TY - JOUR
AU - Fujita, Morihisa
AB - Abstract Glycoprotein therapeutics are among the leading products in the biopharmaceutical industry. The heterogeneity of glycans in therapeutic proteins is an issue for maintaining quality, activity and safety during bioprocessing. In this study, we knocked out genes encoding Golgi α-mannosidase-II, MAN2A1 and MAN2A2 in human embryonic kidney 293 (HEK293) cells, establishing an M2D-KO cell line that can produce recombinant proteins mainly with hybrid-type N-glycans. Furthermore, FUT8, which encodes α1,6-fucosyltransferase, was knocked out in the M2D-KO cell line, establishing a DF-KO cell line that can express noncore fucosylated hybrid-type N-glycans. Two recombinant proteins, lysosomal acid lipase and constant fragment of human IgG1, were expressed in the M2D-KO and DF-KO cell lines. Glycan structural analysis revealed that complex-type N-glycans were removed in both M2D-KO and DF-KO cells. Our results suggest that these cell lines are suitable for the production of therapeutic proteins with hybrid-type N-glycans. Moreover, KO cell lines would be useful as models for researching the mechanism of antimetastatic effects in human tumours by swainsonine treatment. core fucose modification, genome editing, HEK293, hybrid-type N-glycans, therapeutic protein Glycobiologics, such as monoclonal antibodies (mAbs) and recombinant lysosomal enzymes, are increasing used in the biopharmaceutical industry. Mammalian expression systems are generally used as the preferred platforms for manufacturing biopharmaceuticals, as these cell lines are able to produce complex therapeutic proteins with posttranslational modifications similar to those produced in humans (1, 2). Protein glycosylation influences many aspects of protein structure and function, including modified protein folding, stability, molecular recognition and immunogenicity (3). However, there are still drawbacks in the production of recombinant glycoproteins in mammalian cells. One of the challenging issues in the biopharmaceutical industry is the heterogeneity of N-glycosylation on glycoproteins (4). Glycan synthesis is mediated by a series of enzymatic reactions catalyzed by glycosyltransferases and glycosidases. Since glycans are synthesized in a template-independent manner, it is quite difficult to produce proteins with homogenous glycan structures. Heterogeneity directly affects the pharmacodynamics and pharmacokinetic properties of biopharmaceutical proteins, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity, half-life in serum and immunogenic properties (5–7). Glycoengineering is a method to improve the properties of glycoproteins by changing their glycosylation pathways, which has attracted substantial interest over the past few decades (8). Glycoengineering mainly involves the modification of glycan structures and sites on proteins. Genome editing of glycogenes through knockout (KO) and knockin in mammalian cells is a strategy to modify glycan structures on proteins (9). The ADCC of immunoglobulin (IgG) has been enhanced by knocking out α1,6-fucosyltransferase (FUT8) (10–13). IgGs have also been produced with α2,6-sialic acid modification for anti-inflammation by coexpression of α2,6-sialylatransferase and β1,4-galactosyltransferase in Chinese hamster ovary (CHO) cells (14). During protein translocation into the endoplasmic reticulum (ER), an oligosaccharide consisting of Glc3Man9GlcNAc2 (where Glc is glucose; Man is mannose; GlcNAc is N-acetylglucosamine) is transferred to asparagine (Asn) residues of the Asn-X-Ser/Thr (X is any amino acid except proline; Ser is serine and Thr is threonine) motif on proteins (15). The N-glycan structures on proteins are then processed in the ER and the Golgi apparatus, generating heterogeneity of N-glycan structures (Fig. 1). The N-glycan structures are mainly divided into three groups: oligomannose-type, hybrid-type and complex-type. In our previous work, we generated human embryonic kidney 293 (HEK293) cells that only produce oligomannose-type structures as N-glycans by knocking out genes encoding Golgi and ER α1,2-mannosidase-I (16, 17). In this study, we established HEK293 cells that produce hybrid-type N-glycans with or without core fucose. To construct the cells, two genes encoding Golgi α-mannosidase-II were disrupted. Double knockout (M2D-KO) cells defective in two Golgi α-mannosidase-II genes succeeded in the removal of complex-type N-glycans, whereas the major N-glycan structures were hybrid-type N-glycans with core fucose modifications. To produce IgG in the generated cell lines, we further knocked out an FUT8 gene encoding α1,6-fucosyltransferase in M2D-KO cells. In triple-KO (DF-KO) cells, core fucose modification was eliminated. The N-glycans on the recombinant lysosomal acid lipase (LIPA) and constant fragment (Fc) produced in DF-KO cells were hybrid-type without core fucose modification. This is the first report that glycoengineered mammalian cells can produce hybrid-type N-glycans without core fucose modification. The cells should be suitable for therapeutic proteins with hybrid-type N-glycans. Fig. 1. Open in new tabDownload slide N-glycan biosynthetic pathways at the ER and the Golgi in human cell lines. Lipid-linked oligosaccharide (LLO) is biosynthesized by a series of reactions mediated by ALG glycosyltransferases, and the complete LLO with a Glc3Man9GlcNAc2 structure is transferred to nascent proteins in the ER by oligosaccharyltransferase (OST). The terminal and left two Glc residues are trimmed by ER α-glucosidase-I (Glc-I) and α-glucosidase-II (Glc-II), respectively. The Man