The modified glycoprotein then folds with the aid of ER-resident enzymes and chaperones (e.g. Glucosidase, UDP-glucose:glycoprotein glucosyltransferase (UGGT), calreticulin,
calnexin, etc). Properly modified and folded glycoproteins subsequently exit the ER and enter the Golgi apparatus where glycosylation continues. The correctly modified and folded proteins are then successfully secreted outside of the cell. In contrast, the presence of misfolded glycoproteins activates the “unfolded protein response (UPR) and enter ERAD or ER-phagy and are finally degraded in the cytoplasm by proteasomes. This degradation process is referred to as the N-glycan-dependent ER quality control system [8–11]. Most of the information on the N-glycan-dependent ER quality control system has been derived from studies on Saccharomyces cerevisiae or mammalian cells, and many aspects of this system have not been studied in the yeast, P. pastoris. For example, the P. pastoris genome contains the homologous gene of UGGT1, which acts as a central component of the glycoprotein quality control system in mammalian cells [12], but UGGT1 has not yet been identified in S. cerevisiae [13]. Additionally, the self-splicing of HAC1 mRNA, which is the marker transcription factor of UPR, is induced by ER stress in S. cerevisiae and mammalian cells, but that event is constitutive and independent of ER stress in P. pastoris [14]. The two yeast species also have other differences related to glycoprotein quality control, e.g. N-glycosylation type [15,16], Golgi morphology [17], etc. Based on the knowledge of the effect of N-glycosylation on levels of protein secretion, some researchers have explored improving the yield of secreted heterologous glycoproteins in P. pastoris by modifying N-
∗
Corresponding author. E-mail addresses: [email protected] (N. Wang), [email protected] (K.Y. Wang), xffxff[email protected] (F. Xu), [email protected] (G. Li), [email protected] (D. Liu). https://doi.org/10.1016/j.pep.2019.105503 Received 21 March 2019; Received in revised form 22 May 2019; Accepted 20 September 2019 Available online 21 September 2019 1046-5928/ © 2019 Elsevier Inc. All rights reserved.
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glycosylation sites. The number of reports where the modification was successful in improving the yield of secreted protein have been very few, and reports have often been contradictory. The secreted level of heterologous, hydrophobic cutinase was significantly increased in both P. pastoris and S. cerevisiae when an N-glycosylation site was introduced into the N-terminal before the appearance of exposed hydrophobic stretches [18]. An ovalbumin (N292Q) that was mutated by deleting an N-glycosylation site had decreased levels of secretion, but the secretion of this protein was not increased by the addition of a N-glycosylation site close to the N-terminal of the mutant N292Q at position 168 or 236 [15]. The secretion level of a mutated elastase, D11 N, possessing an additional N-glycosylation site at D11, was lower in P. pastoris than of the secretion level of K53S or A95S mutated elastases; even though the N-glycosylation site in the D11 N mutant was closer to the N-terminal end of the protein [19]. Most studies have indicated that reducing the number of native Nglycosylation sites in a glycoprotein results in decreased levels of secretion [20–22], but the opposite results have also been reported in some studies [23–25]. Since the studies on the effect of N-glycosylation on secretion of heterologous glycoproteins in P. pichia or S. cerevisiae have been contradictory or protein-specific, it is obvious that additional studies need to be conducted in order to better understand the relationship between N-glycosylation and the level of protein secretion. The tetanus toxin Fragment C (TetC) from Clostridium tetani is a nontoxic fragment composed of 451 amino acids. TetC was expressed and secreted in S. cerevisiae and was present in two forms; a hyper-glycosylated protein (75–200 kDa) and a core-glycosylated protein (65 kDa) [26]. TetC was also expressed intracellularly in P. pastoris, with an approximate yield of 12 g/L of culture [27], but its secretion outside of the P. pastoris yeast cells has not been reported. In the present study, potential N-glycosylation sites in TeC were predicted in silico and mutated prior to use in the P. pastoris protein expression system. Unaltered TetC (wild-TetC) and eight mutant gene fragments were inserted into P. pastoris. The level of intracellular expressed proteins and secreted proteins were measured and compared among the control (wild-TetC) and eight mutants. The results of this study provide further insight into the relationship between N-glycosylation and the expression and secretion level of heterologous glycoprotein in P. pastoris.
