CHO K1 cells were cultured in Ham's F-12 medium containing 10% fetal calf serum (FCS). The transfectants and mutants derived from CHO K1 cells were cultured in the same medium supplemented with suitable antibiotics. CD59/decay accelerating factor (DAF)-negative human B-cell lymphoma Ramos 517-17 cells were cultured in RPMI 1640 medium containing 10% FCS (Maeda et al., 2001
Establishment of a Novel Class Mutant in CHO Cells
The parental CHO cell line F21, which stably expressed the GPI-anchored protein markers CD59 and DAF and 12 previously known proteins involved in GPI biosynthesis, was established by three cycles of cDNA transfection and clone selection. First, wild-type CHO K1 cells were transfected with pME vectors containing cDNAs of CD59/DAF/neor, FLAG-PIG-L, GST-DPM2, GST-PIG-A, and SL15-FLAG, and then transfectants were selected with 600 μg/ml G418 (Nacalai Tesque, Kyoto, Japan). In the second cycle, cDNAs of PIG-O/hygr, PIG-U-3FLAG, GnTI-FLAG, FLAG-PIG-F, and FLAG-GAA1 were transfected and transfectants were selected with 600 μg/ml G418 and 400 μg/ml hygromycin B (Wako Pure Chemicals, Osaka, Japan). In the third cycle, cDNAs of PIG-N-GST, GST-PIG-V, GST-DPM1, GST-PIG-B, and bsdr were transfected, and the clone F21 was selected with 600 μg/ml G418, 400 μg/ml hygromycin B, and 50 μg/ml blasticidin S (Invivogen, San Diego, CA).
F21 cells (3 × 107) were mutagenized with 1.2 μg/ml N-methyl-N′-nitro-N-nitrosoguanidine (NMMG; Nacalai Tesque) for 2 d and cultured in fresh medium for 7 more days. The cells were then treated with 1 nM proaerolysin (Protox Biotech, Victoria, British Columbia, Canada) for 2 d and/or 1 nM Clostridium septicum α-toxin for 2 d. Surviving cells were isolated by limiting dilution.
Wild-type and mutant CHO cells used in this study are listed in .
CHO cell lines used in this study
Flow Cytometric Analysis
Cells were stained for CD59 and DAF with anti-CD59 (5H8) plus fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (ICN/Cappel, Aurora, OH) and biotinylated anti-DAF (IA10) plus phycoerythrin (PE)-conjugated streptavidin (Biomeda, Foster City, CA). To evaluate the biosynthesis of N-glycan, cells were cultured with or without 10 μg/ml swainsonine (Wako Pure Chemicals) for 4 d, and then stained with 25 μg/ml FITC-conjugated PHA-P lectin (Sigma-Aldrich, St. Louis, MO). Stained cells were analyzed by a FACScan (BD Biosciences, San Jose, CA).
The cloning of p
lycan-class X (PIG-X
) cDNA was carried out by the expression cloning method described previously (Inoue et al., 1993
) with some modifications. The rat C6 glioma cDNA library constructed in the pME-Py vector bearing the polyoma virus origin of replication, and pcDNA-PyT(ori-) for the expression of the polyoma large T, were cotransfected into the mutant CHO2.46 (see Results
for isolation of CHO2.46 cells) by transfection using Lipofectamine 2000 (Invitrogen). After 2 d of cultivation, cells were stained with biotinylated anti-CD59 and PE-conjugated streptavidin, and CD59-positive cells were collected with a cell sorter (FACS-Vantage; BD Biosciences). The plasmids in the sorted cells were rescued in Escherichia coli
. After two cycles of transfection, cell sorting, and plasmid rescue, each plasmid clone was tested by transfection into CHO2.46 cells.
Transfection of Cells
Cells were transfected by either electroporation or lipofection. CHO cells (0.4 × 107) suspended in 0.4 ml of culture medium were electroporated at 260 V and 960 μF with 10 μg of DNA by using a Gene Pulser (Bio-Rad, Hercules, CA). Ramos 517-17 cells (0.4 × 107) suspended in 0.4 ml of HEPES-buffered saline were electroporated under similar conditions. For lipofection, CHO cells were transfected using LipofectAMINE 2000 according to the instruction manual.