residues on Man9GlcNAc2 are trimmed by ER and Golgi α1,2-mannosidase-I (ER-α-MAN and Golgi-α-MAN-I), generating Man5GlcNAc2 structures. A GlcNAc residue is transferred to Man5GlcNAc2 structures on proteins by GlcNAc transferase I (GnT-I). In HEK293 cells, most α1,3- and α1,6-Man residues on GlcNAc1Man5GlcNAc2 structures are eliminated by Golgi α-1,2-mannosidase-II (Golgi MAN-II) to generate GlcNAc1Man3GlcNAc2 structures. These oligosaccharides are further processed and converted to biantennary, triantennary and tetraantennary complex-type N-glycans. Some GlcNAc1Man5GlcNAc2 structures are processed to generate hybrid-type N-glycans. Hybrid-type and complex-type N-glycans can receive core fucosylation. Fig. 1. Open in new tabDownload slide N-glycan biosynthetic pathways at the ER and the Golgi in human cell lines. Lipid-linked oligosaccharide (LLO) is biosynthesized by a series of reactions mediated by ALG glycosyltransferases, and the complete LLO with a Glc3Man9GlcNAc2 structure is transferred to nascent proteins in the ER by oligosaccharyltransferase (OST). The terminal and left two Glc residues are trimmed by ER α-glucosidase-I (Glc-I) and α-glucosidase-II (Glc-II), respectively. The Man residues on Man9GlcNAc2 are trimmed by ER and Golgi α1,2-mannosidase-I (ER-α-MAN and Golgi-α-MAN-I), generating Man5GlcNAc2 structures. A GlcNAc residue is transferred to Man5GlcNAc2 structures on proteins by GlcNAc transferase I (GnT-I). In HEK293 cells, most α1,3- and α1,6-Man residues on GlcNAc1Man5GlcNAc2 structures are eliminated by Golgi α-1,2-mannosidase-II (Golgi MAN-II) to generate GlcNAc1Man3GlcNAc2 structures. These oligosaccharides are further processed and converted to biantennary, triantennary and tetraantennary complex-type N-glycans. Some GlcNAc1Man5GlcNAc2 structures are processed to generate hybrid-type N-glycans. Hybrid-type and complex-type N-glycans can receive core fucosylation. Materials and Methods Cells, antibodies and reagents HEK293 cells were cultured in Dulbecco's modified Eagle’s medium (Biological Industries, Kibbutz Beit Haemek, Israel) with 10% foetal bovine serum (FBS; Biological Industries). OPTI-MEM and Lipofectamine 2000 were obtained from Life Technologies (Carlsbad, CA, USA). Biotin-conjugated concanavalin A (ConA), phytohemagglutinin-L4 (PHA-L4) and Len Culinaris Agglutinin (LCA) were purchased from J-Chemical (Tokyo, Japan). Sequence-grade trypsin was purchased from Promega (Madison, WI, USA). PNGase F and Endo Hf were purchased from New England Biolabs (Ipswish, MA, USA). A Sep-Pak C18 cartridge was obtained from Waters (Milford, MA, USA). Dimethyl sulfoxide (DMSO) was purchased from Solarbio (Beijing, China). Iodomethane was obtained from XIYA Reagent (Shandong, China). Anti-FLAG M2 affinity gel and FLAG peptide were both purchased from Sigma-Aldrich (St. Louis, MI, USA). Protein A Sefinose Resin was obtained from Sangon Biotech (Shanghai, China). Knockout of genes in HEK293 cells The MAN2A1, MAN2A2 and FUT8 genes were knocked out by the CRISPR/Cas9 system. The pX330-EGFP plasmid (18) was cut with BpiI. The KO target sequences were ligated into digested pX330-EGFP. The KO target sequences are listed in Supplementary Table S1. Four days after transfection, GFP-positive cells were sorted by using an S3e cell sorter (Bio-Rad). Sorted cells were further cultured for over 7 days, and then the single colonies were obtained by limiting dilution. Briefly, cells were seeded to 0.3–0.5 cells/well in 96-well plates. During cell growth for 1 or 2 weeks, wells containing cells formed a single colony were carefully picked up. Single clonal cell lines for MAN2A1-KO, MAN2A2-KO, MAN2A1 and A2 double-KO (M2D-KO) and MAN2A1, A2 and FUT8 triple-KO (DF-KO) were obtained. KO of genes was confirmed by PCR and sequencing. The primers used for confirmation are listed in Supplementary Table S2. Flow cytometry Cells (1 × 106) were harvested and washed with ice-cold PBS. Cells were resuspended in 50 μl of FACS solution (PBS with 1% BSA and 0.1% NaN3) containing 10 μg/ml biotin-conjugated lectins and incubated on ice for 15 min. After staining, cells were washed with 200 μl of FACS solution twice. The cell suspensions were then stained with PE-conjugated streptavidin on ice for 15 min and washed twice with 200 μl of FACS solution. The samples were analysed by the BD Accuri C6 flow cytometer (BD, Franklin Lakes, NJ, USA). Release of N-glycans on the cell surface Cell surface N-glycans were prepared according to a previously reported study with minor modifications (19). Approximately 4 × 106 cells were used for the digestion of cell surface glycoproteins. Cells were washed five times with 5 ml of ice-cold PBS on a 10-cm dish, harvested with PBS and washed three times after transfer into a new 1.5 ml tube. The cells were resuspended in 500 μl of PBS containing 12 μg/ml of sequencing grade trypsin and rotated at 150 rpm for 45 min at 37°C. Samples were then centrifuged at 10,000×g and 4°C for 15 min, and the supernatants containing the cell surface glycopeptides were separated from the pellet. Trypsin was inactivated at 100°C for 5 min, and N-glycans were released from glycopeptides overnight at 37°C by 500 U of PNGsae F. Protein expression and purification To express His6-FLAG-tagged lysosomal acid lipase (HF-LIPA), pHEK293Ultra-sHF-LIPA was transfected into HEK293 WT, M2D-KO and DF-KO cells (10-cm dishes). Twelve hours after transfection, the cells were transferred into 15-cm dishes. When the cells reached 50% confluency, the medium was replaced with 25 ml of new medium containing 1% FBS. The cells were further cultured for 48 h. The