Fig. 1. Map of the expression vector used for wild-TetC-9kG or muta-TetC9kG. AOX1 indicates the alcohol oxidase 1 promoter from P. pastoris; α-factor indicates the α-factor secretion signal from S. cerevisiae; Kex2 indicates the signal cleavage site; wild (muta)-TetC indicates the gene coding for wild (native) or mutant TetC protein; TT indicates the alcohol oxidase transcriptional terminator sequence; HIS4 indicates the histidinol dehydrogenase gene; Kan indicates the kanamycin resistance gene.
medium, and cultured for 2 days at 30 °C and 200 rpm. 2.3. Anti-TetC antibody production The TetC gene was amplified by PCR utilizing the primers: TetC-F: 5′-ATGAAAAATCTGGATTGTTGGGTTG-3′ and TetC-R: 5′-TTAATCATT TGTCCATCCTTCATC- 3′, with the wild-TetC-9kG vector serving as a template. The PCR product was cloned into the E. coli expression vector, pEASY-E2, and transformed into cloning competent cells. The TetC gene was confirmed by DNA sequencing. The expression vector was then transformed into E. coli expression competent cells, Transetta (DE3). Synthesis of the recombinant protein was induced by 1 mM IPTG and the production of the recombinant protein was confirmed SDSPAGE. Inclusion bodies were collected from the E. coli cells by breaking the bacterial cell walls ultrasonically and centrifugation of the culture at 12000 rpm at room temperature for 5 min. The inclusion bodies were dissolved in 8 M urea and utilized as an antigen. Polyclonal Anti-TetC antibody was produced in mice by Beijing Protein Innovation (Beijing, China).
2. Material and methods 2.1. Strains, vectors, and reagents E. coli cloning vector, pEASY-T, the expression vector, pEASY-E2, as well as the cloning competent cells and Transetta (DE3) competent cells were purchased from TransGen Biotech Co., Ltd. (Beijing, China). The yeast expression vector, p9K-G, and the yeast, P. pastoris (strain GS115) have been previously described [28]. All of the utilized restriction enzymes, ligase, DNA polymerase, and N-glycosidase F (PNGase F) were obtained from New England Biolabs (Ipswich, MA, USA). A yeast total Protein Extraction Kit was purchased from Sangon Biotech. All other chemicals and reagents were acquired from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China).
2.4. Prediction of glycosylation sites and site-directed mutation Glycosylation sites present in wild-TetC were predicted using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/). Based on the site predictions, the Asn in wild-TetC was mutated into Gln, as Gln has similar structural and chemical properties as Asn. An additional N-glucosylation site was added by replacing the Ile at position 320 with Thr. Various mutated TetC genes were obtained from TransGen Biotech (Beijing, China). The mutated TetC genes were inserted into the expression vector, p9K-G, and subsequently processed and inserted into P. pastoris as described above.
2.2. Construction of expression vectors and yeast transformation The TetC gene (1356bp, GenBank accession no. AF154828.1) was synthesized by Inovogen (Beijing, China). The synthesized gene was inserted into the p9K-G vector between the Xho I and Not I restriction sites to produce the wild-TetC-9kG expression vector (Fig. 1). The wildTetC-9kG expression vector was linearized with Sal I and transformed into P. pastoris GS115 by electroporation according to the manufacturer's instructions (ThermoFisher, Waltham, MA, USA). Positive transformants were identified using a selective YPD agar plates (1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar, and 0.5 mg/mL G418). The positive colonies were inoculated in 5 mL of YPD broth
2.5. Analysis of glycosylation and the expression level of the recombinant proteins The supernatants from cultures of positive colonies were collected by centrifugation at 12000 rpm for 2 min at room temperature. Glycosylation of the recombinant proteins was determined using PNGase F according to the manufacturer's protocol (NEB, CITY, USA). 2
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Both crude and PNGase F-treated supernatants were analyzed by SDS–PAGE and Western blotting [29]. The relative level of secreted recombinant proteins was determined by Enzyme Linked Immunosorbent Assay (ELISA). Accordingly, 50 μL of supernatant from the culture medium containing transformant cell lines was mixed with 50 μL sodium carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, 8 M urea, 0.1 M DTT, pH9.6), and added to 100 μL of substrate in a 96 well plate at 4 °C and incubated overnight. The cells of the 96-well plate were then washed with PBST (137 mM NaCl, 1.8 mM KH2PO4, 8 Mm Na2HPO4·12H2O, 2.7 Mm KCl, 0.05% Tween-20, pH7.4) and then incubated with a blocking solution (PBST with 1% BSA) at 37 °C with shaking for 1 h. The plates were then washed and the anti-TetC antibody solution (the antiserum was diluted 1:5000 by PBST) was added, and the plates were incubated at 37 °C s for 1 h with shaking. The plates were then washed again with buffer and a solution containing the alkaline phosphatase conjugate anti-mouse IgG (diluted 1:5000 by PBST) was added, and the plates were incubated at 37 °C for 1 h with shaking. The plates were then washed with buffer and a detection solution (1 mg/mL pNPP, 10% diethanol amine, 0.5 mM MgCl, pH9.8) was added and the plates were incubated in the dark for 15 min at room temperature. Absorbance of the reaction mixture in each well was measured at 405 nm. Relative intracellular and total expression levels were determined using cell lysate and cell suspensions treating by Yeast total Protein Extraction Kit as the substrates. ELISA was then conducted as described above.
