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Originally published In Press as doi:10.1074/jbc.M408755200 on September 13, 2004

J. Biol. Chem., Vol. 279, Issue 48, 49716-49726, November 26, 2004
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LEC12 and LEC29 Gain-of-Function Chinese Hamster Ovary Mutants Reveal Mechanisms for Regulating VIM-2 Antigen Synthesis and E-selectin Binding*

Santosh K. Patnaik, Barry Potvin, and Pamela Stanley{ddagger}

From the Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461

Received for publication, August 2, 2004 , and in revised form, September 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LEC12 and LEC29 are two gain-of-function Chinese hamster ovary glycosylation mutants that express the Fut9 gene encoding {alpha}(1,3)fucosyltransferase IX ({alpha}(1,3) Fuc-TIX). Both mutants express the Lewis X (LeX) determinant Gal{beta}(1,4)[Fuc{alpha}(1,3)]GlcNAc, and LEC12, but not LEC29 cells, also express the VIM-2 antigen SA{alpha}(2,3)-Gal{beta}(1,4)GlcNAc{beta}(1,3)Gal{beta}(1,4)[Fuc{alpha}(1,3)]GlcNAc. Here we show that LEC29 cells transfected with a Fut9 cDNA express VIM-2, and thus LEC29 cells synthesize appropriate acceptors to generate the VIM-2 epitope. Semiquantitative reverse transcription-PCR showed that LEC12 has 10- to 20-fold less Fut9 gene transcripts than LEC29. However, Western analysis revealed that LEC12 has ~20 times more Fut9 protein than LEC29. The latter finding was consistent with our previous observation that LEC12 has ~40 times more in vitro {alpha}(1,3)Fuc-T activity than LEC29. The basis for the difference in Fut9 protein levels was found to lie in sequence differences in the 5'-untranslated regions (5'-UTR) of LEC12 and LEC29 Fut9 gene transcripts. Whereas reporter assays with the respective 5'-UTR regions linked to luciferase did not indicate a reduced translation efficiency caused by the LEC29 5'-UTR, transfected full-length LEC29 Fut9 cDNA or in vitro-synthesized full-length LEC29 Fut9 RNA gave less Fut9 protein than similar constructs with a LEC12 5'-UTR. This difference appears to be largely responsible for the reduced {alpha}(1,3)Fuc-TIX activity and lack of VIM-2 expression of LEC29 cells. This could be of physiological relevance, because LEC29 and parent Chinese hamster ovary cells transiently expressing a Fut9 cDNA were able to bind mouse E-selectin, although they did not express sialyl-LeX.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There are several established biological functions for mammalian {alpha}(1,3)fucosyltransferases ({alpha}(1,3)Fuc-Ts)1 (1), and it is apparent that regulation of {alpha}(1,3)Fuc-T activities at the transcriptional level may occur (2–6). One approach to identify factors that control {alpha}(1,3)Fuc-T expression at a transcriptional, translational, or activity level is to determine the molecular basis of gain-of-function glycosylation mutants that express an {alpha}(1,3)Fuc-T activity de novo (7). For example, three CHO mutants, termed LEC11, LEC11A, and LEC11B, express {alpha}(1,3)Fuc-TVI from either the Fut6A or Fut6B gene (8). These mutants express sialylated Lewis X (sLeX; SA{alpha}(2,3)Gal{beta}(1,4)[Fuc{alpha}-(1,3)]GlcNAc), VIM-2 (CD65s; SA{alpha}(2,3)Gal{beta}(1,4)GlcNAc{beta}(1,3)-Gal{beta}(1,4)[Fuc{alpha}(1,3)]GlcNAc), and Lewis X (LeX; Gal{beta}(1,4)-[Fuc{alpha}(1,3)]GlcNAc) determinants on cellular glycoproteins, and all bind E-selectin (8, 9). Analyses of gene organization and expression of the CHO Fut6A and Fut6B genes in the LEC11 mutants provided insight into the mechanism of {alpha}(1,3)Fuc-T gene activation in each mutant, including evidence for a negative regulatory factor that represses the Fut6B gene in CHO cells (8). Expression cloning has recently identified three suppressors of {alpha}(1,3)Fuc-T activity in LEC11B cells.2

Four {alpha}(1,3)Fuc-T-encoding genes have been characterized in different CHO gain-of-function mutants. The Fut6A and Fut6B genes appear to be orthologous to the Fut3-Fut5-Fut6 cluster in humans (10) and are active in the LEC11 (Fut6B), LEC11A (Fut6A), and LEC11B (Fut6B) mutants (8). The Chinese hamster Fut4 ortholog is expressed in the LEC30 CHO mutant and the Fut9 ortholog is active in the LEC12, LEC29, and LEC30 CHO mutants (11). Neither of the latter mutants express the Fut4 gene (11). LEC12 and LEC29 do not bind anti-sLeX antibody, and their cell extracts do not fucosylate {alpha}(2,3)sialylated N-acetyllactosamine (LacNAc) in vitro (9), indicating that they do not express the Fut6A, Fut6B, or Fut7 genes. Furthermore, Fut6 gene expression was not detected in LEC12 by RNase protection (8) nor in LEC12 or LEC29 by Northern analysis.3 Thus, Fut9 appears to be the only {alpha}(1,3)Fuc-T-encoding gene expressed in these two mutants. It was therefore surprising to find that LEC12 and LEC29 have very different fucosylation patterns (9). Whereas LEC12 cells express the VIM-2 and LeX epitopes at similar levels, LEC29 cells do not express VIM-2 above background in a binding assay with iodinated antibody (9). LEC29 cells are ~50-fold more sensitive to the toxicity of wheat germ agglutinin than LEC12 cells. They are also 3-fold less resistant than LEC12 to the leukoagglutinin from Phaseolus vulgaris termed L-PHA. In addition, LEC12 extracts have {alpha}(1,3)Fuc-T activity of 85.9 pmol/mg of protein/min compared with 2.4 pmol/mg of protein/min activity in LEC29 extracts (9).

CHO transfectants expressing a CHO Fut9 cDNA behave like LEC12 cells, and bind both anti-LeX and anti-VIM-2 antibodies suggesting that LEC29 cells express a compromised Fut9 (11). However, in vitro mixing experiments provided no evidence for a Fut9-degrading or -inhibiting activity in LEC29 cell extracts (11). In this report, we identify 5'-untranslated region (UTR) sequence differences in Fut9 cDNAs from LEC12 and LEC29 and provide evidence that they are largely responsible for the differences in Fut9 levels and fucosylated antigen expression in LEC12 and LEC29. We also show that regulation of Fut9 levels may have physiological significance in terms of E-selectin binding.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies, Chemicals, and Molecular Biology Reagents—Mouse IgM anti-SSEA-1 (anti-LeX) monoclonal antibody (mAb) (12) was prepared previously in the laboratory (13). Purified, FITC-conjugated, mouse anti-LeX mAb (clone AHN1.1) was obtained from Calbiochem. Mouse IgM anti-VIM-2 mAb (14) was purchased from Bioresearch GmbH. Anti-sialyl-LeX mouse IgM mAb CSLEX-1 (15) was purified from HB-8580 hybridoma medium obtained from ATCC. Human IgG1, rabbit, or goat fluorescein isothiocyanate (FITC)-conjugated anti-human IgG, anti-goat IgG, anti-mouse IgM, anti-mouse IgG+A+M, and horseradish peroxidase-conjugated goat anti-human IgG antibodies were purchased from Zymed Laboratories. Goat allophycocyanin-conjugated anti-mouse IgM (H+L) was from Caltag Laboratories. Purified recombinant mouse E-selectin fused to human IgG1 Fc fragment and produced in J558 myeloma cells was a kind gift of Drs. Martin Wild and Dietmar Vestweber, University of Munster, Germany (16). Mouse mAbs KM8621 and KM2681 (IgG1) raised against human Fut9 protein produced in Escherichia coli and purified from mouse ascites (KM8621) or hybridoma supernatant (KM2681) were a kind gift of Dr. Hisashi Narimatsu, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan. Antibody C-17, an affinity-purified goat polyclonal antibody against a carboxyl-terminal peptide of human Fut9, was purchased from Santa Cruz Biotechnology. Mouse anti-Myc monoclonal antibody 9E10 and an anti-mouse {beta}-tubulin antibody were obtained from Dr. E. Richard Stanley, Albert Einstein College of Medicine. Amino acid mix was purchased from Promega. Coomassie Brilliant Blue, Ponceau S, creatine phosphate, and creatine phosphokinase purified from rabbit muscle were obtained from Sigma. Hanks' balanced salts mix was from Mediatech. Lysophosphocholine was supplied by Avanti%20Polar%20Lipids">Avanti Polar Lipids. Peptide N-glycanase F (PNGase F) purified from Flavobacterium meningosepticum was obtained from New England Biolabs. S-Adenosylmethionine was from ICN. Spermidine was purchased from Fluka. Zeomycin was from Invitrogen.

