Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., New York, NY 10461, USA
Received on August 2, 2002; revised on August 21, 2002; accepted on August 23, 2002
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Abstract |
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Key words: GlcNAc-TI / lectin resistance / Mgat1 gene mutations / site-directed mutagenesis
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Introduction |
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To further enhance the usefulness of CHO Lec1 mutants, it is important to know the molecular basis of mutation in the different Lec1 lines. Point mutations that inactivate or weaken GlcNAc-TI may be interpreted in the context of the crystal structure (Unligil et al., 2000; Chen et al., 2001b
), and mutations that reduce transcription or translation of the Mgat1 gene would identify factors that regulate GlcNAc-TI expression. When the Mgat1 gene coding region was cloned, northern blot analysis revealed that Pro-Lec1.3C cells have normal levels of Mgat1 gene transcripts of apparently full length (Kumar et al., 1990
). The same result was reported by Puthalakath et al. (1996)
, who obtained Pro-Lec1.3C cells from the American Type Culture Collection (ATCC) that had been deposited with the ATCC by this laboratory in 1986. These authors identified three nucleotide differences between Mgat1 cDNAs from Pro-Lec1.3C cells and CHO cells. The change that converted Cys at amino acid 123 to Arg was the only one that inactivated rabbit GlcNAc-TI (Puthalakath et al., 1996
). Although this result clearly identified a missense mutation that abrogates GlcNAc-TI activity, we show here that this is not the molecular basis of the lec1 mutation in Pro-Lec1.3C cells. We found that the Pro-Lec1.3C mutant line obtained from the ATCC and our earliest and recent laboratory stocks do not harbor any of the nucleotide changes reported previously. Rather the Pro-Lec1.3C mutant arose from a single insertion mutation that generates an inactive, truncated GlcNAc-TI of
24 kDa. We have also identified the molecular basis of mutation in five additional CHO glycosylation mutants that carry a lec1 mutation. Each mutation falls in the Mgat1 gene coding exon and five of the six lec1 mutant alleles encode truncated GlcNAc-TI.
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Results |
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Comparison of the wild-type Mgat1 coding sequence with Mgat1 cDNAs from the mutants is shown in Figure 2. The Lec3.2.8.1 Mgat1 coding sequence has a G insertion at nucleotide 1154 that causes a frame shift downstream of amino acid 384 and a stop codon after amino acid 391. The Lec1.1N sequence has a missense mutation at C784 (CT) that introduces a stop codon after amino acid 261. The Lec9.1.3C sequence has a C insertion at nucleotide 310 that causes a reading frame shift after amino acid 103 and introduces a stop codon after amino acid 115. The Lec1.1C sequence has a 33-nucleotide insertion in the Mgat1 gene coding region, consistent with the slightly slower migration of its Mgat1 gene transcripts (Figure 1B). All lec1 mutations were also present in PCR products of genomic DNA from the respective mutants, and there was no indication from sequencing gels of mixtures due to either a mixed cell population, or the presence of wild-type and Mgat1 genes in a cell. Thus only one functional Mgat1 allele is present in the Pro-5 parent CHO line.
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The Mgat1 coding sequence we obtained from Pro-Lec1.3C cDNAs differed significantly from that reported previously (accession number U65792; Puthalakath et al., 1996). Our sequence contained a single C insertion in a run of four cytidines (nucleotides 702705) causing ACCCCT in wild type to become ACCCCCT in Pro-Lec1.3C cells (Figure 4). This leads to a frameshift after amino acid 235 and a stop codon after amino acid 244 of the GlcNAc-TI sequence (Figures 2 and 4). By contrast, Puthalakath et al. (1996)
did not observe the C insertion but found three nucleotide changes in the Pro-Lec1.3C Mgat1 gene coding region (i.e., T367C, Cys123 to Arg123; A1016G, Gln339 to Arg339; and A1202G, Lys401 to Arg401). To investigate the unexpected discrepancy between these findings, we compared Mgat1 cDNA and genomic DNA sequences from three different Pro-Lec1.3C stocks that were frozen on different dates after varying times in culture. Stock 1, cultured for 1.3 months after cloning, had been stored in liquid nitrogen or at -135°C; stock 2, cultured for 1.6 months after cloning, had been stored at -70°C; and stock 3 was obtained from the ATCC. All stocks were derived from the clone described originally as WgaR1.3C (Stanley et al., 1975c
).