culture medium was collected and centrifuged to remove cell pellets, and HF-LIPA was purified by a high-performance His trap column (GE, Boston, MA, USA). The column was washed with wash buffer (25 mM Tris–HCl, 150 mM NaCl, 20 mM imidazole, pH 7.5) and bound HF-LIPA was eluted with elution buffer (25 mM Tris–HCl, 150 mM NaCl, 250 mM imidazole, pH 7.5). The eluted fractions were further incubated with 50 μl of anti-FLAG M2 affinity gel at 4°C overnight. The anti-FLAG gel was washed with Tris-buffered saline (TBS, 25 mM Tris–HCl, 150 mM NaCl, pH 7.5) three times, and HF-LIPA was eluted by TBS containing 500 μg/ml of FLAG peptide. For the expression of Fc, pME-Hyg-sHF-Fc was transfected into HEK293 WT, M2D-KO and DF-KO cells. To obtain cells stably expressing Fc, transfected cells were selected with medium containing 400 μg/ml of hygromycin. The cells stably expressing Fc were cultured in DMEM supplemented with 1% FBS for 3 days. The medium was collected, filtered and incubated with Protein A Sefinose Resin at 4°C for 3 h. The Sefinose Resin was washed with PBS, and the bound Fc was eluted by elution buffer (100 mM citric acid, pH 2.5–3.0). Glycosidase treatment and western blotting The purified HF-LIPA was treated with or without PNGase F or Endo Hf according to the manufacturer’s instructions. Protein samples were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and blotted onto a PVDF membrane. A mouse monoclonal anti-M2 FLAG antibody (5,000-fold dilution) and an HRP-conjugated goat anti-mouse IgG (5,000-fold dilution; TransGen Biotech, Beijing, China) were used as the primary and secondary antibodies, respectively, to detect the HF-LIPA proteins. Release of N-glycans on purified protein The purified HF-LIPA and Fc were electrophoresed by 10% SDS–PAGE and transferred onto a PVDF membrane. The membrane was stained with Direct Blue-71 (Sigma), which did not give any signals derived from the dye during MALDI MS analysis. The bands corresponding to HF-LIPA and Fc were cut and divided into 2 × 2 mm pieces, and these pieces were washed with 1% (w/v) polyvinylpyrrolidone 40000 (PVP-40; Sigma-Aldrich) in 50% methanol and 50 mM ammonium bicarbonate three times. The membrane pieces were put in 50 μl of 50 mM ammonium bicarbonate solution containing 500 U of PNGase F and incubated at 37°C for 1.5 days. Purification and permethylation of N-glycans Released glycans were purified with a Sep-Pak C18 cartridge. The C18 column was conditioned with 5 ml of methanol and 10 ml of 5% acetic acid. Samples were loaded into the column and eluted with 5 ml of 5% acetic acid. The effluents were collected in a glass tube and dried by a rotary evaporator. N-glycans were permethylated as reported previously (20). Briefly, 0.1 g of NaOH solid was mixed with 500 μl of DMSO in a grinding vessel. The dried glycan samples were resuspended in the slurry and mixed with 300 μl of iodomethane. The mixture was then vigorously shaken at room temperature for 25 min. The reaction was quenched by adding 1 ml of water, followed by vortexing for 10 s. The sample was separated into two layers after adding 2 ml of chloroform, and the top aqueous layers were discarded. The addition of 2 ml of water, vortexing and removal of the aqueous layer was repeated 10 times until the water phase was clear. The chloroform was dried under a nitrogen stream. Matrix-assisted laser desorption ionization time of flight mass spectrometry analysis Permethylated glycan solution (1 μl) was mixed with 1 μl of a solution of 20 mg/ml of 2,5-dihydroxybenzonic acid (DHB) in 70% methanol, spotted onto a MALDI target plate, and allowed to air dry. MS spectra were acquired using MALDI-TOF/TOF-MS (New ultrafleXtreme; Bruker Daltonik, Bremen, Germany) in positive-ion reflectron mode. The measurements were repeated at least three times. The MS data were annotated using GlycoWorkbench software. Results Construction of the MAN2A1/A2 M2D-KO cell line HEK293 cells are easy to grow and are widely used in basic and applied researches. The adaptation to suspension serum-free culture, high transfection efficiency and high recombinant protein productivity in HEK293 cells are advantages for producing recombinant pharmaceutical proteins (21). To establish cells that can produce hybrid-type N-glycans, we knocked out genes encoding Golgi α-mannosidase-II, which works after adding a GlcNAc residue to the Man5GlcNAc2 structure and catalyzes the removal of α1,3- and α1,6-mannose residues from the GlcNAc1Man5GlcNAc2 structure during N-glycan processing in the Golgi (22), resulting in the GlcNAc1Man3GlcNAc2 structure (Fig. 1). This step is critical for forming complex-type N-glycan structures (23). In the human genome, two genes (MAN2A1 and MAN2A2) encode Golgi α-mannosidase-II (24). The MAN2A1 and MAN2A2 genes were knocked out in HEK293 cells using the CRISPR/Cas9 system (25, 26). We designed two targets on the same exon of the MAN2A1 or MAN2A2 genes to remove the DNA fragment from the coding sequence of these two genes. After transfection of KO constructs, we selected clonal cells and confirmed the genotypes. Finally, the MAN2A1-KO (A1-KO-3), MAN2A2-KO (A2-KO-2), MAN2A1 and A2 double-KO (M2D-KO-3) cell lines were obtained. A1-KO-3 had a 54-bp deletion between two KO target sequences in the MAN2A1 gene (Fig. 2A and B), whose cleavage was correctly carried out 3 bp upstream of the protospacer adjacent motif (PAM) sequences by Cas9 and connected with nonhomologous end joining. Since of the karyotype of HEK293 cells is near triploid (27), the KO allele sometimes shows three different patterns. In the A2-KO-2 cell line, three