Cell Culture and Transfection—Parent Pro–5 CHO cells (17) and gain-of-function CHO glycosylation mutants LEC11 (18), LEC12 (19), LEC29 (9), and LEC30 (9) CHO cells were isolated previously. Cells were maintained in suspension culture at 37 °C in complete {alpha}-modified Eagle's medium supplemented with 10% fetal bovine serum. Penicillin (100 units/ml) and streptomycin (100 µg/ml) were sometimes added. Cell culture reagents were obtained from Invitrogen and Gemini. Adherent cells were transfected with DNA using Fugene6 (Roche Applied Science) or LipofectAMINE 2000 (Invitrogen) as per protocols suggested by the manufacturers.

Somatic Cell Hybridization—LEC12 and LEC29 cells were transfected with pcDNA3.1 carrying either a neomycin or zeomycin resistance gene (Invitrogen). Stably transfected independent colonies, LEC12-neo, LEC12-zeo, LEC29-neo, and LEC29-zeo, were isolated after selection with neomycin (1.5 mg/ml active weight) or zeomycin (75 µg/ml). LEC12-neo cells were fused with LEC29-zeo cells, and LEC12-zeo cells were fused with LEC29-neo cells as previously described (8). After 8–10 days, hybrid colonies were picked and expanded in culture.

Semi-quantitation of Fut9 Transcripts by RT-PCR—RNA was prepared using 1 ml of TRIzolTM reagent (Invitrogen) per 107 cells. 10 µgof total RNA from LEC12 or LEC29 was reverse-transcribed at 48 °C using 1 µg of oligo(dT) and 100 ng of random hexamers with a mixture of Superscript IITM and avian myeloblastosis virus reverse transcriptases in 40 µl. cDNA products were treated with RNase H (Invitrogen), and 1 µl of cDNA was subjected to PCR for 27, 30, 33, 36, 39, 42, or 45 cycles. Products were electrophoresed on a 1% agarose gel and stained with ethidium bromide. NIH Image software was used to quantitate by densitometry. Fut9 gene-specific primers were: PS394 (5'-TGA CAA CAC AGT GAA GTG GTT CT) and PS229 (5'-CCA CAT GAA TGA ATG AAT CAG CTG G). Glyceraldehyde-3-phosphate dehydrogenase genespecific primers were PS270 (5'-TGA ATT CAT TGA CCT CAA CTA CAT) and PS271 (5'-AGA ATT CTT ACT CCT TGG AGG CCA).

Isolation of Microsomes from Cells—Cells were grown in suspension to a density of 4–5 x 105/ml and pelleted by centrifugation at ~500 g for 10 min at 4 °C. After three washes with isotonic saline, cells were resuspended in 10 mM Tris, pH 7.4, at 6 x 107 cells/ml with 250 mM sucrose and placed on ice for 25 min before being homogenized by seven passages through a Balch ball-bearing homogenizer at 4 °C. Nuclei and unbroken cells were removed by centrifugation at 1,000 x g for 10 min. The post-nuclear supernatant was centrifuged at 10,000 x g for 10 min at 4 °C to pellet the non-microsomal membrane fraction. Microsomes from the supernatant were isolated by centrifugation at 100,000 x g for 1 h at 4 °C. Both microsomal and non-microsomal membrane pellets were resuspended (3 x 105 cell equivalents/µl) in 1.5% Triton X-100 containing Complete EDTA-free protease inhibitor mixture (Roche Applied Science) and stored at –80 °C in 20% glycerol.

Western Analysis—Protein concentrations were determined for membrane fractions and cell lysates by the Bio-Rad Dc assay. Digestion of 100–200 µg of protein with 500 units of PNGase F was performed at 37 °C overnight. Digests were diluted in Laemmli SDS-PAGE sample loading buffer (20) containing {beta}-mercaptoethanol (final concentration, 0.1 M), boiled 5 min, and electrophoresed under reducing conditions on denaturing Tris-glycine SDS-polyacrylamide gels. Gels (7.5% or 10%) were either cast using Mini-Protean (Bio-Rad) or Sturdier (Hoefer Scientific Instruments) apparatus or were purchased from Bio-Rad. Benchmark, MagicMark, or Mark12 protein molecular weight markers (Invitrogen) were loaded in separate lanes. Staining of proteins was with Coomassie Brilliant Blue dye or Gelcode Blue stain (Pierce Biotechnology). For immunoblotting, proteins were electrotransferred overnight to a Polyscreen polyvinylidene difluoride membrane (PerkinElmer Life Sciences) in Tris-glycine buffer containing 8–12% methanol. To assess transfer and protein loading, membranes were sometimes stained with Ponceau S. Subsequent incubations were for 1–2 h at room temperature or overnight at 4 °C. Membranes were blocked in 5% nonfat dry milk (Nestle) in Tris-buffered saline (150 mM NaCl, 10–20 mM Tris, pH 7.3–7.5) with 0.05% Tween 20 or Nonidet P-40 detergent before addition of antibodies. KM8621 and C-17 anti-Fut9 antibodies were used at 1:1,000–2,000 and 1:200–400 dilutions, respectively. Anti-tubulin antibody was used undiluted and antibody KM2681 was diluted 1:1. Secondary antibodies were used at 1:5,000–10,000 dilutions. Chemiluminescent detection was by RenaissanceTM (PerkinElmer Life Sciences) or SuperWest PicoTM (Pierce Biotechnology) reagents and exposure to BiomaxTM MR x-ray films (Kodak).

Anti-VIM-2, Anti-sLeX, and Anti-LeX Binding to Cells by Flow Cytometry—Approximately 2 x 105 cells washed twice with PBS (pH 7.2, calcium- and magnesium-free) or Hanks' buffered salt solution (HBSS, 0.4 g/liter KCl, 0.06 g/liter K2HPO4, 8 g/liter NaCl, 0.048 g/liter Na2HPO4, 1 g/liter D-glucose) were preincubated in 200–400 µl of PBS or HBSS containing 2% BSA at 4 °C for 30 min. Cells were washed once with 3 ml of PBS or HBSS with BSA and resuspended in 200–400 µlof PBS or HBSS with BSA with 1–2 µg of CSLEX-1, anti-SSEA-1, or anti-VIM-2 monoclonal antibody. After incubation at 4 °C for 30 min, cells were washed once and incubated with 1–2 µg of rabbit anti-mouse IgM conjugated to FITC. After 30 min at 4 °C, cells were washed once and resuspended in 200 µl of PBS/BSA for analysis using a FACScan flow cytometer (BD Biosciences). Propidium iodide was added at 1–2 µg/ml just prior to cytometry. All fluorescence data were collected using logarithmic amplification on 10,000–15,000 events (cell counts). Events considered for analysis were gated for either light scatter or low propidium iodide binding, or both. For double staining, an allophycocyanin-conjugated, goat anti-mouse secondary antibody (0.3–1 µg) was used instead of the FITC secondary antibody. Cells were washed with HBSS and incubated with 2 µg of FITC-conjugated anti-LeX antibody in a volume of 400 µl for 1 h at 4 °C. Fluorescence cytometry was performed on a FACSCaliburTM cytometer (BD Biosciences) after a final wash with HBSS.