Fig. 3. Schematic representation of N-glycosylation sites in the wild and mutant TetC proteins that were synthesized in P. pastoris.
3. Results
of the highly glycosylated form of the recombinant wild-TetC protein decreased from 55–100kDa to 50 kDa when the recombinant protein was treated with PNGase F. This result indicates that the wild-TetC glycoprotein possessed all N-linked glycosylation sites.
3.1. Prediction of N-glycosylation sites N-glycosylation sites predominate over predicted O-glycosylation sites in eukaryotic glycoproteins, so the NetNGlyc 1.0 Server was used to predict N-glycosylation sites present in the native TetC protein. A total of five N-glycosylation sites (39NSS, 64NES, 85NFT, 205NIT, and 280NIT) were predicted (Fig. 2). Four of the five N-glycosylation sites were located in the N-terminal section of the TetC protein. In order to reduce the interference of N-glycans in the secretion process, an additional N-glycosylation site was introduced in the C-terminal section at position 320 (Fig. 2). A series of mutants possessing different numbers and locations of N-glycosylation sites were generated (Fig. 3). The results of the immunoblotting (Fig. 4) indicated that the molecular mass
3.2. Effect of N-glycosylation (number and location) on the degree of Nglycosylation in the expressed TetC The degree of N-glycosylation in the mature protein produced by each of the mutant strains was assessed by examining their molecular mass in immunoblots (Fig. 5). Two forms of the wild-TetC protein were detected in the immunoblots, a broad major band of approximately 55–70 kDa and a diffuse fainter band of approximately 75–100 kDa. The broader, major band of the 4Gly (N280Q) mutant was 50–65 kDa,
Fig. 2. Three-dimensional structure of a TetC. Molecule obtained from the Protein Data Bank (http://www.pdb.org PDB ID: 1a8d) and illustrated using PyMOL. The five Asn residues (putative native N-glycosylation sites) are displayed in blue whilean introduced N-glycosylation site is indicated in red. 3
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ratio of hyper-glycosylated form in total secreted target protein increased when the extra N-glycosylation site was introduced at site 320. The sharp band that was evident in the 2Gly/2Gly protein was still present, even though this mutant possessed four N-glycosylation sites just as the 4Gly mutant, which did not produce a sharp band. The shape of the band that was observed in the immunblots of the 1Gly/2Gly mutant was similar to the band produced by the 2Gly/2Gly mutant. The secreted target protein synthesized by the 0Gly/2Gly mutant, however, could not be detected. The collective results described above indicate that the degree of N-glycosylation in the mature TetC protein is related with both the number and the location of the glycosylation sites. 3.3. Effect of N-glycosylation sites on the intercellular residual levels of TetC protein The immunoblots of cell lysates indicated that N-glycosylated target proteins were also present within cells (Fig. 6). Additionally, a slight band appeared at ~25 kDa in immunoblots made from lysates of the 2Gly, 1Gly, and 0Gly mutants, as well as the 2Gly/1Gly, 1Gly/1Gly, and 0Gly/1Gly mutants These slight bands were assumed to represent degradation products of the intracellular taget proteins. The Wild-TetC, 4Gly, and wild/1Gly strains did not exhibit this band and thus suggests that the hyper-glycosylated protein is more resistant to degradation.