To construct pME/PIG-X-3FLAG and pME/puror/PIG-X-3FLAG, we amplified full-length PIG-X cDNA by using pME/PIG-X (clone 63B) as a template and the forward SRα primer 5′-TGACCCTGCTTGCTCAACTCTACG and the specific reverse primer with an MluI site 5′-TTACGCGTTAGGGAAAAATGGCCATATTTGAAAAC (R1), digested with XhoI and MluI, and cloned into XhoI- and MluI-cut pME//3FLAG and pME/puror//3FLAG. To construct pME/PBN1–3FLAG, we amplified full-length PBN1 by using genomic DNA from S. cerevisiae as a template and primers 5′-AACTCGAGCATGGTGACAAGACATAGAGTGACTG (forward) and 5′-TTACGCGTTTCCCGTTTTACTGATCTTTTCTTCTTG (reverse), and cloned it in place of the PIG-X cDNA into pME/PIG-X-3FLAG. For pME/GST-GPI14, the full-length GPI14 gene, amplified using 5′-AACTCGAGCATGACTGGCGAAGAATGGGGCTTG (forward) and 5′-TTACGCGTGTTGTTCTTTTTGTTGGAAACTGTGG (reverse), was cloned in place of the PIG-M cDNA into PME/GST-PIG-M. Various 5′ deletion mutants of PIG-X were amplified with the R1 primer and each of the following forward primers: 5′-AACTCGAGGATGGTATCAGAGAGTTTTAATCTAG for A1, 5′-AACTCGAGCACTTGTTCTGAAATTATTTTG for A2, 5′-AACTCGAGCATGTGTTCTGAAATTATTTTG for A2(ATG) 5′-AACTCGAGTGGGCTGGTCGGCAGGCTCGCC for A3, 5′-AACTCGAGCGTCCTGGCTGCCAGCGCCCTT for A4, and 5′-AACTCGAGCGCTCTGCGTGCTCAGGTTCCTC for A5. Site-directed mutagenesis was carried out using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
Affinity Precipitation and Western Blotting
Cells were lysed in a buffer containing 1.0% Nonidet P-40, 50 mM Tris-HCl, pH 7.7, 5.0 mM EDTA, 150 mM NaCl, and Complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) (TEN buffer), and cell debris was removed by ultracentrifugation (100,000 × g, 1 h). Anti-FLAG M2 agarose (Sigma-Aldrich) or glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ) beads were added to the supernatant and rotated at 4°C for 2 h. The beads were collected and washed with TEN buffer twice, and then absorbed proteins were eluted with reducing SDS sample buffer. An aliquot was subjected to 10–20% gradient SDS-PAGE gel and electroblotted onto a polyvinylidene difluoride membrane. The blot was treated with the primary antibody, either anti-FLAG BioM2 (Sigma-Aldrich) or anti-GST (Amersham Biosciences), and then with horseradish peroxidase-conjugated protein G (Bio-Rad). Detection was carried out using Western lightning chemiluminescence reagents (PerkinElmer Life and Analytical Sciences, Boston, MA).
Metabolic Labeling of GPI Intermediates and Dolichol-linked Oligosaccharides
For labeling with Man, CHO cells (106) were preincubated in medium containing 100 μg/ml Glc and 10 μg/ml tunicamycin (Sigma-Aldrich) for 1 h, and then incubated in the same medium containing 40 μCi/ml d-[2-3H(N-)]Man (American Radiolabeled Chemicals, St. Louis, MO) for 45 min. To test the effect of YW3548/BE49385A, cells were cultured in medium containing 10 μM BE49385A (a gift from Banyu Pharmaceutical, Tokyo, Japan), followed by labeling with Man. For labeling with inositol, cells (106) were cultured in inositol-free DMEM supplemented with 10% dialyzed FCS and 40 μCi/ml myo-[2-3H(N)]inositol (American Radiolabeled Chemicals) for 1 d. Lipids were extracted from cells with chloroform:methanol [2:1 (vol/vol)] and then partitioned into water-saturated 1-butanol. The extracts were applied onto a silica gel thin layer chromatography (TLC) plate (Merck, Whitehouse Station, NJ), and developed with chloroform:methanol:water (10:10:3) for analyses of mannolipids, and with chloroform:methanol:1 N NH4OH (10:10:3) for analyses of the first three products of GPI-anchor. The radiolabeled lipids were detected by a Fuji BAS1500 image analyzer (Fuji Film, Tokyo, Japan).
For analysis of N
-glycan precursors, cells were metabolically labeled with [3
H]Man in the absence of tunicamycin. Dolichol-linked N
-glycan precursors were extracted as described previously (Chantret et al., 2003
) and hydrolyzed with 0.1 M HCl/80% tetrahydrofuran at 65°C for 30 min to release oligosaccharides. After neutralization, oligosaccharides were analyzed by TLC by using 1-propanol:acetic acid:water (3:3:2).
To assay Dol-P-Man synthase and GPI-MT-I activities, microsomes were prepared from cells preincubated for 2 h with 5 μg/ml tunicamycin by hypotonic lysis and disruption with a Teflon homogenizer. The microsomes were incubated at 37°C for 60 min with or without 40 μg/ml GlcN-PI(C8) (a gift from Dr. M. A. Lehrman, Texas Southwestern Medical Center, Dallas, TX) in 100 mM HEPES, pH 7.3, containing 20 μCi/ml GDP-[2-3H]Man (American Radiolabeled Chemicals), 20 μM palmitoyl-CoA, 50 mM KCl, 10 mM MgCl2, 10 mM MnCl2, 2 mM 5′AMP, 2 μg/ml tunicamycin, 4 μg/ml leupeptin, and 0.2 mM N-tosyl-l-lysine chloromethyl ketone. Lipid extraction and TLC analysis were carried out as described above.
Cell Viability Assay
The cell viability assay was carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) as described previously (Hong et al., 2002