{alpha}(1,3)Fucosyltransferase Assay—{alpha}(1,3)Fuc-T activity was measured with LacNAc as acceptor as described previously (9). Each reaction contained 2.5 mmol of 2-morpholinophenylsulfonate buffer (pH 7.0), 5 mmol of NaCl, 0.25 mmol of MnCl2, 2–4 nmol of GDP-[14C]fucose (~5000 cpm/nmol), 0.1 mmol of LacNAc (Dextra Labs), and 10 µl of cell extract (~50–100 µg of protein) prepared in buffer containing 1.5% Triton X-100 in 50 µl. After incubation at 37 °C for 60 min, the reaction was stopped by the addition of 1 ml of ice-cold water, and reaction products were separated on a small AG-1X4 (Bio-Rad) column as described previously (21). Eluates were mixed with Ecolume (ICN Biochemicals) and counted in a scintillation counter (Beckman Coulter). Specific activities were calculated after subtracting the counts per minute incorporated into endogenous acceptors.

Rapid Amplification of cDNA Ends—The GeneRacer kit (Invitrogen) was used for 5'-RACE of LEC12 and LEC29 Fut9 transcripts. Reverse transcription of LEC12 and LEC29 mRNA was performed using a mixture of Superscript IITM and avian myeloblastosis virus reverse transcriptases at 48 °C. RNase H-treated cDNA was amplified with PlatinumTM Pfx polymerase using Fut9 gene-specific primer PS432 (5'-TAG TGA GAT GGC ACC CTT GG) and GeneRacerTM 5'-primer specific for the ligated oligonucleotide (5'-CGA CTG GAG CAC GAG GAC ACT GA). The PCR products were subjected to nested PCR using Fut9-specific primer PS537 (5'-TGT TGG TGG GCT TGA TGT AA) and GeneRacer 5'-nested primer (5'-GGA CAC TGA CAT GGA CTG AAG GAG TA). PCR products were TA-cloned into pCR2.1 vector, and the inserts of at least three clones were sequenced using vector-specific M13R and T7 primers.

Generation of LEC12 and LEC29 Full-length Fut9 cDNA Constructs—Full-length Fut9 cDNA, containing the 5'-UTR, 3'-UTR, and coding regions, was amplified from LEC12 cDNA using Platinum Pfx polymerase with primers PS616 (5'-CAC TCG AAG TTT CTG CCA GCC TGC CCT CTG CTC TCC TAA TGG A) and PS519 (5'-TTT TAA TGA AAG AAT AGA CAT TTC AAG). Taq polymerase was used to add 3'-A overhangs to the purified product, which was cloned into pcDNA3.1/V5-His TOPO vector (Invitrogen). Sequencing confirmed that the 5'-UTR was intact. The plasmid with a Fut9 cDNA insert of 2251 bp was designated pCMV-Full12F9. Similarly, a cDNA containing the 5'-UTR and coding regions of LEC29 Fut9 was obtained using primers PS617 (5'-TCT GCT TCT AGG ACA GCG CCG CCA CCG CCG AAA GC) and PS343 (5'-TTA ATT CCA AAA CCA TTT CTC TAA ATT ACC CAC AG). This cDNA and pCMV-Full12F9 were digested with KpnI and BspEI restriction enzymes. KpnI cuts pcDNA3.1/V5-His TOPO once, just upstream of the cloning site, whereas BspEI cuts both LEC12 and LEC29 Fut9 cDNA once, at positions 793 and 792, respectively. The KpnI/BspEI-released fragment from the LEC29 clone was cloned into KpnI/BspEI-digested pCMV-Full12F9 to generate the LEC29 full-length construct pCMV-Full29F9 with an insert size of 2250 bp. The LEC12 and LEC29 constructs were identical to each other, except for the first exon in their respective 5'-UTRs.

Generation of Myc-tagged Fut9 and Fut11 Constructs—The Fut9 gene ORF was amplified from pCMV-Full12F9 with Platinum Pfx polymerase using primers PS665 (5'-CCA CCA TGG AAC AAA AAC TCA TCT CAG AAG AGG ATC TGA CAT CAA CAT CCA AAG GCA TTC TTC GTC C) and PS343 (5'-TTA ATT CCA AAA CCA TTT CTC TAA ATT ACC CAC AG). The former also encodes the c-Myc peptide sequence EQKLISEEDL. AmpliTaq polymerase was used to add 3'-A overhangs to the purified product, which was then TA-cloned into pCR3.1 (Invitrogen). An N-terminal Myc-tagged mouse Fut11 expression construct was similarly generated by amplification from mouse placenta cDNA using primers PS686 (5'-CCA CCA TGG AAC AAA AAC TCA TCT CAG AAG AGG ATC TGG CTG CTC GCT GTA CCG AGG CGG TGC TGG CCG) and PS553 (5'-ATC CTC GAG GAT TTT TAT TCC GTT TCA TGA AGA TCT).

Generation of Humanized Renilla Luciferase Reporter Constructs— The CMV promoter region of pSecTag2C-hygro vector (Invitrogen) was amplified with Platinum Pfx polymerase using primers PS650 (5'-ATG CAT CTC GAG CGA TGT ACG GGC CAG ATA TAC GCG, with an XhoI restriction site) and PS651 (5'-ATG CAT GGC GCC GTT AGC CAG AGA GCT CTG CTT ATA TAG, with an NarI restriction site). XhoI/NarI-digested PCR products were cloned into phRL-null vector (Promega) between the XhoI and NarI sites to obtain phRL-CMV. The phRL-null vector has a T7 promoter upstream of humanized Renilla luciferase coding region such that the putative transcription start site is 12-nt upstream of the ATG start codon. For phRL-CMV, the CMV promoter-driven transcriptional start site is 260 nt upstream. Complete 5'-UTRs of LEC12 and LEC29 Fut9 were amplified from pCMV-Full12F9 and pCMV-Full29F9 cDNAs, respectively. The forward primers, bearing the T7 promoter region and a NarI restriction site were for LEC12: PS647 (5'-ATG CAT GGC GCC TAA TAC GAC TCA CTA TAG GGA GCA CTC GAA GTT TCT GCC AGC CTG CC); and for LEC29, PS649 (5'-ATG CAT GGC GCC TAA TAC GAC TCA CTA TAG GTC TGC TTC TAG GAC AGC GCC GCC ACC G). The reverse primer PS639 (5'-GCA TGC TAG CAA TTT TTT ACT GTA GAG AAC AGC ATG G) had an NheI restriction site. NarI/NheI-digested PCR products were cloned between the NarI and NheI sites of phRL-null to generate phRL-T7U12 and phRL-T7U29, T7 promoter-driven luciferase constructs fused with LEC12 and LEC29 Fut9 5'-UTRs, respectively. In both of them, the transcription start site is 2 nt upstream of the first base of the 5'-UTR whose end lies 11 nt upstream of the luciferase ATG start codon. To generate the CMV promoter-driven luciferase constructs, phRL-CMVU12 and phRL-CMVU29, which have the luciferase coding region fused with the 5'-UTRs of Fut9, a similar strategy was used, except that the forward primers lacked the T7 promoter region: LEC12, PS646 (5'-ATG CAT GGC GCC GAG CAC TCG AAG TTT CTG CCA GCC TGC C); LEC29, PS648 (5'-ATG CAT GGC GCC TCT GCT TCT AGG ACA GCG CCG CCA CCG). In both these constructs, the transcription start site is 5 nt upstream of the first base of the 5'-UTR whose end lies 11 nt upstream of the luciferase ATG start codon.