Fig. 4. Immunoblot analysis of glycosylated TetC protein and TetC protein that was deglycosylated by treatment with PNGase F. Lane M, protein marker. Lane 1, secreted wild-type TetC protein. Lane 2, supernatant from the control. Lane 3, 500 units of PNGase F. Lane 4, supernatant from the control treated with PNGase F. Lane 5, wild-TetC protein treated with PNGase F.
3.4. Effect of N-glycosylation sites on the expression level of TetC
which slightly smaller than the native wild-TetC protein. The 2Gly (N39/64/85Q) mutant, however, appeared a sharp band with a molecular mass of ~52 kDa and a faint smear at about 60 kDa. The diffuse, fainter bands with higher and broader molecular mass were referred to as hyper-glycosylated forms. The 1Gly (N39/64/85/205Q) mutant was evident as a single sharp band at~52 kDa. The 0Gly mutant was also evident as a sharp band with a molecular mass of 50 kDa which corresponded with the mass of the wild-TetC protein that had been deglycosylated by treatment with PNGase F. Based on the above results, it can be concluded that the degree of N-glycosylation of the TetC protein increased with the number of N-glycosylation sites present in the protein. Importantly, various mutants with the same number of N-glycosylation sites but at different positions were found to have different degrees of N-glycosylation. An additional N-glycosylation site at position 320 was introduced into the Wild-TetC and mutated genes using sitedirected mutation. The results of the immunoblots indicate that the
In order to determine the effect of the level of N-glycosylation on the yield of expressed target protein, the secreted, intracellular, and total protein expression level of the different target proteins were compared by ELISA (Fig. 7). The wild-TetC and the 4Gly mutant had the highest level of secreted protein (Fig. 7A). The level of secreted protein decreased as the number of N-glycosylation sites decreased in the mutated protein. The 0Gly mutant had the lowest level of secreted protein among all of the mutants. The introduction of an N-glycosylation site at position 320, further reduced the level of secreted protein but otherwise followed the same trend; with secretion levels decreasing with a reduction in the number of glycosylation sites. The additional N-glycan at position 320, therefore, appeared to have an inhibitory effect on secretion. The 2Gly and1Gly/1Gly mutants had the same number of Nglycosylation sites but exhibited different levels of secretion. These results indicate that both the number and the location of N-glycosylation sites have an effect on the level of protein that is secreted. Additionally, the 1Gly mutant, with only one N-glycosylation site at
Fig. 5. Immunoblot analysis of the degree of N-glycosylation in the wild- and mutant TetC proteins. Each recombinant strain was cultured for 48 h at 30 °C and 250 rpm. Fifteen microliters of supernatant from each culture was loaded into a lane on a 12% acrylamide gel. 4
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Fig. 6. Immunoblot analysis of the intracellular target proteins of each recombinant strain. Each strain was cultured for 48 h at 30 °C and 250 rpm. The cells were then harvested by centrifugation at 12000 rpm for 2min. Each sample of cells was boiled in 6 × loading buffer for 10min to lyse the cells. After subsequent centrifugation at 12000 rpm for 2min, 15 μL of the supernatant were loaded into a lane of a 12% acrylamide gel.
demonstrated effect of introducing an N-glycosylation site on decreasing the level of secretion confirmed earlier studies indicating that specific locations of N-glycosylation sites have a significant impact on secretion. The present study provides additional insight into the function of N-glycans on secretion in P. pastoris. We also found that the effect of the introduction of a single N-glycan on the secretion of recombinant protein was not independent but rather was dependent on the number and presumably the location of other Nglycans. When a single N-glycosylation site at position 280 was present in the target protein sequence, the intracellular, residual level of protein increased but secretion levels decreased. When this single site was deleted by site-directed mutagenesis, however, the residual and secreted levels of recombinant protein both increased rather than decreased. This result was probably due to the glycan at position 280 being concealed by other glycans. As a result, it can be assumed that the N-glycans in target proteins jointly affect the secretion of recombinant protein. The level and location of N-glycosylation in the translated protein affected the total expression level and the ratio between inter- and extracellular levels of the recombinant protein. The 2Gly, 1Gly, and wild/1Gly mutants all had similar levels of total expressed protein but different ratios of inter-vs. extracellular levels of protein. The total expression levels of protein in the various mutants was also different. Whether the difference in total expression was due to differences in the rate of protein synthesis of the recombinant protein or the degradation rate, however, is not known. More specifically, it is not known if the rate of protein synthesis was different, or whether a feedback mechanism existed in the ER glycosylation quality control system that affected the rate of degradation. If the rate of degradation was different, the question arises of how do N-glycans affect the rate of degradation? Future studies are warranted and necessary in order to address this question. In summary, this paper complements the existing knowledge on the relationship between N-glycosylation and the expression and secretion of heterologous glycoproteins in P. pastoris. Additional research will be needed to determine the optimum combination of the number and location of N-glycosylation sites needed for maximum production of heterologous proteins in P. pastoris.