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Correction of the Pro-Lec1.3C Mgat1 gene mutation
To prove that the single C insertion is the basis of the Pro-Lec1.3C mutant phenotype, site-directed mutagenesis was used to correct the Pro-Lec1.3C mutant cDNA. Primers were designed to delete the extra C and the corrected Mgat1 cDNA was transfected into Lec1 cells that have no GlcNAc-TI activity. Stable G418-resistant colonies were picked and examined for binding of FITC-L-PHA or FITC-WGA by flow cytometry analysis. Lec1 cells expressing a corrected Pro-Lec1.3C Mgat1 cDNA bound L-PHA and WGA similarly to wild-type CHO cells (Figure 3). The GlcNAc-TI activity of Lec1 cells expressing the corrected Mgat1 cDNA was also reverted (Table I). For comparison, ß4galactosyltransferase (ß4GalT) activity was also measured. Wild-type CHO cells had GlcNAc-TI activity of 4 nmol/mg protein/h, whereas Pro-Lec1.3C cells had no GlcNAc-TI activity. Background cpm obtained in the assay is not present in authentic product (Chaney and Stanley, 1986
). The GlcNAc-TI activity of transfectants expressing a corrected Mgat1 cDNA was 813-fold higher than CHO GlcNAc-TI. Therefore the C insertion (Figure 4) is the basis of the Mgat1 gene mutation in Pro-Lec1.3C cells.
Based on the C insertion mutation, Pro-Lec1.3C cells were predicted to synthesize a truncated GlcNAc-TI protein of 24 kDa compared to
48 kDa full-length GlcNAc-TI. Unfortunately, many attempts with three different affinity-purified antibodiesa polyclonal sheep antibody raised against denatured rabbit GlcNAc-TI (Burke et al., 1992
) used by Puthalakath et al. (1996)
, a rabbit polyclonal antibody against a rat GlcNAc-TI fusion protein (Yoshida et al., 1999
), and an affinity-purified chicken antibody against bacterially produced mouse GlcNAc-TI (described in Materials and methods)failed to detect GlcNAc-TI in CHO microsomes by western blot analysis or by immunoprecipitation after prolonged labeling with 35S-Met/Cys. Although each antibody readily detected bacterially produced mouse GlcNAc-TI, no specific signal was obtained from 50 µg CHO microsomes with any affinity-purified preparation. Therefore, detecting endogenous levels of GlcNAc-TI in CHO cells may require the development of antibodies against native CHO GlcNAc-TI.
To detect the protein produced by Pro-Lec1.3C Mgat1 mutant cDNA, a myc epitope sequence was fused in frame with an Mgat1 cDNA from Pro-Lec1.3C cells. After transfection into Lec1 cells, lysates were subjected to western blot analysis and the expected 24-kDa (Pro-Lec1.3C ) or
48-kDa (CHO control) band was obtained (Figure 4). Thus the mutant Mgat1 gene of Pro-Lec1.3C cells generates an
24-kDa protein that is stably expressed in CHO cells, as predicted.