In Vitro Transcription—Capped RNA was produced from linearized plasmid DNA using Ribo m7G cap analog (Promega) with the T7 RNA polymerase-based RiboMAX in vitro transcription kit (Promega). Plasmids were linearized with XhoI for pCMV-Full12F9 and pCMV-Full29F9, and XbaI for phRL-null, phRL-T7U12, and phRL-T7U29. The quality and quantity of purified RNA was assessed by spectrophotometry at 260 nm and electrophoresis on agarose gel followed by ethidium bromide staining.

Preparation of Translational Extracts from CHO, LEC12, and LEC29 Cells—The method of Favre and Trepo (22) that incorporates sucrose and dimeric creatine kinase in lysis buffer was followed. Cells were grown to 90% confluence on a 15-cm plate. After washing once with 12 ml of cold wash buffer (150 mM sucrose, 33 mM NH4Cl, 7 mM KCl, 45 mM Mg[CH3COO]2, 30 mM HEPES, pH 7.4), 4.5 ml of cold wash buffer containing 100 µg/ml 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysophosphocholine) was added evenly over the plate at room temperature. It was aspirated after 90 s and 0.45 ml of cold extraction buffer (100 mM sucrose, 100 mM HEPES-KOH, pH 7.4, 120 mM KCH3COO, pH 7.4, 2.5 mM Mg[CH3COO]2, 1 mM dithiothreitol, 2.5 mM ATP, 1 mM GTP, 0.1 mM S-adenosylmethionine, 1 mM spermidine, 20 mM creatine phosphate, 40 units/ml creatine phosphokinase, 40 µM each amino acid mix) was spread over the cells. After 90 s at 4 °C, cells and buffer were scraped off and passed twice through a 26-gauge needle. After centrifugation at 100 x g for 2 min, the supernatant was aliquoted and stored at –80 °C. Protein concentration was measured using Bio-Rad Dc assay with BSA as standard.

In Vitro Translation—The rabbit reticulocyte lysate-based TNT-coupled transcription-translation kit (Promega) was used as per the manufacturer's protocol with both RNA and DNA templates. Translation reactions using 15 µl of CHO cell extracts were performed in 20 µl containing in vitro transcribed RNA and 1 µl (50 µg) of creatine phosphokinase. When required, [35S]methionine or EasyTag ExpressTM 35S protein labeling mix (PerkinElmer Life Sciences) was added to label-translated products. Translation reactions were directly used in luciferase assays or electrophoresed on SDS-PAGE gels. Gels were either used for immunoblotting or dried and exposed to BioMaxTM MR x-ray films (Kodak).

Luciferase Assays—Cells grown in 6-well plates were transfected with Renilla reniformis (sea pansy) luciferase reporter constructs using Fugene6 DNA transfection reagent. For normalization of transfection efficiency, a firefly (Photinus pyralis) reporter construct containing the Eµ promoter of the mouse immunoglobulin heavy chain gene (a kind gift of Dr. Barbara Birshtein, Albert Einstein College of Medicine) was cotransfected. Activities from both luciferases were measured sequentially from a single sample using the Dual-Luciferase reporter assay system from Promega. Cells were lysed after 2–3 days using 250 µl of Passive Lysis buffer (Promega). Lysates were assayed immediately or after storage at –20 °C. For in vitro translation reactions, samples were diluted 5- to 20-fold in Passive Lysis buffer. Measurements were performed on a Turner Designs TD-20e luminometer with a 2-s pre-read delay and a 10-s measurement period.

Transfection of CHO Cells with RNA—A TransMessengerTM RNA transfection kit from Qiagen was used to transfect adherent cells in 6-well plates as suggested by the manufacturer. For each well, 1–2 µg of RNA was complexed with the lipid-based transfection reagent. Cell monolayers, at 40–60% confluence, were washed once with 4 ml of PBS, and the RNA/transfectant complex was added for 2–3 h. The plates were then washed with 4 ml of PBS before adding 2 ml of {alpha}-minimal Eagle's medium/10% fetal bovine serum. Cells were harvested 18–24 h later.

Binding of Mouse E-selectin-IgG Chimera—To measure mouse E-selectin-IgG binding (16), 8 x 105 cells preincubated in HBSS/2% BSA for 1 h at 4 °C, were incubated with 6 µg of human IgG1 or E-selectin-IgG chimera protein for 1 h at 4 °C in 400 µl of HBSS/2% BSA containing 1 mM CaCl2 or 1 mM CaCl2 and 5 mM EDTA. Cells were washed once with HBSS/2% BSA and resuspended in 400 µl of HBSS/2% BSA containing 3 µg of FITC-conjugated goat anti-human IgG for 1 h at 4 °C. They were washed with HBSS and analyzed for FITC fluorescence on a FACSCalibur instrument.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LEC29 Cells Do Not Possess Inhibitors of VIM-2 Synthesis— LEC29 CHO cells have more Fut9 gene transcripts than LEC12 but ~40-times less {alpha}(1,3)Fuc-T activity (9, 11). LEC12 cells express the VIM-2 epitope on cell surface glycoproteins, but VIM-2 is barely detectable on LEC29. To determine whether activation of a negative factor or repression of a positive factor down-regulates expression of VIM-2 on the LEC29 mutant, somatic cell hybrids formed with LEC12 cells were examined for VIM-2 expression. This approach previously showed that CHO Fut6 genes may become active due to a cis mechanism correlated with gene rearrangement or a trans mechanism due to the loss of a negative regulatory factor that represses Fut6B expression in CHO cells (8). Neomycin or zeomycin resistance genes were introduced into LEC12 and LEC29 cells, allowing post-fusion selection for hybrid colonies in medium containing both antibiotics. Upon analysis by fluorescence cytometry, independent LEC12 x LEC29 hybrids were found to be as positive as LEC12 cells for cell surface VIM-2 expression (Fig. 1). Therefore, LEC29 cells do not express a dominant factor that represses VIM-2 synthesis.



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  		FIG. 1.
Somatic cell hybrids of LEC12 and LEC29 express VIM-2 on the cell surface. LEC12 cells stably transfected with neomycin or zeomycin resistance genes (LEC12-neo and LEC12-zeo) were fused with complementary LEC29 cells (LEC29-zeo and LEC29-neo, respectively). Hybrid clones (LEC12-neo x LEC29-zeo and LEC12-zeo x LEC29-neo) and parent cells (LEC12-neo, LEC12-zeo, LEC29-neo, and LEC29-zeo) were tested for cell surface expression of the VIM-2 antigen by fluorescence cytometry as described under "Materials and Methods."

 
LEC29 Cells Have All the Factors Necessary for Expression of VIM-2—The VIM-2 epitope includes fucose, sialic acid, and two lactosamine units. Thus, the virtual absence of VIM-2 on LEC29 could be due to a defect in the generation of polylactosamines or {alpha}(2,3)sialylation or {alpha}(1,3)fucosylation. It is also possible that one or just a few glycoconjugates carry the VIM-2 epitope. Thus the anti-sLeX mAb CSLEX-1 appears to detect one major sLeX-carrying protein in CHO-K1 cells transfected with a Fut7 cDNA (23). To determine whether overexpression of Fut9 in LEC29 would generate VIM-2 as it does in CHO cells (11), the coding region of the Fut9 gene cloned into pCR3.1 (11) was transiently transfected into LEC29 cells. Fut9 transfectants were positive for cell surface VIM-2 expression by fluorescence cytometry (Fig. 2A). Thus LEC29 cells have the acceptors necessary to generate the VIM-2 antigen. Coexpression of LeX and VIM-2 structures was also examined on individual cells in transfectant populations stably expressing a Fut9 cDNA by fluorescence cytometry. Depending on the site of integration in the genome and number of integrated copies, cells of these stable populations will vary in their expression of Fut9 protein. In Fig. 2B, it can be seen that there was a wide range of expression of fucosylated antigens. However, there was a direct correlation between LeX and VIM-2 expression in both CHO and LEC29 transfectant populations. This indicates that the level of Fut9 must be increased in LEC29 cells to allow synthesis of VIM-2.



