Homogeneous production and characterization of recombinant N-GlcNAc-protein in Pichia pastoris
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Therapeutic glycoproteins have occupied an extremely important position in the market of biopharmaceuticals. N-Glycosylation of protein drugs facilitates them to maintain optimal conformations and affect their structural stabilities, serum half-lives and biological efficiencies. Thus homogeneous N-glycoproteins with defined N-glycans are essential in their application in clinic therapeutics. However, there still remain several obstacles to acquire homogeneous N-glycans, such as the high production costs induced by the universal utilization of mammalian cell expression systems, the non-humanized N-glycan structures and the N-glycosylation microheterogeneities between batches.
In this study, we constructed a Pichia pastoris (Komagataella phaffii) expression system producing truncated N-GlcNAc-modified recombinant proteins through introducing an ENGase isoform (Endo-T) which possesses powerful hydrolytic activities towards high-mannose type N-glycans. The results showed that the location of Endo-T in different subcellular fractions, such as Endoplasmic reticulum (ER), Golgi or cell membrane, affected their hydrolytic efficiencies. When the Endo-T was expressed in Golgi, the secreted IgG1-Fc region was efficiently produced with almost completely truncated N-glycans and the N-GlcNAc modification on the glycosite Asn297 was confirmed via Mass Spectrometry.
This strategy develops a simple glycoengineered yeast expression system to produce N-GlcNAc modified proteins, which could be further extended to different N-glycan structures. This system would provide a prospective platform for mass production of increasing novel glycoprotein drugs.
KeywordsPichia pastoris Endoglycosidase N-GlcNAc-protein Glycoprotein Homogeneous protein production
endo-beta-N-acetylglucosaminidase or endoglycosidase
buffered minimal glycerol medium
buffered minimal methanol medium
polypeptide N-acetylgalactosaminyltransferase 1
phosphate buffered saline
rich yeast medium
N-Linked glycosylation is a fundamental co- and/or posttranslational modification, regulating glycoprotein folding and functions. N-Glycosylation is evolutionarily conserved in all domains of life, including all eukaryotes, some bacteria  and many archaea . In mammalian cells, most of the membrane-bound and secreted proteins are generally N-glycosylated and involved in many essential biological processes [3, 4]. In the classical pathway of N-linked glycosylation, the assembled oligosaccharide (GlcNAc2Man9Glc3) is transferred onto the asparagine (Asn) residue in the NXS/T (X ≠ Pro) context of the polypeptides from dolichol pyrophosphate by the oligosaccharyltransferases (OST) in endoplasmic reticulum [5, 6, 7] and glycans are subsequently maturated in the Golgi compartment .
At present, therapeutic glycoproteins have occupied an increasing proportion in the market of biopharmaceuticals. Glycoprotein drugs have been widely used to fight against diverse diseases, such as pathogenic microbial invasive diseases, autoimmune disorders and cancers. It has been shown that N-glycosylation and N-glycan structures can affect the biophysical and pharmacokinetic properties of therapeutic glycoproteins [9, 10, 11]. Several novel approaches have been attempted to engineer N-glycosylation pathway to decrease the microheterogeneity of therapeutic proteins via in vitro chemoenzymatic methods or in vivo engineered expression systems [11, 12, 13, 14, 15, 16, 17, 18].
The endo-N-acetyl-β-D-glucosaminidase (endoglycosidase or ENGase) specifically cleave the diacetylchitobiose core [GlcNAc β (1–4) GlcNAc] of N-linked glycans between the two N-acetylglucosamine (GlcNAc) residues  to release an N-GlcNAc-carrying peptides/proteins and an intact oligosaccharide group . Some ENGases or mutants also have potent transglycosylation activity [21, 22, 23, 24, 25, 26] and were utilized in N-glycoprotein remodeling . Wang and collaborators used an Endo-A mutant (N171A) to glycosylate IgG1-Fc region [21, 23, 28, 29], and further used the mutants of Endo-S (D233A and D233Q) or Endo-S2 (D184M and D184Q) for full-length antibody glycosylation remodeling with three major types (complex, high-mannose, and hybrid type) of N-glycans for modulating IgG effector function [14, 22, 30]. This chemoenzymatic glycosylation method utilizing ENGases provides an efficient way to introduce complex N-glycans onto polypeptides, which was valuable for glycoprotein drug production [13, 31]. In this method, N-GlcNAc modified proteins were essential as acceptors for the production of glycoproteins with different glycans. However, the direct transfer of a single GlcNAc moiety has only been found in the modification of specific serines or threonines catalyzed by O-linked GlcNAc transferase (OGT) . Recently, N-Glycosyltransferase AaNGT and ApNGTQ469A were reported to transfer GlcN and produce N-GlcNAc glycans by coupling with GlmA [16, 33].