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Discussion |
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The Mgat1 gene in the most commonly used Lec1 mutant, Pro-Lec1.3C from the ATCC, is shown here to contain a C insertion between nucleotides 702 and 705 causing a run of four cytidines to become five, and an 24-kDa catalytically inactive GlcNAc-TI to be produced. This type of sequence change can quite readily be missed (e.g., see Ruiz-Gomez et al., 2000
), which may explain why it was not observed in the Pro-Lec1.3C Mgat1 cDNA sequence of Puthalakath et al. (1996)
. The region of the gel at
24 kDa, where truncated GlcNAc-TI would migrate is not shown in Puthalakath et al. (1996)
. In subsequent studies with CHO cells, myc-tagged GlcNAc-TI was used (Opat et al., 2000
, 2001
) rather than the sheep antibody that bound many nonspecific bands (Puthalakath et al., 1996
; present study). It is less easy to understand why Puthalakath et al. (1996)
found three nucleotide differences that we did not find in Mgat1 cDNAs or genomic DNA from either the ATCC stock or early and more recent stocks of Pro-Lec1.3C. We also determined by genomic DNA sequencing that clone 15B, a GlcNAc-TI mutant used frequently by cell biologists (reviewed in Kornfeld and Kornfeld, 1985
; Stanley, 1984
; Brandli, 1991
), did not arise from the Cys123 to Arg123 mutation of Puthalakath et al. (1996)
(data not shown). Puthalakath et al. (1996)
confirmed the presence of three nucleotide changes in genomic DNA from their stock of Pro-Lec1.3C cells, and thus it seems likely that mutations had accumulated during cell culture. Pro-Lec1.3C arose spontaneously and contains only one functional Mgat1 allele because mixed sequences were not observed in uncloned RT-PCR or PCR products. Therefore, we conclude that the Pro-Lec1.3C mutant available from the ATCC carries a single point mutation in the Mgat1 gene coding regiona C insertion giving rise to truncated, inactive GlcNAc-TI of
24 kDa that lacks 203 C-terminal amino acids.
It is interesting that none of the six lec1 mutations we analyzed arose from a missense inactivating mutation, despite the fact that three of them were isolated following chemical mutagenesis (see Materials and methods). Such mutations would be helpful in designing structurefunction studies of GlcNAc-TI in the context of the crystal structure (Unligil et al., 2000). Several mutations that inactivate or weaken GlcNAc-TI have already been described: the change of amino acid 123 from Cys to Arg inactivates rabbit GlcNAc-TI (Puthalakath et al., 1996
) as does the mutation Gly320Asp in GlcNAc-TI from the BHK mutant RicR14 (Opat et al., 1998
). Removal of the N-terminal 106 amino acids has no effect, although further removal of 14 amino acids results in complete loss of GlcNAc-TI activity (Sarkar et al., 1998
). Two point mutations in Lec1A CHO glycosylation mutants (D212N and R303W) weaken GlcNAc-TI by altering its kinetic properties (Chen et al., 2001b
). D212 is the central residue of the motif E211DD213 present in all GlcNAc-TIs. This mutation may perturb interactions that both D212 and R117 make with UDP-GlcNAc. R117 also participates in the loop structuring required for acceptor binding (amino acids 318330). A large panel of Lec1 and Lec1A mutants could easily be obtained using a specific lectin selection protocol described previously (Stanley, 1981
) because Lec1 mutants arise at a frequency of
10-3 after mutagenesis (Stanley et al., 1975a
).
Overexpression of GlcNAc-TI deletion mutants has been effectively used to characterize the role of the cytoplasmic tail, transmembrane domain, and stem region of GlcNAc-TI in Golgi localization and in interactions with other Golgi enzymes, a process referred to as kin recognition (Burke et al., 1992; Nilsson et al., 1996
). Recently, evidence that medial Golgi enzymes, including GlcNAc-TI, exist as complexes formed via their lumenal domains was reported (Opat et al., 2000
). The residues in the stem region of GlcNAc-TI thought to be critical for kin recognition (Nilsson et al., 1996
) were shown not to be required for Golgi localization, complex formation, or the transferase function of GlcNAc-TI (Opat et al., 2000
, 2001
). The five Lec1 mutants with endogenous levels of C-terminally truncated inactive GlcNAc-TI described here (Figure 2) represent tools for studying the effects of physiological levels of GlcNAc-TI of different lengths on medial Golgi enzyme complex formation and potentially other aspects of Golgi function.
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Materials and methods |
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Cell culture
Parental CHO (Pro-5 and Gat-2) and the Lec1 mutants (Pro-Lec1.3C, Pro-Lec1.1C, and Gat-Lec1.1N (Stanley et al., 1975c), Pro-Lec9.1.3C (Ripka et al., 1986
), Pro-Lec3.2.8.1, and Pro-Lec3.2.1.15C (Stanley, 1989
) from laboratory stocks and Pro-Lec1.3C from the ATCC (CRL 1735) were cultured in suspension or monolayer at 37°C in
medium containing 10% FBS. Among the six Lec1 mutants, Lec9.1.3C, Lec3.2.8.1, and Lec3.2.1.15C were selected from chemically mutagenized cell populations, and the remainder arose spontaneously. Transfectants carrying cDNA constructs in pcDNA3.1(+) (Invitrogen, Carlsbad, CA) were grown in the same medium containing G418 at 1.0 mg/ml active weight.