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  		FIG. 2.
A, the VIM-2 antigen is generated on LEC29 cells expressing a Fut9 cDNA. LEC29 cells were transiently transfected with either pCR3.1 vector (LEC29 + vector) or pCR3.1-Fut9 plasmid carrying the LEC29 Fut9 coding region (LEC29 + Fut9). Cell surface expression of VIM-2 was determined after 72 h for transfectants compared with untransfected LEC12 and LEC29 cells by fluorescence cytometry. B, the expression of VIM-2 and LeX correlate in Fut9 cDNA transfectants. CHO and LEC29 cell populations stably transfected with a Fut9 coding region cDNA were examined for the expression of LeX and VIM-2 antigens by fluorescence cytometry. The LeX antibody was conjugated with FITC. VIM-2 antigen was detected by an allophycocyanin-conjugated secondary antibody. Dotplot diagrams show simultaneous binding of both antibodies to individual cells. The secondary (2°) antibody control had no primary antibodies.

 
Fut9 Gene Transcripts in LEC12 and LEC29 Cells—Although Fut9 gene transcripts can be detected by RT-PCR in LEC12 and LEC29, a signal was not observed for LEC12 by Northern blot analysis (11). Therefore, a semi-quantitative RT-PCR study was performed to compare the relative levels of Fut9 gene transcripts in the two cell lines. Total RNA of LEC12 or LEC29 was reverse-transcribed, and the cDNA was used as template for amplification of glyceraldehyde-3-phosphate dehydrogenase or Fut9 genes by PCR for different numbers of cycles. Forward and reverse primers were designed to recognize sequences on separate exons to ensure amplification of cDNA but not genomic DNA sequences. Glyceraldehyde-3-phosphate dehydrogenase products were detected in the 27-cycle reactions for both LEC12 and LEC29, and product increased almost linearly to 42 cycles (Fig. 3A). Fut9 cDNA products were easily detected in the 39-cycle reaction for LEC29 but not until the 45-cycle reaction for LEC12. After a longer exposure (Fig. 3A), LEC12 products were visible at 33 cycles. Thus, LEC12 expresses ~10- to 20-fold fewer Fut9 gene transcripts than LEC29.



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  		FIG. 3.
LEC12 cells have fewer Fut9 transcripts but more Fut9 protein than LEC29 cells. A, semi-quantitative RT-PCR of Fut9 RNA. LEC12 or LEC29 cDNA was amplified using glyceraldehyde-3-phosphate dehydrogenase or Fut9 gene-specific primers in reactions with different numbers of PCR cycles. Products were separated on an agarose gel and stained with ethidium bromide for imaging. The lowest panel is a longer exposure to better reveal LEC12 Fut9 transcripts. B, Western analysis was performed after electrophoresis in a 7.5% reducing SDS-PAGE gel and transfer to membrane, using anti-human Fut9 mAb KM8621. Each lane had 100 µg of protein from 1.5% Nonidet P-40 detergent extracts made from CHO, LEC12, or LEC29 cells. PNGase F treatment was used prior to electrophoresis to remove N-glycans. The star indicates a non-Fut9 cross-reacting band present in all CHO cells. C, microsomes and a non-microsomal fraction were prepared from CHO, LEC12, and LEC29 cells as described under "Materials and Methods," and 50 µg of protein from each were treated with PNGase F overnight. Immunoblotting to detect Fut9 was performed with mAb KM8621 after electrophoresis in a 7.5% reducing SDS-PAGE gel and transfer to membrane. The lower panel shows a longer exposure of the microsomal fraction. D, Fut9 protein is not selectively degraded in LEC29 cells. CHO, LEC12, and LEC29 cells were transiently cotransfected with expression plasmids for mouse Fut9 and Fut11 proteins with Myc tag at the N terminus. Cell extracts in 1.5% Nonidet P-40 were prepared 3 days after transfection. Western analysis using an anti-Myc antibody was performed after electrophoresis of 100 µg of protein in a 7.5% reducing SDS-PAGE gel and transfer to polyvinylidene difluoride membrane.

 
LEC12 Cells Have Higher Levels of Fut9 Protein Than LEC29 —The CHO Fut9 protein has a deduced molecular mass of 42.2 kDa and three predicted N-glycosylation sites (11). Anti-Fut9 KM8621 antibody identified two major proteins in a reducing SDS-polyacrylamide gel (Fig. 3B). A Fut9 band at ~51 kDa was readily detected in LEC12, was present at a very low level in LEC29, and was missing from CHO lysates. The ~55-kDa protein marked with a star in Fig. 3B is a nonspecific band unrelated to Fut9, because it is present in CHO cells that do not express the Fut9 gene (11). Fut9 was further resolved from the nonspecific protein by treatment with PNGase F to remove N-glycans prior to SDS-PAGE. The position of the nonspecific band was not altered by this treatment while the Fut9 band moved to ~43 kDa (Fig. 3B). A probable Fut9 dimer at ~80 kDa was present only in LEC12. Further evidence for Fut9 assignment was obtained by cell fractionation. Microsomal membranes from post-nuclear cell extracts were enriched in Fut9 protein and contained much less of the ~55-kDa unknown protein compared with the non-microsomal fraction (Fig. 3C). This was the expected location of Fut9, which functions in the Golgi compartment. The amount of Fut9 protein was at least 20-fold higher in the LEC12 microsomal fraction compared with LEC29, and it was not detected in CHO. One explanation for this difference is that Fut9 protein in LEC29 gets degraded fast. Because available antibodies against Fut9 protein were ineffective for immunoprecipitation, pulse-chase studies to estimate the half-life of Fut9 protein in cells could not be performed. However, transfected myc-tagged Fut9 protein was found not to be selectively degraded in LEC29 cells (Fig. 3D). Differences at the level of translation were therefore investigated.

Sequence Differences in the 5'-UTR of Fut9 Transcripts in LEC12 and LEC29 —The coding region (1080 bp) of Fut9 cDNA from LEC12 and LEC29 have an identical sequence (11). Sequences of the 3'-UTR (969 bp) of Fut9 cDNA from LEC12 and LEC29 and the 92 bases immediately upstream of the coding region were also identical for the two sequences. This region of the hamster 5'-UTR is 70–80% identical to 5'-UTR sequences of mouse and rat Fut9 genes. However, the sequence further 5' was completely different in LEC12 and LEC29 cDNAs (Fig. 4A). The segment (1–109 nt) of the LEC29 5'-UTR sequence is most similar (80–90%) to 5'-UTRs of Fut9 genes from mouse and rat, strongly suggesting that the LEC29 sequence represents the 5'-UTR of the Chinese hamster Fut9 gene. By contrast the LEC12 sequence (1–110 nt) appears to have come from another chromosome. A BLAST query showed that regions on mouse chromosome 6 and on rat chromosome 4 have significant similarity (75–85%) to the first 110 bp of the LEC12 5'-UTR sequence. Because the Fut9 gene is located on a different chromosome from this sequence in mouse and rat (chromosomes 4 and 5, respectively), it appears that a translocation occurred upstream of the Fut9 gene in LEC12 cells.