different types of KO genotypes were determined. The first one contained a 76-bp deletion between two target sequences with correct cleavage. The second and third genotypes contained a deletion between two KO targets but had 1- and 71-bp insertions at the cleavage site, respectively (Fig. 2C and D). All the deletions cause a frameshift, suggesting that the MAN2A2 alleles are mutated. In M2D-KO-3 cells, the MAN2A1 gene was cleaved 1 bp upstream of the target site, whereas there were three mutant patterns in the MAN2A2 gene (76-bp deletion with correct cleavage, 76-bp deletion plus 438-bp insertion, 76-bp deletion plus 890-bp insertion) (Fig. 2B and D). Since all the mutations cause frameshifts, both the MAN2A1 and MAN2A2 genes were knocked out in M2D-KO-3 cells. Fig. 2. Open in new tabDownload slide Establishment of the KO MAN2A1 and/or MAN2A2 cell lines. (A and C) Genomic DNA was extracted from HEK293 WT, MAN2A1-KO clone 3 (A1-KO-3), MAN2A2-KO clone 2 (A2-KO-2) and MAN1A1 and MAN1A2 double-KO clone 3 (M2D-KO-3) cells. The KO region was amplified using primer sets to check KO of MAN2A1 and MAN2A2. The size of the designed DNA fragment for MAN2A1 is 326 bp for WT. When MAN2A1 was correctly deleted by CRISPR/Cas9, the size was 272 bp (A). The size of the DNA fragment for MAN2A2 is 237 bp for WT and 161 bp for KO (C). (B and D) Patterns of KO in MAN2A1 (B) and MAN2A2 (D) genes from the WT, A1-KO-3, A2-KO-2 and M2D-KO-3 cell lines were analysed by sequencing. Capital bold letters indicate the target sequences of guide RNAs; underlined text indicates the PAM sequence. The KO of MAN2A2 in A2-KO-2 and M2D-KO-3 cells had three variations (D). Fig. 2. Open in new tabDownload slide Establishment of the KO MAN2A1 and/or MAN2A2 cell lines. (A and C) Genomic DNA was extracted from HEK293 WT, MAN2A1-KO clone 3 (A1-KO-3), MAN2A2-KO clone 2 (A2-KO-2) and MAN1A1 and MAN1A2 double-KO clone 3 (M2D-KO-3) cells. The KO region was amplified using primer sets to check KO of MAN2A1 and MAN2A2. The size of the designed DNA fragment for MAN2A1 is 326 bp for WT. When MAN2A1 was correctly deleted by CRISPR/Cas9, the size was 272 bp (A). The size of the DNA fragment for MAN2A2 is 237 bp for WT and 161 bp for KO (C). (B and D) Patterns of KO in MAN2A1 (B) and MAN2A2 (D) genes from the WT, A1-KO-3, A2-KO-2 and M2D-KO-3 cell lines were analysed by sequencing. Capital bold letters indicate the target sequences of guide RNAs; underlined text indicates the PAM sequence. The KO of MAN2A2 in A2-KO-2 and M2D-KO-3 cells had three variations (D). Cell surface N-glycan profile To analyse the cell surface N-glycan profiles, KO cells were stained using the fluorescent-conjugated lectins PHA-L4, ConA and LCA. PHA-L4 recognizes complex-type N-glycans containing the β1,6-linked N-acetyllactosamine structure (28), while ConA mainly binds to oligomannose-type and hybrid-type N-glycans (29). Compared to HEK293 wild-type (WT) cells, PHA-L4 staining was not changed in A1-KO-3 and A2-KO-2 cells. On the other hand, PHA-L4 staining was almost diminished in M2D-KO-3 cells (Fig. 3A and B). ConA staining was slightly increased in A1-KO-3 cells compared to WT and A2-KO-2 cells, while M2D-KO-3 cells had significantly increased ConA staining (Fig. 3C and D). The staining of LCA, which binds with the core fucose on N-glycans (30), was increased in M2D-KO-3 cells compared to WT cells (Fig. 3E and F), suggesting that core fucosylation on N-glycans is increased in M2D-KO-3 cells. These results indicate that MAN2A1 and MAN2A2 have overlapping functions and that complex-type N-glycans on the cell surface were changed to oligomannose-type or hybrid-type N-glycans in the M2D-KO-3 cell line. Fig. 3. Open in new tabDownload slide Lectin staining of the HEK293 WT, A1-KO, A2-KO and M2D-KO cell lines. (A, C and E) Flow cytometric analysis is shown of cell surface glycans on the HEK293 WT, A1-KO, A2-KO and M2D-KO cell lines using PHA-L4 (A), ConA (C) and LCA (E) lectins. The results are representative of >3 independent experiments. (B, D and F) Relative fluorescence intensities of PHA-L4 (B), ConA (D) and LCA (F) staining on the cell surface of HEK293 WT, A1-KO, A2-KO and M2D-KO lines are plotted. The average fluorescence intensity of each type of lectin staining in HEK293 WT cells was set to 1. Means of the relative fluorescence intensity with SD of three independent measurements are plotted. *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001; ns, not significant (one-tailed Student’s t test). Fig. 3. Open in new tabDownload slide Lectin staining of the HEK293 WT, A1-KO, A2-KO and M2D-KO cell lines. (A, C and E) Flow cytometric analysis is shown of cell surface glycans on the HEK293 WT, A1-KO, A2-KO and M2D-KO cell lines using PHA-L4 (A), ConA (C) and LCA (E) lectins. The results are representative of >3 independent experiments. (B, D and F) Relative fluorescence intensities of PHA-L4 (B), ConA (D) and LCA (F) staining on the cell surface of HEK293 WT, A1-KO, A2-KO and M2D-KO lines are plotted. The average fluorescence intensity of each type of lectin staining in HEK293 WT cells was set to 1. Means of the relative fluorescence intensity with SD of three independent measurements are plotted. *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001; ns, not significant (one-tailed Student’s t test). To determine the precise structures in M2D-KO (M2D-KO-3) cells, the N-glycans on the cell surface were detected using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). In WT cells, at least 60 different types of glycan structures, including oligomannose types, hybrid types and complex types, were