Northern blot analysis and RT-PCR
Northern analysis of total RNA was performed as described (Chen et al., 2001b). Blots were hybridized with a Mgat1 gene coding region probe from a mouse Mgat1 cDNA (Kumar et al., 1992
). RT-PCR was performed on total RNA as described using an oligodT12 primer and Superscript II reverse transcriptase to generate cDNA and primers 150 (forward), 5'CCAAGCTTCCTCCCCKGY-GGGGGCCAGG3' and 154 (reverse), 5'GGCTCGAG-CCCAGRARGGAMAGGCAGGWGCT3' that span the Mgat1 gene coding region. The PCR reaction was performed as described elsewhere (Chen et al., 2001b
).
Site-directed mutagenesis
Site-directed mutagenesis was performed on cDNA clones in pcDNA3.1(+) using the QuikChange Site-Directed Mutagenesis Kit and pfu DNA polymerase. The primers used to correct the C702705 insertion in Pro-Lec1.3C were forward, 5' GCTCAGAACAGACC-CCTCCCTTTGG3', and reverse, 5'CCAAAGGGAGGGGTCTGTTCTGAGC3'. The primers used to correct C784 to T784 in Lec1.1N were forward, 5'CCTGAGCTGCTCTATCGAACAGAC-TTT-TTTCC3', and reverse, 5'GGAAAAAAGTCTGTT-CGATAGAGCAGCTCAGG3'. The primers used to correct the C310 insertion in Lec9.1.3C were forward, 5'GTGTGCCTGCGACCCC-CTCCCAG3', and reverse, 5'CTGGTGAGGGGGTCGCAGGCACAC3'. Cycling conditions were 95°C for 2 min, 18 cycles at 95°C for 30 s, 55°C for 1 min, 68°C for 12 min. Corrected sequences were confirmed by sequencing both strands.
Sequence analysis
For Pro-Lec1.3C Mgat1 gene sequencing, two cDNA clones from each stock were sequenced in both directions, and the region spanning nucleotides 702706 was sequenced five times for the ATCC stock and four times for each of the two laboratory stocks. Both RT-PCR and genomic DNA PCR products from each Pro-Lec1.3C stock were sequenced at least twice over the 702706 area. For all other Lec1 mutants at least two cloned Mgat1 cDNAs were sequenced in both directions. All mutations found in a cDNA were confirmed by sequencing total RT-PCR products and PCR products from the corresponding genomic DNA in both directions. Sequencing was performed by the Sequencing Facility at the Albert Einstein College of Medicine.
Transfection of Lec1 cells and flow cytometry analysis
Transfection of cDNA into Lec1 cells was performed using Polybrene, as described (Chen et al., 2001b). After transfection, colonies resistant to G418 were picked, expanded in suspension culture, and tested for binding of L-PHA and WGA by flow cytometry as described previously (Chen et al., 2001b
).
GlcNAc-TI and ß4GalT assays
GlcNAc-TI was assayed in a detergent extract as described (Chen et al., 2001b) using Man5GlcNAc2Asn as acceptor and UDP-3H-GlcNAc as donor. ß4GalT activity was assayed in 50 µl final volume. The reaction contained 5 µmol 2-(N-morpholino) ethanesulfonate buffer (pH 6.5), 3 µmol MnCl2, 1.2% Triton X-100, 25 µmol UDP-[6-3H]-Gal (
10,000 cpm/nmol), and 50100 µg protein. Reactions lacking acceptor were used to determine incorporation into endogenous acceptors and degradation of donor sugar. Reactions were stopped by adding 1 ml cold water after incubation at 37°C for 2 h. Reactions were then passed through a 1-ml column of AG1-X4 (Cl- form) that was subsequently washed with 2 ml water to obtain unbound products. Radioactivity was measured by liquid scintillation counter.