Pichia pastoris, which was reassigned to the genus Komagataella spp. in 1995 , is an organism commonly employed to produce a variety of active proteins [35, 36, 37] with N- and/or O-linked glycans [38, 39, 40]. The N-linked glycans of the P. pastoris-produced proteins was high-mannose type without core fucose , which leads to reduced in vivo half-life and therapeutic function. The engineered P. pastoris have been constructed to produce glycoproteins with N-glycosylation profiles similar to human [39, 42], but the products are still heterogeneous with lower yield [39, 40, 43].
In this study, we construct a P. pastoris system expressing truncated N-GlcNAc-modified recombinant proteins through introducing an ENGase isoform (Endo-T) which possesses powerful hydrolytic activities towards high-mannose type N-glycan in intracellular environment, into different subcellular fractions. We believe the application of this easy and low-cost glycoprotein synthetic method would provide a prospective platform for mass production of increasing novel glycoprotein drugs with diverse homogeneous N-glycan structures.
Expression of Endo-T on the surface of Pichia pastoris
Expression of ENGase in the ER or Golgi of Pichia pastoris
Characterization of IgG1-Fc region with N-GlcNAc
Structural conformation of N-GlcNAc IgG1-Fc
The secondary structures of IgG1-Fc regions expressed in P. pastoris were determined using far-UV circular dichroism (CD) spectroscopy (Fig. 4b). The IgG1-Fc region purified from P. pastoris WT strain and engineered P. pastoris were tested and compared. The secondary structure of the Fc fragment at 25 °C is populated primarily of beta-strands and a wavelength of 218 nm was chosen for unfolding by CD measurement . For the WT-Fc, the spectra obtained at 25 °C showed a maximum negative peak at 218 nm, which was similar with previous reports . Moreover, the CD spectrum of N-GlcNAc-Fc showed only minor differences to the WT spectrum (Fig. 4b), which was consistent with deglycosylated IgG  or aglycosylated Fc . It can be seen that the Fc fragments with truncated glycans have intact secondary and tertiary structures that are very similar to the wild-type Fc fragment, with a characteristic minimum at 218 nm.
Glycoproteins are an important class of biomolecules involved in many physiological and pathological processes. Several strategies have been developed to produce glycoproteins with homogeneous glycan structures [11, 12, 13, 14], of which ENGase-mediated N-glycan remodeling was a powerful approach to prepare defined glycoconjugates. The major limitation of this method is the difficulty to obtain N-GlcNAc proteins in large quantities. In this study, we constructed a P. pastoris expression system, which localized recombinant ENGases in the cell membrane, ER or Golgi, to produce secreted N-GlcNAc-modified proteins. Our results showed the location of ENGase in different subcellular fractions affected their hydrolytic efficiencies.
Pichia pastoris is an expression strain widely utilized to produce functional N-glycoproteins [35, 36, 37] with high yields . The expression levels of recombinant proteins in P. pastoris were even up to 10 g/L . The N-linked glycans from P. pastoris are of high mannose type without core fucose, which could be preferred as substrates by a variety of ENGase isoforms. We attempt to build up an expression system, which localized the recombinant ENGases in the cell surface membrane, ER or Golgi. As an immobilized enzyme on cell surface, the ENGase could hydrolyze glycans from N-glycoproteins in in vitro reaction system, while few deglycosylated proteins were found in the cultured medium containing methanol. When the ENGase was expressed in Golgi or ER, the secreted target glycoprotein could be efficiently deglycosylated. Fused with MNN9, the hydrolysis activity of ENGase against IgG Fc domain and GalNAc-T1 proteins is higher than fused with MNS1. It is assumed that the Endo-T preferred the microenvironment of yeast Golgi, such as the intracellular pH, as well as the glycan structure.
Human IgG1 carries a conserved N-glycan at Asn-297 of its Fc region. The presence and precise structures of this N-glycan plays an important role in determining antibody’s structure and effector functions. For example, the deglycosylated IgG1 are highly flexible and more prone to aggregation [59, 60]; removal of the core fucose from N-glycans increases the Fc’s affinity towards FcγRIIIA [14, 61, 62, 63]; the terminal α2, 6-sialylation is critical for its anti-inflammatory activity [64, 65, 66]. Fc region-containing fusion proteins are also influenced by the structure of N-glycans [67, 68, 69]. Both full length of human IgG1 and the IgG1-Fc region have been expressed in P. pastoris for glycan remodeling, in which the N-glycans need removing by in vitro reactions [14, 28]. When IgG1-Fc was expressed in our engineered strain (MNN9-EndoT), > 95% of secreted IgG1-Fc harbored only one GlcNAc moiety. Our results also showed that the total yield, the secondary structure and the protein conformation were not affected by the removal of the N-glycans. As the secreted proteins have been folded to the native state in the ER apparatus, the deglycosylation in the Golgi should only slightly affect the secretion of glycoproteins. Thus, N-GlcNAc IgG1-Fc protein produced from engineered P. pastoris should have the same properties as the in vitro deglycosylated proteins used for further N-glycans remodeling [14, 27, 30]. In our strategy, the N-GlcNAc proteins could be obtained with high yield via simple purification step from the culture medium.