observed (Fig. 4A). Complex-type N-glycans contained biantennary, triantennary and tetraantennary structures with or without sialic acids and/or fucoses. On the other hand, in M2D-KO cells, the types of glycan structures decreased and complex-type N-glycan structures were not detected, which is consistent with lectin staining (Fig. 4B). Furthermore, the major glycan structure was a hybrid-type N-glycan with sialic acid and a core fucose modification. In addition, other hybrid-type glycans that were not detected in WT cells were observed. The oligomannose-type N-glycans remained in the M2D-KO cell line. The results indicate that complex-type N-glycans were eliminated by knocking out Golgi α-mannosidase-II; instead, hybrid-type N-glycans were clearly increased in M2D-KO cells. A signal presented at m/z 2,244, which represents HexNAc2Hex2Man3GlcNAc2, was detected in both the WT and M2D-KO cell lines (Fig. 4). The MS/MS results showed that the peak at m/z 2,244 was different between WT and M2D-KO cells. In WT, it should be a complex-type N-glycan without sialylated modification, while it should be a hybrid-type N-glycan in M2D-KO cell lines (Supplementary Fig. S1). Fig. 4. Open in new tabDownload slide MALDI-TOF-MS spectra of N-glycans from HEK293 WT and M2D-KO cell surfaces. N-glycans prepared from the cell surface of HEK293 WT (A) and M2D-KO (B) cell were analysed by MALDI-TOF-MS. N-glycans were permethylated during sample preparation as described under ‘Materials and Methods’ section. MALDI-TOF-MS was performed in positive-ion mode. Possible N-glycan structures from mass peaks are represented with standard cartoon symbolic representations. The observed N-glycan structures with a composition matching the glycan masses and their relative intensities are shown in Supplementary Table S3. Fig. 4. Open in new tabDownload slide MALDI-TOF-MS spectra of N-glycans from HEK293 WT and M2D-KO cell surfaces. N-glycans prepared from the cell surface of HEK293 WT (A) and M2D-KO (B) cell were analysed by MALDI-TOF-MS. N-glycans were permethylated during sample preparation as described under ‘Materials and Methods’ section. MALDI-TOF-MS was performed in positive-ion mode. Possible N-glycan structures from mass peaks are represented with standard cartoon symbolic representations. The observed N-glycan structures with a composition matching the glycan masses and their relative intensities are shown in Supplementary Table S3. Construction of the MAN2A1/A2/FUT8 DF-KO cell line The defucosylated IgG antibodies produced by FUT8-KO or SLC35C1-KO cells showed increased binding affinities to human FcγRIIIa, resulting in higher ADCC activities than conventional fucosylated IgG (10–13). Lectin staining using LCA and N-glycan analysis using mass spectrometry showed that core fucosylated N-glycans were major in M2D-KO cells (Fig. 4B). To eliminate this modification, we disrupted the FUT8 gene in M2D-KO cells. After transfection of the FUT8 KO constructs, the genotypes in the clonal KO cell lines were analysed (Fig. 5A and B). We obtained a clone cell that had a 2,259-bp deletion in the FUT8 gene and named it the DF-KO (DF-KO-11) cell line. Fig. 5. Open in new tabDownload slide Establishment of MAN2A1, A2 and FUT8 triple-KO cells. (A) Genomic DNA was extracted from HEK293 WT and MAN2A1/MAN2A2/FUT8 triple-KO (DF-KO-11) clones. The KO region was amplified using primer sets to check FUT8-KO and analysed by agarose gel electrophoresis. The expected size of the designed DNA fragment for FUT8 was 2,476 bp for WT HEK293 cells and 216 bp for FUT8 KO cells. (B) Sequences of target regions in the FUT8 gene from the WT and DF-KO-11 cell lines are shown. Capital bold letters indicate target sequences of guide RNA; underlined text indicates the PAM sequence. The KO of FUT8 in M2D-KO cells had only one variation. (C) Flow cytometric analysis is shown of cell surface glycans of the M2D-KO-3 and DF-KO-11 cell lines using LCA lectin. The results are representative of three independent experiments. (D) Relative fluorescence intensities of LCA staining on the cell surface of the HEK293 WT, M2D-KO-3 and DF-KO-11 cell lines were plotted. The average fluorescence intensity of LCA staining in HEK293 WT cells was set to 1. Means of the relative fluorescence intensity with SD of three independent measurements were plotted. *P < 0.05; ****P < 0.0001 (one-tailed Student’s t test, compared to WT). (E) N-glycans prepared from the cell surface of the DF-KO cell line were analysed by MALDI-TOF-MS. Possible N-glycan structures from mass peaks are represented with standard cartoon symbolic representations. The observed N-glycan structures with a composition matching the glycan masses and their relative intensities are shown in Supplementary Table S3. Fig. 5. Open in new tabDownload slide Establishment of MAN2A1, A2 and FUT8 triple-KO cells. (A) Genomic DNA was extracted from HEK293 WT and MAN2A1/MAN2A2/FUT8 triple-KO (DF-KO-11) clones. The KO region was amplified using primer sets to check FUT8-KO and analysed by agarose gel electrophoresis. The expected size of the designed DNA fragment for FUT8 was 2,476 bp for WT HEK293 cells and 216 bp for FUT8 KO cells. (B) Sequences of target regions in the FUT8 gene from the WT and DF-KO-11 cell lines are shown. Capital bold letters indicate target sequences of guide RNA; underlined text indicates the PAM sequence. The KO of FUT8 in M2D-KO cells had only one variation. (C) Flow cytometric analysis is shown of cell surface glycans of the M2D-KO-3 and DF-KO-11 cell lines using LCA lectin. The results are representative of three independent experiments. (D) Relative