Production of recombinant GlcNAc-TI
A Mgat1 cDNA from mouse (Kumar et al., 1992) was cloned into the pRSETB (Invitrogen) vector to express GlcNAc-TI with a His6 tag. Recombinant GlcNAc-TI protein was produced in Escherichia coli BL21 (DE3) (Invitrogen), bound to HisBind resin (Novagen, Madison, WI), and eluted with 500 mM imidazole. The eluted protein was treated with enterokinase (Biozyme) and electrophoresed on a 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) gel. The gel was stained with Coomassie Blue and the
45-kDa band was cut out and stored in phosphate buffered saline (PBS) containing 0.5 mM Ca2+ and 0.5 mM Mg2+ at 4°C.
Preparation of chicken anti-GlcNAc-TI IgY
About 300 µg recombinant GlcNAc-TI in gel pieces was homogenized in 2 ml PBS and 2 ml Freund's complete adjuvant (Pierce, Rockford, IL). Chickens were injected with 90 µg protein under each wing once every 2 weeks for 6 weeks. Subsequently, two more injections were given in incomplete Freund's adjuvant. Eggs collected daily were processed in batches of five to prepare IgY (Gassmann et al., 1990
). IgY obtained 3 months after the first injection gave an OD405 nm in an enzyme linked immunosorbent assay three- to five-fold higher than pre-immune sera at a dilution of 1:5000. This IgY at a 1:1000 dilution readily detected 100 ng of recombinant GlcNAc-TI by western blot analysis. Affinity-purified antibody was prepared by incubating serum at 1:500 dilution in Tris-buffered saline (TBS) containing 0.05% NP-40 (TBSN) and 5% nonfat dry milk powder for 3 h at room temperature with
100 µg recombinant GlcNAc-TI that had been gel-purified and transferred to polyvinylidene fluoride (PVDF) transfer membrane (PolyScreen, NEN Life Science Products). After four 5-min washes with TBSN and two with TBS, antibodies were eluted in 200 µl 100 mM glycine, pH 2.5, at 4°C for 10 min and neutralized with 1 M Tris-HCl, pH 8.0. This antibody was used at a dilution of 1:50 or 1:100 for western blot analysis.
Western blot analysis
Microsomes were prepared from cells washed in hypotonic lysis buffer (10 mM Tris, pH 7.4, 0.25 mM sucrose) containing protease inhibitors. After 20 min at 4°C, the swollen cells were passed seven times through a cell homogenizer at 4°C. Lysates were centrifuged at low speed (300 rpm) for 10 min to remove nuclei and unbroken cells, at medium speed (2000 rpm) for 10 min to remove lysosomes and mitochondria, and at 100,000xg for 2 h at 4°C to obtain microsomes. Microsomes were dissolved in 1.5% NP-40 in saline with protease inhibitors and 20% glycerol and stored at -80°C. Cell-free extract prepared as for enzyme assay (50 µg protein) or microsomes (50 µg protein) were electrophoresed on a 7.510% SDS-PAGE gel under reducing conditions and transferred to PVDF membrane. After blocking the membrane in 5% nonfat dry milk for 2 h at room temperature or overnight at 4°C, first antibody was added and incubated with the membrane for 1.5 h at room temperature followed by washing with TBSN, five times for 5 min each. The membrane was subsequently incubated with secondary antibody conjugated to horseradish peroxidase at room temperature for 1.5 h. After washing with TBSN five times and TBS twice for 5 min, bound antibody was visualized by incubating the membrane for exactly 1 min with Western Blot Chemiluminescence Reagent (Renaissance, NEN Life Science). A polyclonal sheep antibody raised against denatured rabbit GlcNAc-TI was a gift from Drs. Harry Schachter and Paul Gleeson (Burke et al., 1992; Puthalakath et al., 1996
), a rabbit polyclonal antibody raised against a recombinant rat GlcNAc-TI fusion protein was from Dr. Tohru Komano (Yoshida et al., 1999
), and the chicken antibody to bacterially produced mouse GlcNAc-TI was already described.
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Acknowledgements |
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Footnotes |
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1 To whom correspondence should be addressed; e-mail: stanley{at}aecom.yu.edu
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Abbreviations |
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References |
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