position 280, had the highest level of intracellular, residual protein among all of the mutant strains; even higher than the 0Gly mutant (Fig. 7B). When the N-glycosylation site at 280 was deleted from the 4Gly mutant, however, the intracellular residual level of protein in the 4Gly mutant was still higher than in the wild-TetC strain. This indicates that the native N-glycan at position 280 had an inhibitory effect on protein secretion and this effect was not independent but rather associated with the impact of other N-glycan sites. Lastly, the level of total expressed protein (combined intracellular, residual and secreted protein) in the different strains is presented in Fig. 7C. The trend of the total expression level was similar to the secreted protein levels. Total levels of expressed protein differed among the different strains. Interestingly, some strains had similar levels of secreted protein but different levels of total expressed protein, as evidenced in the 0Gly and 1Gly/1Gly mutants. These results indicate that N-glycosylation affects not only the secreted level but also the total level of expressed proteins. 4. Discussion Over the past few decades, many researchers have studied the function of N-glycans in the quality control of glycoproteins as they are processed in the ER [4,9]. Quality control in the ER is closely related to the secretion of heterologous proteins and it is known that the specific location of N-glycan has a significant effect on the secretion of recombinant protein in P. pastoris [3,15,18,19,22], and other eukaryotic cells [5,20,23]. The details of how N-glycan affects secretion, however, have not been clearly elucidated. Our current study mainly focused on the influence of the number of N-glycosylation sites on protein expression and secretion levels rather than the specific location of N-glycosylation sites. Native wild-TetC and eight other mutated genes in which the number of glycosylation sites that would be present in the translated protein were either decreased or increased, were inserted into P. pastoris and the resulting levels of expression and secretion of recombinant protein were monitored. Results indicated that increases in the number of N-glycosylation sites increased the level of N-glycosylation in the translated protein which also enhanced the yield of secreted protein. Although the level of secreted protein decreased when a N-glycosylation site was added at position 320, the overall trend still existed. At the same time, however, the 5
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Conflicts of interest The authors declare that they have no competing interests. Authors’ contributions NW- performed experiments, analyzed data, and wrote manuscript. KYW- Analyzed data, designed experiments and prepare manuscript. GL- Performed experiments, ran bioreactor, FX- Analyzed data, performed study. DL- Conceived of study, analyzed data, and wrote manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by the National Biotechnology Development Plan (2016ZX08005-004), and the Researcher Foundation of the Chinese Academy of Agricultural Sciences. References [1] M. Ahmad, M. Hirz, H. Pichler, H. Schwab, Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production, Appl. Microbiol. Biotechnol. 98 (12) (2014) 5301–5317. [2] L.M. Damasceno, H. Chung Jr., C.A. Batt, Protein secretion in Pichia pastoris and advances in protein production, Appl. Microbiol. Biotechnol. 93 (1) (2012) 31–39. [3] M. Han, X. Yu, Enhanced expression of heterologous proteins in yeast cells via the modification of N-glycosylation sites, Bioengineered 6 (2) (2015) 115–118. [4] A. Tannous, G.B. Pisoni, D.N. Hebert, M. Molinari, N-linked sugar-regulated protein folding and quality control in the ER, Semin. Cell Dev. Biol. 41 (2015) 79–89. [5] J.H. Li, W. Huang, P. Lin, B. Wu, Z.G. Fu, H.M. Shen, et al., N-linked glycosylation at Asn 152 on CD147 affects protein folding and stability: promoting tumour metastasis in hepatocellular carcinoma, Sci. Rep. 6 (2016) 35210, https://doi.org/10. 