fluorescence intensities of LCA staining on the cell surface of the HEK293 WT, M2D-KO-3 and DF-KO-11 cell lines were plotted. The average fluorescence intensity of LCA staining in HEK293 WT cells was set to 1. Means of the relative fluorescence intensity with SD of three independent measurements were plotted. *P < 0.05; ****P < 0.0001 (one-tailed Student’s t test, compared to WT). (E) N-glycans prepared from the cell surface of the DF-KO cell line were analysed by MALDI-TOF-MS. Possible N-glycan structures from mass peaks are represented with standard cartoon symbolic representations. The observed N-glycan structures with a composition matching the glycan masses and their relative intensities are shown in Supplementary Table S3. In DF-KO (DF-KO-11) cells, core fucose modification will not occur. To confirm this, DF-KO cells were stained with fluorescently conjugated LCA lectin. Compared to parental M2D-KO cells, which were obviously stained by LCA, only background LCA staining was detected in DF-KO cells (Fig. 5C and D). We also analysed the N-glycans from the DF-KO cell surface using MALDI-TOF-MS. The major glycan was a sialylated hybrid-type N-glycan without core fucose (Fig. 5E). Although there were some fucosylated N-glycan structures presented at m/z 1,999, 2,203, 2,244 and 2,375, the fucose was modified to the branched structure according to the LCA staining and MS/MS results (Supplementary Fig. S2). Taken together, we succeeded in establishing a cell line that produces noncore fucosylated hybrid-type structures as the main N-glycans. Expression of recombinant LIPA in KO cell lines Next, we expressed recombinant LIPA in our KO cells. LIPA deficiency causes lysosomal storage diseases, including Wolman disease and cholesteryl ester storage disease (31). Recombinant LIPA is used for enzyme replacement treatment of diseases. His6-FLAG-tagged LIPA (HF-LIPA) was expressed in HEK293 WT, M2D-KO and DF-KO cells. The purified HF-LIPA secreted into the medium was treated with either PNGase F or Endo Hf to analyse the glycan structures on LIPA. PNGase F can cleave all mammalian N-glycans, whereas Endo Hf cleaves oligomannose-type and hybrid-type but not complex-type N-glycans. The HF-LIPA expressed in WT cells showed resistance to Endo Hf treatment (Fig. 6). In M2D-KO cells, the majority of N-glycans on HF-LIPA were sensitive to Endo Hf treatment, whereas a small fraction of HF-LIPA had Endo Hf resistance. It was previously reported that incomplete digestion could be attributed to the presence of core fucose on hybrid-type N-glycans, which limits cleavage by Endo Hf (32). In contrast, Endo Hf-treated HF-LIPA prepared from DF-KO cells was completely shifted to the same position as that treated with PNGase F (Fig. 6), suggesting that all the N-glycans on HF-LIPA produced in DF-KO cells were digested by Endo Hf. Fig. 6. Open in new tabDownload slide Glycan changes of recombinant HF-LIPA. The plasmid expressing His6-FLAG-tagged LIPA (HF-LIPA) was transiently expressed in HEK293 WT, M2D-KO and DF-KO cells. Secreted HF-LIPA was precipitated with an anti-FLAG M2 affinity gel and eluted with the FLAG peptide. Partially purified HF-LIPA was treated with or without glycosidases (PNGase F or Endo Hf). Proteins were electrophoresed by SDS–PAGE and detected by anti-FLAG as the primary antibody. Fig. 6. Open in new tabDownload slide Glycan changes of recombinant HF-LIPA. The plasmid expressing His6-FLAG-tagged LIPA (HF-LIPA) was transiently expressed in HEK293 WT, M2D-KO and DF-KO cells. Secreted HF-LIPA was precipitated with an anti-FLAG M2 affinity gel and eluted with the FLAG peptide. Partially purified HF-LIPA was treated with or without glycosidases (PNGase F or Endo Hf). Proteins were electrophoresed by SDS–PAGE and detected by anti-FLAG as the primary antibody. N-glycan structures on recombinant LIPA and Fc To analyse the glycan structures on recombinant HF-LIPA, N-glycans released from purified HF-LIPA by PNGase F were permethylated and detected by MALDI-TOF-MS. In the HF-LIPA prepared from HEK293 WT cells, >20 different types of N-glycan structures, which contain oligomannose types, hybrid types and complex types, were observed (Fig. 7, upper panel). In contrast, the N-glycan structures on HF-LIPA prepared from M2D-KO cells were simplified, and the main glycan structures were hybrid-type N-glycans with fucose modifications (Fig. 7, middle panel). Furthermore, oligomannose-type N-glycans were inevitably detected. In HF-LIPA expressed in DF-KO cells, hybrid-type N-glycans with no core fucose were the major structures observed, as expected (Fig. 7, lower panel). Fig. 7. Open in new tabDownload slide MALDI-TOF-MS spectra of N-glycans on transiently expressed HF-LIPA. HF-LIPA was transiently expressed in the HEK293 WT, M2D-KO and DF-KO cell lines. After purification, HF-LIPA was analysed by SDS–PAGE, transferred to a PVDF membrane and stained with Direct Blue-71. The N-glycans were released from bands corresponding to HF-LIPA, permethylated and analysed by MALDI-TOF-MS. MALDI-TOF-MS was performed in positive-ion mode with DHB as the matrix. Possible N-glycan structures from mass peaks are represented with standard cartoon symbolic representations. All the observed N-glycan structures with a composition matching the glycan masses and their relative intensities are shown in Supplementary Table S4. Fig. 7. Open in new tabDownload slide MALDI-TOF-MS spectra of N-glycans on transiently expressed HF-LIPA. HF-LIPA was transiently expressed in the HEK293 WT, M2D-KO and DF-KO cell lines. After purification, HF-LIPA was analysed by SDS–PAGE, transferred