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  		FIG. 4.
LEC12 and LEC29 5'-UTR sequences differ in exon 1. A, alignment of 5'-UTR sequences of Fut9 cDNAs from LEC12 and LEC29. Residues shown in black belong to the putative first exon of the CHO Fut9 gene. Translation start and stop codons are underlined. B, the diagram shows the three exons of the CHO Fut9 gene as a cDNA with the positions of the primers used. Products obtained from reverse transcription reactions of LEC12 or LEC29 total RNA (+ and – RT enzyme) were subjected to PCR using reverse primer PS229 (specific to the Fut9 coding region) and forward primers PS616 specific for exon 1 of LEC12 Fut9 or PS617 specific for exon 1 of LEC29 Fut9. Genomic DNA (gDNA) gave no product as expected.

 
A 217-nt 5'-UTR from the Fut9 gene expressed in the LEC30 CHO mutant (11) was also obtained by the GeneRacer strategy. This mutant has more Fut9 gene transcripts and ~5x more Fut9 protein than LEC12 (11).3 The first eight bases upstream of the LEC30 Fut9 coding region were identical to the 5'-UTR sequence of LEC12 and LEC29. However, the next stretch of 180 bp is 87% identical to the 5'-region of the 6.4-kb Chinese hamster Intracisternal A Particle 34 element sequence (24) postulated to be a defective retroviral sequence. It is estimated that there are 80 copies of the Chinese hamster Intracisternal A Particle element per haploid genome of CHO cells (24).

The identity of the first eight bases upstream of the coding region of the Fut9 gene in all three mutants, and of the next 84 bases in LEC12 and LEC29, indicates that the CHO Fut9 gene is composed of at least three exons, similar to human, mouse, and rat, which have three Fut9 exons separated from each other by large introns (~90–120 kb). The coding region and 3'-UTR lie in the third exon in each species. RT-PCR using a forward primer specific for either the LEC12 or LEC29 Fut9 exon 1 (PS616 or PS617, respectively) and a reverse primer specific from the Fut9 coding region showed that the LEC12 and LEC29 5'-UTRs differ from each other in the first exon (Fig. 4B). No products were obtained using the same primers with genomic DNA as template. A newly identified 121-nt exon that is present in mouse cDNAs from embryonic stem cells (GenBankTM accession number AK049122 [GenBank] ) and cecum (AK033607 [GenBank] ) between mouse exons 1 and 2 was not present in the LEC12 or LEC29 cDNAs.

The sizes of the LEC12 and LEC29 5'-UTR sequences obtained using the GeneRacer 5'-RACE strategy to amplify cDNA molecules containing a capped 5'-end are consistent with the sizes of full-length Fut9 transcripts in CHO cells (11). In addition, primer extension analysis performed on LEC29 mRNA with two primers gave major products that were ~10 bases longer or ~35 bases shorter than obtained by 5'-RACE (data not shown). Fut9 RNA from both LEC12 and LEC29 was found equivalently associated with polysomes by RT-PCR of fractions across a polysome gradient (data not shown), and thus effects of the different 5'-UTRs on translation were investigated.

The LEC29 Fut9 5'-UTR Efficiently Translates a Reporter Transcript—To investigate effects of the different LEC12 and LEC29 5'-UTRs on translation efficiency, reporter cDNA constructs containing the respective 5'-UTRs upstream of Renilla luciferase and downstream of a T7 or CMV promoter were generated. CHO, LEC12, or LEC29 cells were transfected, and cell lysates were analyzed after 2–3 days. Surprisingly, all three cell types expressing the LEC29 5'-UTR construct had 3–5 times more activity than those transfected with the LEC12 5'-UTR reporter (Fig. 5A). There was no apparent difference in reporter gene transcripts between transfectants based on Northern analysis (data not shown). When the same cDNA constructs were assayed by coupled transcription-translation using the rabbit reticulocyte lysate-based TNT system, the presence of either the LEC12 or LEC29 5'-UTR inhibited translation ~4–5 times compared with a construct that had no 5'-UTR. However, in this case there was no significant difference between luciferase activity generated from LEC12 and LEC29 5'-UTR constructs (data not shown).



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  		FIG. 5.
Effect of LEC12 and LEC29 Fut9 5'-UTRs on the translation of Renilla luciferase. A, CHO, LEC12, and LEC29 cells were transiently transfected with an expression plasmid containing the Renilla luciferase coding region downstream of either the LEC12 or LEC29 Fut9 5'-UTR. A firefly luciferase construct was cotransfected to normalize variability in transfection efficiency. After 48 h cell extracts were assayed for both luciferase activities. Data are plotted as the ratios of Renilla:Firefly luciferase activity. B, in vitro translation of Renilla luciferase reporter RNA by CHO, LEC12, and LEC29 translation extracts. RNA containing no 5'-UTR or the entire LEC12 or LEC29 Fut9 5'-UTR was transcribed from linearized plasmids as described under "Materials and Methods." Capped RNA (1.5 µg) was subsequently translated in cell extracts at 30 °C for 2 h. A 2-µl aliquot from the 25-µl reaction was assayed for luciferase activity. For both A and B error bars indicate mean and range of data acquired in duplicate transfections or translation reactions. Similar results were obtained in two independent experiments in A and B.

 
Similar results were obtained from translation of in vitro generated RNA by translation extracts prepared from CHO, LEC12, or LEC29 cells, respectively. m7G-capped reporter RNA synthesized in vitro using T7 RNA polymerase was translated by each translation extract (Fig. 5B) and by a rabbit reticulocyte lysate (data not shown). As expected, the absence of a 5'-UTR improved translation considerably. However, the different LEC12 and LEC29 5'-UTRs did not differentially affect translation of the in vitro synthesized reporter luciferase RNA by any of the cell extracts (Fig. 5B).

The LEC29 5'-UTR Inhibits Translation from Full-length Fut9 cDNA Constructs—The absence of an inhibitory effect of the LEC29 Fut9 5'-UTR in luciferase reporter studies suggested that the 5'-UTR may interact with other regions of the transcript to regulate Fut9 mRNA translation. To address this, full-length Fut9 cDNA from LEC12 and LEC29 containing the 5'-UTR, coding region, and 3'-UTR, and differing only in the region corresponding to exon 1, were cloned into the pcDNA3.1/V5-His TOPO mammalian expression vector. As shown in Fig. 6A, lysates from LEC29 cells transfected with vector had a small amount of Fut9 protein detected by a polyclonal antibody. There was no nonspecific band with this commercial antibody. LEC29 cells transfected with the Fut9 ORF had the largest amount of Fut9 protein, including the dimer form. However, full-length LEC29 cDNA gave rise to much less Fut9 protein than observed in LEC29 cells transfected with full-length LEC12 cDNA. The levels of {beta}-tubulin in each lysate were equivalent. In vitro {alpha}(1,3)Fuc-T enzyme activity in cell lysates were 90.2 (Fut9 coding region), 17.2 (full-length LEC12), and 5.3 (full-length LEC29 cDNA) pmol/mg of protein/min, respectively, correlating approximately with differences in Fut9 protein levels.



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  		FIG. 6.
The LEC29 Fut9 5'-UTR inhibits the translation of full-length Fut9 cDNA. LEC29 cells were transiently transfected with pCR3.1 vector alone, or vector containing the Fut9 gene coding region, or with a pcDNA3.1/V5-His TOPO vector containing full-length (5'-UTR plus coding region plus 3'-UTR) LEC12 or LEC29 Fut9 cDNA. A, lysates (~100 µg of protein) from LEC29 transfectants prepared in 1.5% Triton X-100 were analyzed for Fut9 protein level by immunoblotting with polyclonal C-17 anti-Fut9 antibody followed by anti-{beta}-tubulin antibody. B, diagram of the CHO Fut9 cDNA coding transcript. There are six ATGs that are in-frame with the stop codon. Sizes of products arising from initiation of translation from each of the six ATGs are indicated as number of amino acid residues and predicted molecular weight. 0.5 or 1 µg of Fut9 coding region (Fut9 ORF), full-length LEC12 Fut9 (LEC12Fut9), or full-length LEC29 Fut9 (LEC29Fut9) cDNA was used as template in in vitro coupled transcription-translation reactions containing [35S]methionine. Reaction products were electrophoresed on a 7.5% reducing SDS-PAGE gel and treated for autoradiography. Exposure to film was for 36 h at –80 °C. The dash indicates no cDNA added. C, Western analysis of in vitro translation reactions. In vitro transcribed, capped, full-length LEC12 (12) or LEC29 (29) Fut9 RNA (1.5 µg) was translated in translation extracts prepared from CHO, LEC12, or LEC29 cells, and products were separated by SDS-PAGE, transferred to membrane, and immunoblotted with monoclonal KM2681 anti-Fut9 mAb. The star identifies the nonspecific band identified by this antibody. D, LEC29 cells were transfected with in vitro transcribed, full-length, LEC12, or LEC29 Fut9 RNA (2 µg) using the TransMessenger reagent. Cell lysates in 1.5% Triton X-100 were prepared after 20 h (50 µl of lysate per plate) and assayed for {alpha}(1,3)Fuc-T activity using LacNAc as substrate. Error bars indicate mean and range of data acquired in duplicate assays of lysates from duplicate transfections. Similar results were obtained in two experiments.

 
To determine if the reduced translation of LEC29 Fut9 cDNA observed in transfectants was reflected by in vitro assays, full-length Fut9 cDNAs were subjected to in vitro transcription/translation in the coupled TNT reticulocyte lysate system. The coding region of the hamster Fut9 gene has six in-frame translation start codons that could generate multiple products (Fig. 6B). Three major species were obtained from the Fut9 coding region construct. Both LEC12 and LEC29 full-length Fut9 constructs gave fewer products, with a major band at 37.4 kDa in each case. The intensity of this band was less for full-length LEC29 Fut9 cDNA.

To investigate further, in vitro translation of full-length, capped Fut9 RNAs was examined using translation extracts prepared from CHO, LEC12, or LEC29 cells (Fig. 6C). Addition of in vitro synthesized RNAs to each translation extract resulted in several translation products of different sizes. The nonspecific band at ~55 kDa detected by this mAb (Fig. 3B) is marked with a star and provides evidence for relative loading. The ~42-kDa band present only in LEC12 extracts probably represents endogenous, unglycosylated Fut9 protein, because it was absent in the "-RNA" reactions of CHO and LEC29 extracts. Also, the intensity of this band was not increased when RNA was added, apparently because translation of this species did not occur efficiently in the extracts. Signals from the ~37-, ~32-, and ~26-kDa species with full-length LEC12 Fut9 RNA were more intense than those with full-length LEC29 Fut9 RNA in reactions with all three translation extracts, even though the CHO extract was not as active as LEC12 or LEC29 extracts. It is clear from these in vitro translation assays that full-length LEC29 Fut9 RNA with 5'- and 3'-UTRs is translated less efficiently than full-length LEC12 Fut9 RNA.

To assay full-length Fut9 RNAs in vivo, LEC29 cells were transfected with capped, in vitro synthesized, full-length LEC12 and LEC29 Fut9 RNA. Cell lysates prepared 20 h after transfection were assayed for {alpha}(1,3)Fuc-T activity (Fig. 6D). LEC12 RNA gave more {alpha}(1,3)Fuc-T activity than the LEC29 RNA. Taken together, these findings suggest that the 5'-UTR of LEC29 Fut9 mRNA acting together with its coding or 3'-UTR region reduces translation of Fut9 gene transcripts giving rise to less Fut9 protein and less {alpha}(1,3)Fuc-T activity.

CHO Cells Expressing Fut9 Bind a Mouse E-selectin-IgG Chimera—LEC11 CHO cells express sialyl-LeX (9, 13, 25) and bind E-selectin (8, 26). Although LEC12 cells do not express sialyl-LeX (9, 13, 25) and bind at only background levels to activated human endothelial cells (26), LEC12 cells bind to human E-selectin overexpressed in CHO cells (albeit less well than LEC11 (27), and a small subset of glycoproteins from LEC12 bind a mouse E-selectin-IgG chimera (28). Flow cytometry with the latter mouse E-selectin-IgG chimera showed that LEC12 CHO cells bound mouse E-selectin (Fig. 7A),4 and LEC12 cells bound ~2% of the amount of E-selectin bound by LEC11 cells (Fig. 7A). LEC29 cells that make good amounts of LeX but almost no VIM-2 (Fig. 2) did not bind mouse E-selectin (Fig. 7A). However, when CHO or LEC29 cells were transiently transfected with an Fut9 coding region cDNA, binding of mouse E-selectin was induced. Control human IgG1 did not bind to the transfectants. Furthermore, E-selectin binding was abolished in the presence of 1 mM CaCl2 and 5 mM EDTA, a condition that does not induce membrane shedding of glycoproteins (data not shown). Both mouse and human Fut9 have been shown not to generate sLeX following transfection, nor to fucosylate {alpha}(2,3)sialylated LacNAc structures in vitro (29, 30). In agreement with this, LEC29 cells overexpressing Fut9 did not bind the CSLEX-1 mAb that recognizes sLeX, although LEC29 cells were capable of synthesizing sLeX if transfected with a Fut6 cDNA (Fig. 7B). This shows that levels of Fut9 expressed in LEC12 cells and induced by transfection of a Fut9 cDNA in CHO or LEC29 cells cause the synthesis of a fucosylated ligand for mouse E-selectin likely to be VIM-2.



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  		FIG. 7.
A, mouse E-selectin binds to CHO cells overexpressing Fut9. LEC11 and LEC12 CHO mutants and LEC29 or CHO cells transiently transfected with empty vector (+ vector), or with vector containing the coding region of Fut9 (+ Fut9) were analyzed by fluorescence cytometry for binding of 6 µg of mouse E-selectin-IgG fusion protein or human IgG1 detected with anti-human IgG1 secondary antibody. LEC29+Fut9 cells were also tested in the presence of EDTA as described under "Materials and Methods." The inset enlarges the area of difference in the LEC29 + Fut9 panel. B, sialyl-LeX is not generated on LEC29 cells overexpressing a Fut9 cDNA. LEC29 cells that were transiently transfected with empty vector (+ vector), or with vector containing the coding region of the Fut6B (8) or Fut9 Chinese hamster genes (LEC29 + Fut9, left panel; LEC29 + Fut6, right panel) were analyzed by fluorescence cytometry for binding of 3 µg of CSLEX-1 mAb.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
When the LEC12 and LEC29 CHO mutants were initially characterized, their glycosylation phenotypes were different, and it was considered unlikely that they expressed the same Fut gene (9). They have different lectin resistance phenotypes, express a different spectrum of {alpha}(1,3)fucosylated cell surface antigens, and have very different levels of {alpha}(1,3)Fuc-T activity in cell extracts. Thus it was surprising to find that both mutants express the Fut9 gene (8, 11). However, the mutant with the most Fut9 gene transcripts, LEC29, had the least {alpha}(1,3)Fuc-T activity. Here we identify a molecular basis for the discrepant phenotypes of LEC12 and LEC29, provide insight into translational control of Fut9 gene transcripts, and reveal potential biological consequences of regulated expression of {alpha}(1,3)Fuc-TIX. A comparison of LEC12 and LEC29 properties is summarized in Table I.


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  		TABLE I
Characteristics of CHO, LEC12, and LEC29 cells

Properties of LEC12, LEC29, and CHO cells are summarized from this study (Figs. 2, 3A, 3C, and 7A) and Refs. 8 and 10. –, negative; +, positive; the number of + indicates relative quantitation.

 
Here we show that LEC12 and LEC29 Fut9 genes differ in the sequence of their first exon. The LEC29 Fut9 exon 1 is highly related to exon 1 in mouse and rat Fut9 and human FUT9 and thus most likely represents the endogenous hamster Fut9 sequence. The LEC12 Fut9 exon 1 sequence differs completely, and, based on sequence comparisons, probably arose from a chromosomal translocation, as did the exon 1 of Fut9 expressed in LEC30 CHO cells. Fut9 mRNA in LEC12 and LEC29 associated to the same extent with polysomes, and there was no apparent difference in the intracellular stability of Myc-tagged Fut9 protein in these cells (Fig. 3D). Therefore, the translational abilities of the respective 5'-UTRs were compared. Interestingly, the LEC29 5'-UTR ligated to a luciferase reporter facilitated translation better than the LEC12 5'-UTR, whereas the opposite result was expected. It was subsequently found that the LEC29 5'-UTR is inhibitory to translation if it is expressed in the context of a full-length Fut9 cDNA or RNA. In this context the LEC29 5'-UTR inhibited in vitro translation of Fut9 transcripts and produced less Fut9 protein in transfectants than the LEC12 5'-UTR in full-length constructs. These results are summarized in Table II.


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  		TABLE II
Effect of Fut9 5'-UTRs on the translation of downstream coding regions

The data in Figs. 5 and 6 are summarized. –, negative; +, positive; the number of + sings indicates relative quantitation. ND, not done.

 
The characteristics of 5'-UTR sequences that affect translation include their length, Kozak consensus residues near the ATG start codon, secondary structure, and upstream ORFs (31). LEC12 and LEC29 Fut9 5'-UTR sequences are 210 and 209 nucleotides, respectively, but differ in sequence in exon 1. LEC12 exon 1 is 55% GC-rich, whereas LEC29 exon 1 is 68% GC-rich. Based on the mFold algorithm (32), LEC12 exon 1 has a {Delta}G value of –46.33 kCal/mol, whereas the LEC29 exon 1 has a {Delta}G value of –50.36 kCal/mol. The same start codon appears to be used in both cells, because Fut9 proteins from the two cell lines are indistinguishable in molecular weight. The LEC12 5'-UTR has three translation start codons, only one of which lies in a Kozak consensus sequence. The small upstream ORF of 20 codons does not have ATG in a Kozak consensus sequence. The LEC29 5'-UTR has four upstream ATGs. The only one in a Kozak consensus sequence initiates a small ORF of 15 nucleotides. The ability of eukaryotic ribosomes to reinitiate translation at a downstream ATG is limited by the size of upstream ORFs. Luukkonen and colleagues (33) have estimated the cut-off length as 30 codons. The LEC29 upstream ORF of 5 codons is 29 codons upstream of the start codon of the Fut9 coding region, and it is possible that it inhibits reinitiation of translation from that codon. The LEC29 exon 1 sequence could bind factors that inhibit translation or it could interact with the coding or 3'-UTR region to form an inhibitory structure. For example, cis inhibition of p53 RNA translation in yeast is caused by a pseudoknot structure that forms by interactions between the 5'-UTR and the ORF (34). The cis regulatory sequences that affect the translation of mRNA are generally located in the 3'-UTR and recruit factors that repress translation by different mechanisms (35).

Differential expression of {alpha}(1,3)Fuc-TIX activity has profound consequences for the expression of the VIM-2 oncofetal antigen at the cell surface. The low levels of Fut9 in LEC29 result in almost undetectable levels of VIM-2. However, the level of VIM-2 is significantly increased when the cells are transfected with a Fut9 cDNA (Fig. 2). This suggests that expression of VIM-2 is dependent on the actual levels of active {alpha}(1,3)Fuc-TIX protein. This is consistent with previous data showing that Fut9 is not very efficient in generating VIM-2 in vitro. For example, with nLc6 glycolipid (Gal{beta}(1,4)GlcNAc{beta}-(1,3)Gal{beta}(1–4)GlcNAc{beta}(1,3)Gal{beta}(1,4)Glc{beta}(1,1)-ceramide) as a substrate, the {alpha}(1,3)Fuc-TIX activity in LEC12 cell lysates acts preferentially on the terminal (distal) GlcNAc (13). Partially purified human {alpha}(1,3)Fuc-TIX has a higher activity of ~2-fold with LacNAc compared with di-LacNAc, and an increased preference for fucosylating of ~14-fold for the terminal GlcNAc over the internal (proximal) GlcNAc of di-LacNAc (30). When presented with a tri-LacNAc (36) or a tetra-LacNAc (30), human {alpha}(1,3)Fuc-TIX also preferentially fucosylated the terminal GlcNAc residue. However, with {alpha}(2,3)sialylated polylactosamine acceptors, the preference for fucosylation shifted toward internal GlcNAc residues, and residues farthest from the sialic acid were more likely to be fucosylated (30).

Based on databases searches, human and mouse Fut9 gene transcripts include 5'-splice variants that might be differentially translated and result in variable levels of {alpha}(1,3)Fuc-TIX activity in vivo. {alpha}(1,3)Fuc-TIX is primarily responsible for the synthesis of the LeX determinant in mature granulocytes (37), in brain (38), and in the mouse embryo (39). However, our results show that sufficient levels of {alpha}(1,3)Fuc-TIX may also synthesize VIM-2 in a cell, and this could be biologically relevant in vivo. Thus, LEC12 CHO cells that express VIM-2 bind low levels of mouse E-selectin (39 and Fig. 7), whereas LEC29 cells that express only LeX do not (Fig. 7). In addition high level expression of Fut9 in CHO transfectants correlates with increased levels of VIM-2 expression (Fig. 2) and induces a low degree of binding of mouse E-selectin (Fig. 7). VIM-2 on glycolipids has been identified as a binding determinant for E-selectin (40–42). It seems possible, therefore, that VIM-2 glycans mediate binding of E-selectin to the CHO Fut9 cDNA transfectants and thus may also do so under certain circumstances in vivo. However, regardless of the nature of glycans involved, the data presented here suggest that {alpha}(1,3)Fuc-TIX, like {alpha}(1,3)Fuc-TIV and {alpha}(1,3)Fuc-TVII, may act as a regulator of E-selectin-mediated processes.


   FOOTNOTES
 
* This work was supported by Grant NCI RO1 30645 from the National Institutes of Health (NIH) (to P. S.), and partial support was provided by Cancer Center Grant PO1 13330 from the NCI, NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY628212 [GenBank] and AY628213 [GenBank] . Back

{ddagger} To whom correspondence should be addressed: Dept. of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., New York, NY 10461. Tel.: 718-430-3346; Fax: 718-430-8574; E-mail: stanley{at}aecom.yu.edu.

1 The abbreviations used are: {alpha}(1,3)Fuc-T, {alpha}(1,3)fucosyltransferase; CHO, Chinese hamster ovary; mAb, monoclonal antibody; LeX, Lewis X; sLeX, sialyl Lewis X; LacNAc, N-acetyllactosamine; UTR, untranslated region; FITC, fluorescence isothiocyanate; PNGase F, peptide N-glycanase F; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution; RT, reverse transcription; BSA, bovine serum albumin; RACE, rapid amplification of cDNA ends; CMV, cytomegalovirus; nt, nucleotide(s). Back

2 W. Chen, J. Tang, and P. Stanley, submitted for publication. Back

3 S. K. Patnaik, B. Potvin, and P. Stanley, unpublished observations. Back

4 M. Asada and P. Stanley, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Dietmar Vestweber and Martin Wild for mouse E-selectin-IgG chimera, Dr. Hisashi Narimatsu for monoclonal antibodies to Fut9, and Dr. Olga Blumenfeld for helpful comments on the manuscript.



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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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S. K. Patnaik, B. Potvin, S. Carlsson, D. Sturm, H. Leffler, and P. Stanley
Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells
Glycobiology, April 1, 2006; 16(4): 305 - 317.
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