1038/srep35210. [6] D. Skropeta, The effect of individual N-glycans on enzyme activity, Bioorg. Med. Chem. 17 (7) (2009) 2645–2653. [7] N. Mitra, S. Sinha, T.N. Ramya, A. Surolia, N-linked oligosaccharides as outfitters for glycoprotein folding, form and function, Trends Biochem. Sci. 31 (3) (2006) 156–163. [8] J.J. Caramelo, A.J. Parodi, A sweet code for glycoprotein folding, FEBS Lett. 589 (22) (2015) 3379–3387. [9] Q. Wang, J. Groenendyk, M. Michalak, Glycoprotein quality control and endoplasmic reticulum stress, Molecules 20 (8) (2015) 13689–13704. [10] Y. Chen, D. Hu, R. Yabe, H. Tateno, S.Y. Qin, N. Matsumoto, et al., Role of malectin in Glc(2)Man(9)GlcNAc(2)-dependent quality control of alpha1-antitrypsin, Mol. Biol. Cell 22 (19) (2011) 3559–3570. [11] C. Galli, R. Bernasconi, T. Solda, V. Calanca, M. Molinari, Malectin participates in a backup glycoprotein quality control pathway in the mammalian ER, PLoS One 6 (1) (2011) e16304. [12] S.P. Ferris, N.S. Jaber, M. Molinari, P. Arvan, R.J. Kaufman, UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum, Mol. Biol. Cell 24 (17) (2013) 2597–2608. [13] F.S. Fernández, S.E. Trombetta, U. Hellman, A.J. Parodi, Purification to homogeneity of UDP-glucose:glycoprotein glucosyltransferase from Schizosaccharomyces pombe and apparent absence of the enzyme fro Saccharomyces cerevisiae, J. Biol. Chem. 269 (48) (1994) 30701–30706. [14] M. Guerfal, S. Ryckaert, P.P. Jacobs, P. Ameloot, K. Van Craenenbroeck, R. Derycke, et al., The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins, Microb. Cell Factories 9 (2010) 49, https://doi.org/10.1186/1475-2859-9-49. [15] K. Ito, T. Ishimaru, F. Kimura, N. Matsudomi, Importance of N-glycosylation positioning for secretion and folding of ovalbumin, Biochem. Biophys. Res. Commun. 361 (3) (2007) 725–731. [16] M. Delic, M. Valli, A.B. Graf, M. Pfeffer, D. Mattanovich, B. Gasser, The secretory pathway: exploring yeast diversity, FEMS Microbiol. Rev. 37 (6) (2013) 872–914. [17] S. Mogelsvang, N. Gomez-Ospina, J. Soderholm, B.S. Glick, L.A. Staehelin, Tomographic evidence for continuous turnover of Golgi cisternae in Pichia pastoris, Mol. Biol. Cell 14 (6) (2003) 2277–2291. [18] C.M.J. Sagt, B. Kleizen, R. Verwaal, M.D.M.D. Jong, W.H. Müller, A. Smits, et al., Introduction of an N-glycosylation site increases secretion of heterologous proteins in yeasts, Appl. Environ. Microbiol. 66 (11) (2000) 4940–4944. [19] M. Han, W. Wang, G. Jiang, X. Wang, X. Liu, H. Cao, et al., Enhanced expression of recombinant elastase in Pichia pastoris through addition of N-glycosylation sites to the propeptide, Biotechnol. Lett. 36 (12) (2014) 2467–2471. [20] K.M. Turner, L.C. Wright, T.C. Sorrell, J.T. Djordjevic, N-linked glycosylation sites affect secretion of cryptococcal phospholipase B1, irrespective of glycosylphosphatidylinositol anchoring, Biochim. Biophys. Acta 1760 (10) (2006) 1569–1579. [21] Q. Yan, X.P. Li, N.E. Tumer, N-glycosylation does not affect the catalytic activity of ricin a chain but stimulates cytotoxicity by promoting its transport out of the endoplasmic reticulum, Traffic 13 (11) (2012) 1508–1521. [22] M. Han, X. Wang, H. Ding, M. Jin, L. Yu, J. Wang, et al., The role of N-glycosylation
Fig. 7. Relative expression of recombinant protein in the Wild and various TetC mutants of P. pastoris. The secreted (A), intracellular (B) and total (C) protein expression levels produced by the various yeast strains were determined by measuring the OD405 value in an ELISA assay system. The left and right sides of the dotted line indicate the protein expression level of the target protein containing native and introduced N-glycosylation sites.
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