to a PVDF membrane and stained with Direct Blue-71. The N-glycans were released from bands corresponding to HF-LIPA, permethylated and analysed by MALDI-TOF-MS. MALDI-TOF-MS was performed in positive-ion mode with DHB as the matrix. Possible N-glycan structures from mass peaks are represented with standard cartoon symbolic representations. All the observed N-glycan structures with a composition matching the glycan masses and their relative intensities are shown in Supplementary Table S4. Because the FUT8 gene is disrupted in DF-KO cells, IgG with N-glycans without core fucose could be produced. To check the N-glycans on IgG1, we expressed a Fc of human IgG1 in the HEK293 WT, M2D-KO and DF-KO cell lines. The N-glycans prepared from recombinant human Fc were analysed by MALDI-TOF-MS. Compared to the N-glycans from LIPA, no oligomannose-type N-glycan was detected from Fc in the HEK293 WT, M2D-KO or DF-KO cell lines. Biantennary complex-type N-glycans without sialylated modification were observed as the major N-glycans from Fc expressed in WT (Fig. 8, upper panel). Furthermore, the main N-glycan structures on Fc prepared from M2D-KO cells were hybrid-type N-glycans with core fucose modification (Fig. 8, middle panel). As expected, Fc was expressed in DF-KO cells, and hybrid-type N-glycans with no core fucose were the major structures observed (Fig. 8, lower panel). Fig. 8. Open in new tabDownload slide MALDI-TOF-MS spectra of N-glycans on stably expressed HF-Fc. HF-Fc was stably expressed in the HEK293 WT, M2D-KO and DF-KO cell lines. The permethylated N-glycans were prepared and detected as described in Fig. 7. Possible N-glycan structures from mass peaks are represented with standard cartoon symbolic representations. The observed N-glycan structures with a composition matching the glycan masses and their relative intensities are shown in Supplementary Table S5. Fig. 8. Open in new tabDownload slide MALDI-TOF-MS spectra of N-glycans on stably expressed HF-Fc. HF-Fc was stably expressed in the HEK293 WT, M2D-KO and DF-KO cell lines. The permethylated N-glycans were prepared and detected as described in Fig. 7. Possible N-glycan structures from mass peaks are represented with standard cartoon symbolic representations. The observed N-glycan structures with a composition matching the glycan masses and their relative intensities are shown in Supplementary Table S5. Open in new tabDownload slide Open in new tabDownload slide Discussion Glycosylation of biopharmaceutical proteins is necessary for protein structure, function, stability, targeting and immunogenicity (3, 33). Mammalian glycoproteins possess different glycoforms, and in the context of protein therapeutics, heterogeneity leads to complications in downstream processing and product characterization (2, 8, 34, 35). Heterogeneity in glycans arises because glycosylation is not a template-driven process and is not directly controlled by transcriptional regulation; instead, it is guided by the organization of the glycosylation machinery in the Golgi cisternae (36, 37). Here, we established HEK293 cell lines that produce hybrid-type N-glycans with or without core fucose as major N-glycan structures. Previously, Golgi α-mannosidase-II was disrupted or inhibited in HEK293T and A431 cells (32, 38), and an increase in hybrid-type glycosylation was observed. In our flow cytometric and MS analyses, N-glycan profiles of the M2D-KO cell surface showed that complex-type N-glycans were completely removed, whereas hybrid-type N-glycans accounted for a large proportion of all glycans. Furthermore, we knocked out the FUT8 gene in M2D-KO and then established the three-gene KO cell line DF-KO. Compared to M2D-KO cells, the major glycans in DF-KO cells became hybrid-type N-glycans without core fucose. Compared to HEK293 WT cells, the increased LCA lectin staining was observed in M2D-KO cell line. It would be the substrate specificity of FUT8. Once a β1,6-GlcNAc is modified to N-glycans by GnT-V, the structures are not the substrates for the FUT8 (39). Moreover, in the MS result of cell surface N-glycans prepared from HEK293 WT cells, several triantenary or tetraantenary N-glycan structures without a fucose were observed (Fig. 4A). Based on the previous report and our results, since GnT-V substrates are not generated in M2D-KO cells, core fucosylation on N-glycans would be increased in M2D-KO cell line. In the cell surface N-glycan profiles from M2D-KO and DF-KO cells, although the main glycan structures were hybrid-type N-glycans, some oligomannose-type N-glycans, such as Man8GlcNAc2, Man7GlcNAc2 and Man6GlcNAc2, were still detected. Since N-glycan processing starts from oligomannose-type N-glycans, which are converted to hybrid-type or complex-type structures (40, 41), oligomannose-type N-glycans inevitably remain in the M2D-KO and DF-KO cell lines. Because the precursor structure for hybrid-type glycans is Man5GlcNAc2, we will try to reduce the proportion of oligomannose-type N-glycans by introducing α1,2-mannosidases in KO cells. The N-glycan profiles of LIPA and Fc produced in the WT, M2D-KO and DF-KO cell lines showed some differences. Compared to LIPA, no oligomannose-type N-glycans were observed on Fc, indicating that oligomannoses on Fc N-glycans are more efficiently trimmed by mannosidases than those on LIPA. It is caused by the difference of properties in each protein. LIPA is originally a lysosomal protein, which preferentially receives mannose-6-phosphates on oligomannose-type N-glycan structures, for their transport to lysosomes. Since phosphate group is easily lost during sample preparation