Combined with the in vitro glycan remodeling or enzymatical elongation methods, this engineered P. pastoris system provides a prospective platform for powerful production of recombinant glycoprotein drugs. On the other hands, this system was not efficient enough to remove all the N-glycans when more than one oligosaccharide was attached on the target proteins. Some reasons might be responsible for the decrease of ENGase hydrolysis activity, such as (1) the spatial hindrance caused by localization expression; (2) the intracellular pH in Golgi was a non-optimal pH for Endo-T; (3) the cultured temperature (20–25 °C) was too low. But, the lower pH (pH 6.0) of the medium and the lower cultured temperature (20–25 °C) were important for higher yields of secreted recombinant proteins. The precise optimum pH of ENGases generally corresponds with the catalytic carboxylic acid residues in the enzyme active sites [70, 71, 72], and depends on the individual ENGase isoform . The hydrolytic activity of ENGase was pH-dependent and drops rapidly as the pH is either higher or lower than the optimum pH . The temperature was another factor to affect ENGases’ hydrolytic activity. Most of the novel ENGase isoforms are derived from microbes. Thus the optimum temperature is 30–37 °C and the lower temperature would decrease the hydrolytic activity. We supposed the temperature was the major reason for the lower deglycosylation efficiency of the fungal ENGase (Endo-T) in P. pastoris than in mammalian cells or plant cells. In the further work, we would screen and apply some novel ENGase isoforms which possess powerful hydrolytic activities towards high-mannose type N-glycan in the cultured condition of P. pastoris, such as pH 6.0, 20–25 °C.
In this work, we developed a simple glycoengineered yeast expression system to efficiently produce homogeneous N-GlcNAc modified glycoproteins which could be further elongated to different N-glycan structures. We believe the application of this easy and low-cost glycoprotein synthetic method would provide a prospective platform to efficiently produce a growing number of novel glycoprotein drugs.
Materials and methods
Bacterial strains, media and chemicals
Pichia pastoris GS115 (his4−), pGAPZa and pPIC9K used for the protein expression were obtained from Invitrogen (Thermo Fisher Scientific). Escherichia coli TOP10 or DH5α strain was used as the host for recombinant DNA construction work. E. coli was grown in Luria–Bertani (LB) medium at 37 °C with 100 μg/mL ampicillin or 50 μg/mL zeocin where necessary. Buffered minimal glycerol (BMGY) medium, buffered minimal methanol (BMMY) medium and minimal dextrose (MD) medium were prepared following the P. pastoris expression manual (Invitrogen). Mouse anti-His monoclonal antibody and mouse anti-Flag monoclonal antibody were purchased from Genscript Bio-Technologies (Nanjing, China). Con A-Biotin was purchased from Vector Laboratories. HRP-conjugated secondary antibody and HRP- conjugated Streptavidin was purchased from ZSGB-Bio (Beijing, China). All other chemicals and solvents were bought from Sangon-Biotech (Shanghai, China).
Plasmid construction and transformation
The primers used in this study
The cDNA encoding the human GalNAc-T1 and IgG1-Fc region were subcloned into the pPIC9K vector respectively. Resultant clones, named pPIC9k-GALNT1 and pPIC9K-Fc, were selected and confirmed by DNA sequencing. The plasmid pPIC9k-GALNT1 and pPIC9K-Fc were linearized with SacI and introduced into P. pastoris GS115 WT and obtained pGAPZa-MNN9-EndoT and pGAPZa-MNS1-EndoT strains. The multicopy insert of transformants were selected with MD plates and subsequently YPD plates containing different concentrations of G418 (0.5 mg/mL, 1 mg/mL, 2 mg/mL or 4 mg/mL). The G418-resistant clones were confirmed by PCR with GalNAc-T1-F or Fc-F and 3′-AOXI primers. The PCR-positive clones from 4 mg/mL G418 plates were selected for the expression. Besides, the pET28a-IgG1-Fc was transferred into E. coli BL21 (DE3) as a control.
Analysis of engineered P. pastoris strains
The engineered P. pastoris Pir1-EndoT strains were cultured in BMMY medium with 0.5% methanol (v/v) for 12 h and washed with PBS. For immunofluorescence staining, the P. pastoris WT and Pir-EndoT strains were incubated with anti-Flag antibody and subsequently FITC-conjugated rabbit antibody against mouse Ig for 45 min and mounted with antifade reagent (BBI Life Sciences). Fluorescence microscopy was performed using a Zeiss Axioskop 2 plus with an AxioCam MR3. Bit depth and pixel dimensions were 36 bits and 1388 × 1040 pixels, respectively. For western blot, the P. pastoris strains were lysed with glass beads and analyzed by Western blot with anti-Flag antibody.
Expression and purification of recombinant proteins
Recombinant yeast clones were grown at 30 °C in 50 mL BMGY until the OD600 reached 2–6. For the fermentation condition screen, Cells were harvested and cultured in BMMY (with pH 6.0, 6.5 or 7.0) for 4–5 days at different temperature (20 °C or 25 °C) and 0.5% or 1% methanol (v/v) was added to the culture every 24 h. The fermentation culture was precipitated by cold acetone after 2–5 days respectively and Coomassie-stained SDS-PAGE was used to test the production of total and glycosylated proteins.
After fermentation, secreted recombinant proteins were purified using Ni–NTA agarose (for GlalNAc-T1) or Protein G column (for IgG1-Fc region). For GalNAc-T1, the cell-free supernatant was loaded onto the Ni–NTA column pre-equilibrated with binding buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 20 mM imidazole). After washed with 30 mL of binding buffer, the purified proteins were eluted with binding buffer containing 250 mM imidazole. For IgG1-Fc region, the cell-free supernatant was diluted 5 times by PBS buffer, and was loaded onto the Protein G column pre-equilibrated with PBS buffer. After washed with 30 mL of PBS buffer, the purified proteins were eluted with 0.1 M Glycine Buffer pH 2.7. The eluted protein was neutralized immediately with 1 M Tris–HCl (pH 7.0). The positive fractions (determined by SDS-PAGE) were desalted and stored at − 20 °C. Recombinant IgG1-Fc region produced in E. coli was purified following the same Ni–NTA protocol.
SDS-PAGE and western blot
Purified IgG1-Fc region and GalNAc-T1 proteins were treated with peptide N-glycosidase F (PNGase F, New England Biolabs), following the manufacturer’s protocol. Samples were run on 12% SDS-PAGE gels with or without DTT reduction, and transferred onto polyvinylidene fluoride membranes for 90 min. After blocked in 5% BSA or 1% polyvinylpyrrolidone (Sigma) the membranes were incubated with His-tag antibody or ConA-B respectively at 4 °C overnight. Blots were developed with DAB Substrate kit (Solarbio, China) following incubation with HRP-conjugated secondary antibody for 1 h at room temperature.
Mass spectrometric analysis of IgG1-Fc protein
Approximately 20 μg of Fc protein was reduced with 10 mM DTT in 50 mM ammonium bicarbonate (AmBic) for 45 min at 60 °C and alkylated by 20 mM iodoacetamide at room temperate for 30 min. Then, 10 mM DTT was added to terminate alkylation before the protein was subjected to proteolysis by Glu-C (Promega). The treatment was terminated by boiling, and the digested peptides were desalted via a standard C18 Zip-Tip procedure and analyzed by MALDI-TOF MS (Shimadzu, Tokyo, Japan) or LCMS-IT-TOF system (Shimadzu, Tokyo, Japan) operated in the positive linear mode.
Circular dichroism spectroscopy
The secondary structure of the IgG1-Fc domian (from P. pastoris WT and MNN9-EndoT strains) were determined by circular dichroism using J-815 Jasco spectropolarimeter (Jasco Co., Tokyo, Japan) equipped with a PTC-348 WI thermostat under a constant nitrogen flow. A 0.1-cm path length cell was used to collect data in the far ultraviolet region (200–250 nm) at a scan speed of 20 nm/min and a response time of 1 s. Spectra were acquired at 25 °C and measured in PBS buffer. The spectrum of a blank containing buffer alone was subtracted from all spectra. The CD data were analyzed using the CDtoolX and online tools dichroweb (http://dichroweb.cryst.bbk.ac.uk/).
SJW, YHR, YGW and YK designed and performed experiments, analyzed data and wrote the manuscript; DCK, MC and PGW contributed to the design of experiments and edited the manuscript. All authors read and approved the final manuscript.
This work was supported by Shandong province Key R&D Program (No. 2019GSF107048), the National Natural Science Foundation of China Grants (No. 31500648), and Open Projects Fund of Shandong Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University (No. 2019CCG08).
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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