and ionization in MS analysis, the phosphorylated glycans were not detected. On the other hand, most literatures reported that IgG predominantly receives a N-glycan structure with biantennary complex-type structures with or without galactose, which is consistent with our results. The major glycans were changed to hybrid-type N-glycans in both LIPA and Fc prepared from the M2D-KO cell line, while the major glycans from DF-KO cells were hybrid-type N-glycans without core fucose. N-glycans lacking core fucose on antibodies have been shown to enhance binding to FcγRIII receptors on natural killer cells, improving ADCC activity (10–13). It is worth analysing the ADCC activity of antibodies produced from DF-KO cells in future analyses. For recombinant lysosomal enzymes such as LIPA, mannose-6-phosphate modification of oligomannose-type N-glycans is utilized for cellular targeting of enzyme replacement therapy of lysosomal storage diseases (42, 43). The disadvantages of the proteins having oligomannose-type N-glycans are their lower half-life in serum in vivo compared to those that have sialylated complex-type N-glycans (44–46). Since hybrid-type N-glycan structures could possess both sialic acid and mannose-6-phosphate, it might be possible to produce recombinant proteins having both sialylated and mannose-6-phosphorylated glycans in M2D-KO or DF-KO cells. The biological significance of glycoproteins carrying hybrid-type N-glycans is less clear. It is because that amounts of hybrid-type N-glycans are very low in normal cells, and it may be considered that the glycan structures are simply ‘intermediates’ on the glycan processing. Recently, however, it is reported that HIV-1 broadly neutralizing antibodies including PG9, PG16, VRG26.25 and VRC26.09, strongly bind with sialylated hybrid-type N-glycan structures, proposing that the hybrid-type N-glycans serve as epitopes for the design of vaccines against HIV (47). In addition, hybrid-type N-glycans change protein properties such as binding of IgG1 to Fcγ receptor IIIa and cell–cell interaction mediated by E-cadherin (48, 49). Therefore, the cell lines established in this study, which are suitable for production of glycoproteins or antibodies that require hybrid-type N-glycans with or without core fucose modification, are important for designing therapeutic proteins with improved characteristics. On the other hand, swainsonine, an indolizidine alkaloid, is an inhibitor of Golgi α-mannosidase-II (50, 51), which has been reported to have anticancer activity and can activate natural killer cells (52). Swainsonine has been confirmed to have inhibitory and antimetastatic effects in human tumours, including hepatoma, spongiblastoma, breast carcinoma and melanoma cells (53, 54). It also reduces the survival of cancer cells and inhibits lung carcinoma A549 xenograft tumours (55). In preliminary clinical trials with late-stage cancer patients, specific inhibition of Golgi α-mannosidase-II by swainsonine resulted in reduced tumour growth and metastasis, with minimal side effects (56). The KO cell lines constructed in this study could use not only for production of glycoproteins, but also for studying the anticancer mechanism of swainsonine treatment as model cells. Since our KO cell lines do not need to use any inhibitor to produce hybrid-type N-glycans, they would be much stable than the inhibitor-treated cells for production of glycoproteins or analysing cell properties. The Golgi α-mannosidase-II-deficient M2D-KO and DF-KO cell lines described herein are important resources for functional analysis of hybrid-type N-glycans and for elucidating their role in cells. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements We thank Dr. Weijie Dong (Dalian Medical University) for mass spectrometric analysis and Dr. Ning Wang (Jiangnan University) for discussion. Funding This work was supported by grants-in-aid from the National Natural Science Foundation of China [32071278 and 31770853], the Program of Introducing Talents of Discipline to Universities [111-2-06], National first-class discipline program of Light Industry Technology and Engineering [LITE2018-015], Top-notch Academic Programs Project of Jiangsu Higher Education Institutions and the International Joint Research Laboratory for Investigation of Glycoprotein Biosynthesis at Jiangnan University. Conflict of Interest None declared. References 1 Zhu J. 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Cancer Res . 3 , 1077 Google Scholar PubMed OpenURL Placeholder Text WorldCat Abbreviations Abbreviations ADCC antibody-dependent cell-mediated cytotoxicity CHO Chinese hamster ovary ConA concanavalin A DHB 2,5-dihydroxybenzonic acid DMSO dimethyl sulfoxide FBS foetal bovine serum Fc constant fragment ER endoplasmic reticulum HEK293 human embryonic kidney 293 IgG immunoglobulin G KO knockout LCA Len culinaris agglutinin LLO Lipid-linked oligosaccharide LIPA lysosomal acid lipase mAbs monoclonal antibodies MALDI-TOF-MS matrix-assisted laser desorption ionization time of flight mass spectrometry OST oligosaccharyltransferase PAM protospacer adjacent motif PHA-L4 phytohaemagglutinin-L4 SDS–PAGE sodium dodecyl sulphate–polyacrylamide gel electrophoresis © The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Cell engineering for the production of hybrid-type N-glycans in HEK293 cells
JO - The Journal of Biochemistry
DO - 10.1093/jb/mvab051
DA - 2021-04-20
UR - https://www.deepdyve.com/lp/oxford-university-press/cell-engineering-for-the-production-of-hybrid-type-n-glycans-in-hek293-p51DAJssSO
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -