Reduction of CMP N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy

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Biochimica et Biophysica Acta 1622 (2003) 133–144

Reduction of CMP-N-acetylneuraminic acid hydroxylase activity in

engineered Chinese hamster ovary cells using an antisense-RNA strategy

Stephane Chenua, Anne Gregoirea, Yanina Malykhb, Athanase Visvikisc, Lucia Monacod,1,Lee Shawb, Roland Schauerb, Annie Marca, Jean-Louis Goergena,*

aLaboratoire des Sciences du Genie Chimique, CNRS-ENSAIA, 2, av. de la Foret de Haye, F-54505 Vandoeuvre-les-Nancy, FrancebBiochemisches Institut, Christian-Albrechts-Universitat zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany

cFaculte de Pharmacie, Univ. H. Poincare, 30 rue Lionnois, F-54000 Nancy, FrancedDIBIT–San Raffaele Scientific Institute, I-20132 Milan, Italy

Received 2 April 2003; received in revised form 5 June 2003; accepted 18 June 2003

Abstract

Rodent cells, widely used for the industrial production of recombinant human glycoproteins, possess CMP-N-acetylneuraminic acid

hydroxylase (CMP-Neu5Ac hydroxylase; EC 1.14.13.45) which is the key enzyme in the formation of the sialic acid, N-glycolylneuraminic

acid (Neu5Gc). This enzyme is not expressed in an active form in man and evidence suggests that the presence of Neu5Gc in recombinant

therapeutic glycoproteins may elicit an immune response. The aim of this work was, therefore, to reduce CMP-Neu5Ac hydroxylase activity

in a Chinese Hamster Ovary (CHO) cell line, and thus the Neu5Gc content of the resulting glycoconjugates, using a rational antisense RNA

approach. For this purpose, the cDNA of the hamster hydroxylase was partially cloned and sequenced. Based on the sequence of the mouse

and hamster cDNAs, optimal antisense RNA fragments were selected from preliminary in vitro translation tests. Compared to the parental

cell line, the new strain (CHO-AsUH2), which was transfected with a 199-bp antisense fragment derived from the mouse CMP-Neu5Ac

hydroxylase cDNA, showed an 80% reduction in hydroxylase activity. An analysis of the sialic acids present in the cells’ own

glycoconjugates revealed a decrease in the percentage of Neu5Gc residues from 4% in the parental cells to less than 1% in the CHO-AsUH2

cell line.

D 2003 Elsevier B.V. All rights reserved.

Keywords: CMP-Neu5Ac hydroxylase; Sialylation; N-glycolylneuraminic acid; CHO cell; Antisense RNA

1. Introduction of N-glycolylneuraminic acid (Neu5Gc). This sialic acid is

Chinese Hamster Ovary (CHO) cells are widely used for

the production of recombinant glycoproteins employed in

human therapy. Although the posttranslational modifica-

tions of recombinant glycoproteins generated in CHO cells

are very similar to that present on human proteins, these

rodent cells do not perfectly mimic the human sialylation

pattern required for bioactivity, stability, and absence of

antigenicity [1,2]. One of the most significant differences

between rodent and human sialylation is the incorporation

0304-4165/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0304-4165(03)00137-5

* Corresponding author. Tel.: +33-383-59-5844; fax: +33-383-59-

5804.

E-mail address: [email protected]

(J.-L. Goergen).1 Present address: Keryos SpA, via Maritano 26, I-20097 San Donato

Milanese, Italy.

present in the glycoconjugates of virtually all vertebrates,

with the notable exception of man and chicken. In animals,

Neu5Gc is formed as the sugar-nucleotide cytidine mono-

phosphate-N-glycolylneuraminic acid (CMP-Neu5Gc) by

the action of CMP-Neu5Ac hydroxylase (EC 1.14.13.45)

[3]. In man, the gene encoding this enzyme lacks an exon,

resulting in an incomplete mRNA with a premature termi-

nation signal [4,5]. Due to this mutation, Neu5Gc cannot be

formed by CMP-Neu5Ac hydroxylase in man and is prac-

tically undetectable in healthy human tissues [6], though

small amounts of Neu5Gc are found in gangliosides and

glycoproteins of certain human tumors [7]. Glycoconjugates

containing this common sialic acid are therefore foreign to

humans and can induce an immune response, causing the

formation of so-called Hanganutziu–Deicher antibodies

[8,9]. The appearance of these ‘‘serum sickness’’ antibodies

was observed after inoculation of patients with animal

S. Chenu et al. / Biochimica et Biophysica Acta 1622 (2003) 133–144134

antisera containing glycoconjugates sialylated with Neu5Gc

[10,11]. The titer of the Hanganutziu–Deicher antibodies

seems to be dependent on the presentation of the antigen.

Thus, the injection of recombinant human erythropoietin,

containing about 1% of Neu5Gc in total sialic acids, failed

to produce immune response in humans [12,13], while small

amounts of GM1 (Neu5Gc) introduced with bovine brain

gangliosides are immunogenic in man [14].

Most of CHO cell lines produce recombinant glycopro-

teins with low levels of Neu5Gc comprising approximately

3% of total sialic acid [15]. However, the amount of

Neu5Gc in recombinant glycoproteins may be dependent

on the nature of the protein [16], the place of the insertion of

the recombinant gene in the host genome [17], and the

cultivation conditions [18]. In some CHO clones, Neu5Ac is

almost completely replaced by Neu5Gc genome [17]. All

these facts lead to the possibility that the injection of

Neu5Gc-containing recombinant glycoproteins produced

in CHO-cells could lead to an immunogenic reaction in

man. The generation of a CHO cell line lacking hydroxylase

activity and hence Neu5Gc would therefore be desirable in

order to abolish this potential hazard.

The relatively recent cloning of the mouse CMP-Neu5Ac

hydroxylase cDNA [19] has provided the necessary infor-

mation for a rational genetic approach to suppress or abro-

gate the expression of this gene in biotechnologically

important mammalian cell lines. Gene knock-out, random

mutagenesis and antisense oligonucleotide strategies can be

envisaged to inhibit the expression of CMP-Neu5Ac hy-

droxylase [20]. Due to its selectivity and ease of handling,

antisense RNA provides an accessible approach to this

problem. Antisense oligonucleotides can be introduced into

the cell by transcription of a transfected cDNA fragment or

by exogenous antisense oligonucleotides. The latter method

has the advantage of allowing rapid testing of the antisense

efficiency, but remains transient. However, the expression of

antisense RNAs within the cell can lead to the generation of a

cell line of industrial interest with stable modified properties.

Although several examples of the manipulation of gly-

cosylation in CHO cells using antisense technology have

been published [21–24], only empirical approaches, or the

generation of unstable cell lines were reported. Since the

main difficulty of this strategy is in the choice of antisense

sequences, an in vitro translation assay is necessary for

selecting an antisense fragment that will efficiently hybrid-

ize to its target mRNA [25,26].

In this paper, we describe the generation of a stable CHO

cell line with significantly reduced levels of CMP-Neu5Ac

hydroxylase activity and Neu5Gc amounts, using a rational

antisense RNA strategy. The first part of the paper presents in

vitro assays where different antisense fragments were tested

for their efficacy to inhibit the hydroxylase translation. The

efficiency of a selected antisense fragment was then assessed

in cultured CHO cells by measuring the resulting intracellu-

lar CMP-Neu5Ac hydroxylase activity and the amount of

Neu5Gc present on cellular glycoconjugates.

2. Materials and methods

2.1. Partial cloning and sequencing of cDNA for CMP-

Neu5Ac hydroxylase from CHO-cells

All DNA and RNA manipulations were performed

according to Ref. [27]. Total RNA was prepared from

5� 106 CHO-UH cells (SV Total RNA isolation system,

Promega). RNA integrity was checked by electrophoresis

on a 1% agarose gel. Total cellular RNA (1 Ag) was

reverse-transcribed (Thermoscript-RT, Gibco-BRL) with

random hexamer primers. Two microliters of the reverse-

transcribed mixture was used as a template for PCR

amplification of hamster cDNAs in a mixture containing:

5 Al of 10� PCR buffer, 1 Al of 10 AM dNTPs (Euro-

gentec, Belgium), 20 pmol of each primer (Eurogentec, for

sequences see below) and 5 units of AmpliTaq polymerase

(Perkin-Elmer) in a final volume of 50 Al. Oligonucleotideprimers for amplification of CHO CMP-Neu5Ac hydroxy-

lase were designed from the published mouse CMP-

Neu5Ac hydroxylase cDNA sequence [19]. Sense primer

(5V-GAAACAGACAGCTGAGACCCTGTTGTC-3V) and an-

tisense primer (5V-GGGTGGTGTTAGAGGGAGTTT-

TATCTG-3V) were used to amplify a 1691-bp fragment of

the hamster sequence. Amplification was carried out with

35 cycles of 94 jC for 1 min, 58 jC for 1 min and 72 jCfor 2 min. The PCR products were purified by electropho-

resis on a 1.5% agarose gel (QIAEX, Qiagen), prior to

subcloning into pCR2.1 TOPO (Invitrogen). The plasmid

containing the hamster hydroxylase cDNA cloned in

pCR2.1 TOPO was termed pCRCNAH. These constructs

were introduced in DH5a E. coli cells by electroporation

and three clones were isolated and sequenced (MWG-

Biotech, Germany). The hamster CMP-Neu5Ac hydroxy-

lase cDNA partial sequence reported in this paper has been

submitted to the EMBL Data bank with the accession

number AJ242835.

2.2. Plasmid constructs

For in vitro translation tests, mouse or hamster antisense

fragments were generated from pBSCNAH (complete

mouse CMP-Neu5Ac hydroxylase cDNA sequence cloned

into the pBluescript II K + vector) [19] and pCRCNAH

(hamster hydroxylase cDNA partial sequence cloned into

the pCR2.1 TOPO vector), respectively. The fragments were

subcloned either into pGEM3Z (Promega) or pCR2.1

TOPO. The mouse 403 bp antisense cDNA is a XbaI/SacI

pBSCNAH fragment (nucleotides 1–403 according to Fig.

1) inserted into pGEM3Z. The mouse 714 bp antisense

cDNA (nucleotides 99–812 according to Fig. 1) was

amplified by PCR using pBSCNAH as the template with

sense (5V-GATGATGGACAGGAAACAGACAGC-3V) and

antisense (5V-ACCTGGATC-AGAGTGGCGTCGGGT-3V)primers, and cloned into pCR2.1 TOPO. The mouse and

hamster 199 bp fragments (nucleotides 112–309 according

50 100 mouse ggcagtctaa agtcatcctc gccatcctcg ccttcctggt gtgattcgca gaagtagaaa gaacaccagc agctgctttg aaataccctg gagctggcag hamster ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 101 150 200 mouse ATGATGGACA GGAAACAGAC AGCTGAGACC CTGCTGACCC TGTCTCCTGC TGAAGTTGCC AACCTCAAGG AAGGGATCAA TTTTTTTCGA AATAAGACTA hamster ~~~~~~~~~~ ~GAAACAGAC AGCTGAGACC CTGTTGTCTC TGTCACCTGC TGAAACTGCC AACCTCAAGG AAGGAATCAA TTTTTTTCGA AATAAGACTA 201 250 300 mouse CTGGGAAAGA GTACATTTTA TACAAGGAGA AGGACCATCT AAAGGCATGC AAGAACCTCT GCAAGCACCA GGGAGGCCTG TTCATGAAAG ACATCGAGGA hamster CTGGCAAAGA GTACATTTTA TACAAGGAGA AAAACCACCT AAAGGCATGC AAGAACCTCT GCAAGCACCA AGGAGGCCTG TTCATAAAAG ACATTGAGGA 301 350 400 mouse TTTAGATGGA AGGTCCGTTA AATGCACAAA GCACAACTGG AAGTTAGACG TGAGCACCAT GAAATATATC AACCCTCCAG GGAGCTTCTG TCAAGACGAG hamster TTTAGACGGA AGGTCCGTTA AATGTACAAA GCACAACTGG AAGTTAGATG TGAGCACCAT GAAGTACATC AACCCTCCAG GGAGCTTCTG TCAGGACGAG 401 450 500 mouse CTCGTTATTG AAATGGATGA AAACAATGGG CTTTCCCTGG TAGAACTGAA CCCTCCTAAC CCCTGGGACT CTGATCCCAG GTCTCCTGAA GAATTAGCTT hamster CTGGTTGTCG AAATGGATGG AAACGATGGG CTTTTCCTTA TAGAATTGAA TCCTCCTAAC CCTTGGGACT CTGATCCCAG GACTCCTGAA GAGTTGGCTT 501 550 600 mouse TTGGGGAAGT ACAGATAACA TATCTCACTC ATGCCTGCAT GGACCTCAAG TTGGGAGACA AGCGAATGGT ATTTGACCCT TGGTTAATTG GCCCTGCTTT hamster TTGGGGAAGT ACAGATAACA TATCTCACTC ATGCCTGCAT GGACCTCAAG TTGGGAGACA AGCGGATGGT GTTTGACCCT TGGTTAATTG GCCCTGCTTT 601 650 700 mouse TGCCCGAGGA TGGTGGTTGC TACATGAGCC TCCATCTGAC TGGTTGGAGA GGCTGTGCAA AGCAGACCTC ATTTATATCA GCCACATGCA CTCAGACCAC hamster TGCCCGAGGA TGGTGGTTGC TACATGAGCC TCCATCTGAT TGGCTGGAGA GGCTGTGCAA AGCAGACCTC ATTTACATCA GTCACATGCA CTCAGACCAC 701 750 800 mouse CTGAGCTACC CTACCCTGAA GCAGCTTTCC CAGAGACGAC CAGACATTCC CATTTATGTT GGCGACACAG AAAGGCCTGT GTTTTGGAAC CTGGATCAGA hamster CTGAGTTACC CCACTCTGAA GCAGCTTTCC CAGAGACGAC CAGACATTCC CATATATGTT GGTGACACGG AAAGGCCTGT GTTTTGGAAT CTGGACCAGA 801 850 900 mouse GTGGCGTCGG GTTAACTAAC ATCAACGTGG TTCCATTTGG AATATGGCAA CAGGTAGACA AAAGTCTGCG GTTCATGATC TTGATGGACG GCGTTCATCC hamster GCGGTGTTCA GCTAACTAAC ATCAATGTGG TACCATTTGG AGTGTGGCAA CAGGTAGACA AAAATCTCCG ATTCATGATC TTGATGGATG GCGTTCATCC 901 950 1000 mouse TGAGATGGAC ACATGCATTA TCGTGGAGTA CAAAGGTCAT AAAATACTCA ACACAGTGGA CTGCACCAGA CCCAATGGGG GAAGGCTTCC TGAGAAAGTT hamster TGAAATGGAC ACATGCATTA TCGTAGAGTA CAAAGGTCAT AAAATACTCA ACACAGTGGA TTGCACCAGA CCCAATGGGG GAAGGCTGCC TGAGAAAGCT 1001 1050 1100 mouse GCTCTAATGA TGAGTGATTT CGCAGGAGGT GCATCAGGCT TTCCAATGAC TTTCAGTGGT GGAAAATTTA CTGAGGAATG GAAAGCCCAG TTCATTAAGG hamster GCTCTAATGA TGAGTGATTT TGCTGGAGGA GCATCAGGCT TTCCAATGAC TTTCAGCGGT GGAAAATTTA CTGAGGAATG GAAGGCCCAG TTCATTAAGG 1101 1150 1200 mouse CTGAAAGAAG AAAGCTTCTG AATTACAAAG CTCAGCTGGT GAAGGACCTG CAGCCCCGAA TCTACTGTCC GTTTGCTGGG TACTTTGTGG AGTCTCACCC hamster CGGAAAGAAG AAAGCTTCTG AATTACAAAG CTCAGCTCGT GAAGGACCTC CAGCCTCGAA TCTACTGTCC CTTTGCTGGG TATTTTGTGG AATCTCATCC 1201 1250 1300 mouse ATCTGACAAG TACATTAAGG AAACAAACAC CAAAAATGAC CCAAATCAGC TCAACAATCT TATCAGGAAA AACTCTGACG TGGTGACATG GACCCCACGA hamster ATCTGACAAG TATATTAAGG AAACAAACAT CAAAAATGAC CCGATTCAAC TCAACAATCT CATCAAGAAA AACTGTGATG TGGTGACATG GACCCCACGA 1301 1350 1400 mouse CCTGGCGCTG TCCTCGACCT TGGCAGGATG CTGAAGGACC CAACAGACAG CAAGGGCATT GTGGAGCCTC CAGAGGGGAC AAAGATTTAC AAGGATTCCT hamster CCTGGAGCTA CTCTTGACCT GGGCAGGATG CTGAAGGACC CAACAGACAG CCAGGGCATC ATAGAGCCTC CAGAAGGGAC AAAAATTTAC AAGGATTCAT 1401 1450 1500 mouse GGGACTTTGG CCCGTACCTG GAGATCTTGA ATTCTGCTGT CAGAGATGAA ATCTTCTGTC ATTCATCCTG GATTAAAGAG TACTTCACGT GGGCTGGATT hamster GGGACTTCGG CCCATACCTG AGCACCTTGC ACTCTGCTGT AGGAGATGAA ATCTTCCTTC ACTCGTCCTG GATAAAAGAG TACTTCACTT GGGCTGGATT 1501 1550 1600 mouse TAAGAATTAC AACCTGGTGG TCAGGATGAT TGAAACAGAT GAAGATTTCA GCCCTTTTCC TGGAGGGTAC GACTATCTGG TGGACTTTCT AGATTTATCC hamster TAAGAGTTAC AACTTGGTGG TCAGGATGAT TGAAACAGAT GAAGACTTCA ACCCTTTTCC TGGAGGGTAT GACTATCTGG TGGACTTTCT AGATTTGTCT 1601 1650 1700 mouse TTTCCGAAAG AAAGACCCAG CCGGGAGCAT CCTTATGAAG AAATCCATAG CCGGGTGGAT GTCATCAGGT ACGTGGTGAA GAACGGCCTG CTGTGGGATG hamster TTTCCAAAAG AAAGACCAAG CAGGGAGCAT CCCTATGAAG AAATCCGTAG CCGTGTGGAT GTCGTCAGGT ACGTGGTGAA GCACGGTCTG CTGTGGGATG 1701 1750 1800 mouse ATCTGTATAT TGGATTCCAG ACCCGATTGC TGCGGGACCC TGATATATAC CATCATCTGT TTTGGAATCA TTTTCAGATA AAACTCCCTC TAACACCACC hamster ACCTGTACAT TGGCTTCCAG ACCCGGTTGC AGCGGGACCC TGACATATAC CATCATCTGT TTTGGAATCA TTTTCAGATA AAACTCCCTC TAACACCACC 1801 1850 mouse CAACTGGAAG TCGTTCCTAA TGCACTGTGA TTAGtctgga cctggtaagt ccca hamster CA~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~

Fig. 1. Hamster and mouse CMP-Neu5Ac hydroxylase cDNA sequence alignment: shaded letters show differences between the mouse and hamster sequences.

Start and stop codons are boxed.

S. Chenu et al. / Biochimica et Biophysica Acta 1622 (2003) 133–144 135

to Fig. 1) were amplified by PCR using identical sense (5V-TTTCTAGAGAAACAGACAGCTGAGACCC-3V) and anti-

sense (5V-TTTGAGCTCC CGTCTAAATCCTCAATGTC-3V)primers. The XbaI/SacI digested PCR products were then

subcloned into pGEM3Z. For cell transfections, the mouse

199, 403 and 714 bp antisense cDNAs were subcloned into

the EcoRI/XbaI-cleaved pClSfiT [28] vector and the ham-

ster 199 bp antisense was subcloned into the same vector

digested with EcoRI and SalI. For selection of stable clones,

plasmid pSfiSVdhfr [29], carrying the dehydrofolate reduc-

tase selectable marker, was used.

2.3. In vitro transcription and translation assays

In vitro transcription reactions were performed using the

‘‘Riboprobe in vitro Transcription System’’ (Promega). T7

RNA polymerase was used for transcription of the mouse

CMP-Neu5Ac hydroxylase cDNA from pBSCNAH linear-

ized with XhoI, as well as the 714, 403 and 199 bp antisense

DNAs generated by cleavage of the appropriate plasmid with

BamHI,XbaI and SalI, respectively. T3 RNApolymerase was

used to produce a full-length antisense mouse CMP-Neu5Ac

hydroxylase RNA from pBSCNAH linearized with NotI.


In vitro translation reactions were performed using the

‘‘Rabbit Reticulocyte Lysate System’’ (Promega) and

chemiluminescent detection of the translation products

was performed using the ‘‘Transcendk non-radioactive

Translation Detection System’’ (Promega).

2.4. Generation of stable CHO-AsUH2 cells expressing

CMP-Neu5Ac hydroxylase antisense RNA

The cell line (CHO-UH cell line) used for expression of

antisense fragments results from the co-transfection of a

plasmid bearing the rat a2,6-sialyltransferase cDNA and a

plasmid carrying the neomycin selectable marker into the

Dux-B11 CHO (dhfr-) cell line [2]. CHO-UH cells were

used to generate a second stable cell line producing CMP-

Neu5Ac hydroxylase antisense RNAs. CHO-UH cells were

transfected using a method derived from the in vitro

amplification system described previously [30], in which

lipofection was replaced by electroporation (1400 V, 25 AF)with 10 Ag of a concatenamer composed of the cDNA

coding for the tested antisense RNA and the cDNA coding

for dhfr at a ratio of 10:1. The selective medium used for

CHO cells was aMEM (without ribo- and deoxyribonucleo-

sides) (Gibco-BRL) + 5% dialyzed heat inactivated fetal

calf serum (FCS) (ATGC, France). Methotrexate (MTX)

was used for gene amplification at a starting concentration

of 50 nM and was increased progressively to a final

concentration of 500 nM. Clones were isolated after a 9-

week period in the selection medium. For sialic acid

analyses, cells were maintained in the same selection

medium except that calf serum was replaced by 4% human

serum.

2.5. Preparation of high-speed supernatants from different

cell lines for CMP-Neu5Ac hydroxylase assays

Cell pellets (about 108 cells) were thawed, resuspended

in one volume (ml/g wet mass) of ice-cold 50 mM HEPES/

NaOH, pH 7.4 containing Triton X-100 (5% by mass) and

incubated for 20 min at 4 jC with permanent agitation. The

resulting homogenates were centrifuged for 15 min at

100000� g and 4 jC and the clear high-speed supernatants

were removed and used for determination of the hydroxy-

lase activity.

2.6. Assay of CMP-Neu5Ac hydroxylase activity

The activity of CMP-Neu5Ac hydroxylase was detected

by incubation of 15 Al of the high-speed supernatants with 1

mM NADH, 0.5 mM FeSO4, pig liver microsomes (60 Agprotein solubilised in a final concentration of 0.5% w/v

Triton X-100), and CMP-[4,5,6,7,8,9-14C]Neu5Ac (Amer-

sham) in 50 mM HEPES/NaOH, pH 7.4 at 37 jC, the finalvolume of the assay being 25 Al. The concentration of CMP-

Neu5Ac in the test varied from 2.1 to 18 AM and the

quantity of the radioactivity was 12.5–15.6 nCi per assay.

To obtain substrate concentrations above 12 AM the re-

quired amount of non-radioactive CMP-Neu5Ac was added.

Incubation times are as indicated in Results and all enzyme

tests were performed in duplicate. The assays were stopped

by the addition of 5 Al 1 M trichloroacetic acid, and

precipitated proteins were removed by centrifugation for 3

min at 14000� g. Released [14C]Neu5Gc and [14C]Neu5Ac

were separated and quantified by radio thin-layer chroma-

tography, as described previously [31]. Minute amounts of

activity due to traces of hydroxylase in the pig microsomes,

were subtracted from the sample activity. With this method,

the minimum detectable conversion of CMP-Neu5Ac to

CMP-Neu5Gc was about 1–2%. The activity of each

sample was estimated using two incubation times, both of

which were within the linear region of the reaction time

course (see Results). The apparent KM for CMP-Neu5Ac of

the enzyme in the extracts of CHO-UH cells was determined

using the non-linear regression program Enzfitter (Elsevier

Biosoft, UK).

2.7. RNA analysis by RT-PCR

Total RNA was prepared from CHO-UH and CHO-

AsUH2 cells (SV Total RNA isolation system, Promega)

and the first cDNA strand synthesis was performed using

the AMV reverse transcriptase and random nonamers.

Oligonucleotides specific for the hamster CMP-Neu5Ac

hydroxylase and the expression vector pCISfiT sequences

were used to amplify either the CMP-Neu5Ac hydroxylase

cDNAs or the 199 bp fragments present in the cell lines.

PCR amplification was performed in a final volume of 50

Al, using 0.8 units of AccuTaq polymerase (Sigma) and 1 Agof template cDNA. Amplification was carried out using 35

cycles with a temperature profile of: 40 s at 94 jC, 1 min at

58 jC and 1.5 min at 68 jC. The PCR products were

analysed by electrophoresis on a 1.5% agarose gel. A

similar protocol was used to amplify a h-actin sequence,

with the help of standard oligonucleotides, except that the

temperature of the annealing step was increased to 68 jC.

2.8. Sialic acid analysis

Cells (6–10� 107) were thawed, resuspended in two

volumes (ml/g wet mass) of distilled water and homoge-

nised on ice for 3� 10 s using a Branson B-12 sonicator (40

W power) fitted with a microtip, allowing 15 s cooling

between bursts. To release sialic acids, the resulting homog-

enate (0.1 ml) was mixed with the same volume of 0.2 M

HCl and incubated for 2 h at 80 jC. The samples were

centrifuged for 20 min at 14000� g and the resulting pellets

were washed with 0.1 ml ice-cold water followed by

centrifugation at 14000� g. After both centrifugation steps,

the supernatants were pooled and filtered with a Centricon

10 ultrafiltration unit (Amicon). The retained material was

washed twice with 0.2 ml water followed by ultrafiltration.

The filtrates were lyophilised, and the released sialic acids


were purified by passage through 2 ml cation- and anion-

exchange columns as described by Ref. [32]. The adsorbed

sialic acids were eluted from the latter resin with seven

column volumes of 1 M formic acid, lyophilised and

resuspended in 1 ml water. The enriched sialic acids were

derivatized with 1,2-diamino-4,5-methylene dioxybenzene

(DMB) and the resulting derivatives were analysed by

reversed-phase HPLC with fluorimetric detection [33].

Quantification of sialic acids was performed by integration

of the peaks in the chromatogram using a calibration curve

obtained by DMB-derivatization of known Neu5Ac

amounts.

2.9. Protein concentration

Protein concentrations were measured using the BCA

protein assay reagent (Pierce) with bovine serum albumin as

a standard.

3. Results

3.1. PCR amplification and cloning of the partial CMP-

Neu5Ac hydroxylase cDNA from CHO-cells

Using primers designed from the nucleotide sequence of

the mouse CMP-Neu5Ac hydroxylase cDNA [19], two

products of 1691 and 1250 bp were amplified by RT-PCR

using total RNA from CHO-UH cells as a template (data not

shown). These fragments were subcloned into pCR2.1

TOPO and transformed into E. coli. Three colonies from

each transformation were randomly selected, their plasmids

isolated and the DNA inserts sequenced. The amino acid

sequence deduced from the ORF of the 1250-bp product

exhibited a 98% similarity to fibronectin from CHO, a

protein which is abundantly expressed in CHO cells. The

nucleotide sequence of the 1691-bp product was 94%

identical to that of the mouse CMP-Neu5Ac hydroxylase

cDNA. The deduced sequence of the 563 amino acids was

97% homologous to the mouse enzyme, indicating that

Fig. 2. Schematic representation of the mouse CMP-Neu5Ac hydroxylase cDNA cl

translation inhibition tests.

several nucleotide exchanges are silent (Fig. 1). Due to

the fact that oligonucleotides from the mouse hydroxylase

open reading frame were used to amplify the PCR frag-

ments, the given sequence is only partial, covering 98% of

the mouse CMP-Neu5Ac hydroxylase cDNA. The high

degree of homology between the sequence presented here

and the primary structure of CMP-Neu5Ac hydroxylase

from mouse and other sources [34] confirms its assignment

as CMP-Neu5Ac hydroxylase, presumably the main en-

zyme responsible for the biosynthesis of Neu5Gc in CHO

cells.

3.2. Selection of CMP-Neu5Ac hydroxylase antisense

constructs

Since initial experiments [35,36] showed that artificial

antisense oligonucleotides may be used to interfere with

gene expression, there has been a growing body of literature

on antisense nucleic acids [22,23,37–39]. Due to the lack of

generally applicable rules for the design of efficient anti-

sense RNAs, the ability of different antisense RNAs to

inhibit the translation of the mouse CMP-Neu5Ac hydrox-

ylase mRNA was tested in vitro. The approach adopted

involved examining the efficiency of antisense RNAs of

different lengths by generating fragments from the mouse

CMP-Neu5Ac hydroxylase cDNA. Four different antisense

RNAs of 714, 403, and 199 nucleotides (nt) plus the full-

length of the mouse cDNA were generated (for exact

position on the hydroxylase cDNA see materials and meth-

ods). In addition, a 199 nt fragment from the hamster CMP-

Neu5Ac hydroxylase cDNA spanning the same region as

the mouse 199 nt fragment was tested (Fig. 2).

The mouse CMP-Neu5Ac hydroxylase cDNA and the

antisense fragments were transcribed in vitro with either T7

or T3 RNA polymerases and the formation of the respective

RNA was confirmed by agarose gel electrophoresis by

comparison with four standard RNAs of 250, 1065, 1525,

and 2346 nt (data not shown). The band intensity reflected

the amount of RNA obtained, allowing an estimation of the

sense and antisense RNA quantities used in the in vitro

oned into pGEM3Z and of the sense and antisense RNAs used in the in vitro

Fig. 4. pCISfiT plasmid used for the cloning and the in vitro amplification

of the antisense RNAs. pCISfiT possesses a multiple cloning site terminated

by a SfiI cloning site; the gene of interest is under the control of the strong

CMV promoter; a SV40 late polyadenylation sequence and a transcription

terminator from the human gastrin gene are located downstream to the

cDNA coding for the antisense.

Fig. 3. In vitro CMP-Neu5Ac hydroxylase translation inhibition tests. Proteins translated using rabbit reticulocyte lysates were loaded on a SDS

polyacrylamide gel, then transferred on an Immobilon P membrane. Incorporation of biotinylated lysine during translation was detected as described in the

‘‘Transcendk non-radioactive translation detection’’ kit. Panels A and B: antisense/sense RNAs molar ratio = 1:1. Panels C and D: antisense/sense RNAs

molar ratio = 1:2. For all panels, T(– ): proteins naturally biotinylated in the reticulocyte lysate. Luciferase: positive control resulting from the in vitro luciferase

mRNA translation. CMP-Neu5Ac hydroxylase: in vitro translation of the CMP-Neu5Ac hydroxylase mRNA; Full-length, 714mAs, 403mAs, 199mAs and

199hAs: in vitro translation of the CMP-Neu5Ac hydroxylase mRNA in the presence of the mentioned antisense RNA fragments (numbers show the length of

fragments; m and h indicate the mouse or hamster source of the hydroxylase cDNA, used to design the fragments; arrows indicate the position of the CMP-

Neu5Ac hydroxylase).


translation tests. Fig. 3A and B present the proteins trans-

lated in vitro with different levels of sense and antisense

RNAs. Luciferase is the positive control giving rise to a

band of 61 kDa. As can be seen, when the same amount of

sense and antisense RNA were mixed in the translation

cocktail, no band corresponding to the CMP-Neu5Ac hy-

droxylase protein (66 kDa) appeared on SDS-polyacryl-

amide gel electrophoresis of the products, indicating that

the five antisense RNAs were able to inhibit the translation

of CMP-Neu5Ac hydroxylase mRNA. After a 50% reduc-

tion in the quantity of the antisense RNAs, the full-length,

the 403 and the 199 nucleotides still led to a very efficient

inhibition of translation, whereas a strong reduction of the

inhibition was observed with the 714 nt antisense RNA (Fig.

3C and D). Interestingly, both the mouse and hamster 199

bp antisense fragments were equally effective in inhibiting

hydroxylase translation, despite 15 nt differences. In view of

these results, the shorter and most efficient antisense frag-


ments were selected and cloned into eucaryotic expression

vectors for the in vivo inhibition of CMP-Neu5Ac hydrox-

ylase translation.

3.3. Generation of CMP-Neu5Ac hydroxylase depleted cell

lines

EcoRI–XbaI antisense fragments of 199 and 403 bp

were cloned into the pCISfiT vector (Fig. 4) and expressed

under the control of the CMV promoter. Each of these

constructs was combined with the pSfiSVdhfr plasmid,

which bears the DHFR selectable marker, according to the

in vitro amplification method [30], for the insertion of

multiple copies of the antisense fragment into the genome

Fig. 5. Radio thin-layer chromatograms of [14C] sialic acids released by acid trea

lines with 2.1 AM CMP-[14C]Neu5Ac and cofactors, as described in Materials and

Al of high-speed supernatant of OKT3 mouse hybridoma cells. Panel B (negative c

supernatant. Panels C and D: analysis of the parental CHO-UH cell line for 60 and

and 403m2 cell lines, respectively. The numbers above the peaks identify the nat

of CHO-UH cells. DHFR-positive transformants were

obtained after 9 weeks by initial selection in aMEM

medium lacking ribo- and deoxyribonucleosides, followed

by exposure to progressively increasing concentrations of

MTX up to 500 nM. One of the main advantages of the in

vitro amplification system is the high rate of good producer

clones generated and then a reduction of the clones to be

screened. The combined in vitro/in vivo amplification

procedures was already used for the production of granulo-

cyte colony-stimulating factor (G-CSF) [29]: a 30-fold

improvement of the protein production was obtained com-

pared to the in vitro amplification protocol alone.

A total of approximately 15 clones was obtained, of

which two to three stable clones from each of the three

tment of incubation mixtures of high-speed supernatants of the various cell

methods. The time of incubation is indicated. Panel A (positive control): 15

ontrol): 15 Al of the 50 mM HEPES/NaOH was used instead of high-speed

120 min, respectively. Panels E and F: analysis for 120 min of the 199m2

ure of the sialic acid ((1) Neu5Gc; (2) Neu5Ac).


antisense constructs were tested for their CMP-Neu5Ac

hydroxylase activity.

3.4. Assay of CMP-Neu5Ac hydroxylase activity in

engineered cell lines

Representative radio thin-layer chromatograms of the

enzyme tests for several cell lines are given in Fig. 5. The

activity of the enzyme in the parental CHO-UH cells was

very low in comparison with other cell lines, such as OKT3

mouse hybridoma cells which are known to produce large

amounts of Neu5Gc (Fig. 5A and C). To obtain a high

turnover of the radioactive substrate and thus the required

sensitivity, it was necessary to use non-isotopically diluted

CMP-[14C]Neu5Ac in low concentrations with long incu-

bation times. For example, after a 2-h incubation, the high-

speed supernatant of CHO-UH cells in the assays containing

2.1 and 18 AM substrate gave rise to 14% and 3%

[14C]Neu5Gc, respectively.

The apparent KM for CMP-Neu5Ac determined for the

crude extracts of CHO-UH cells was about 8 AM (data not

shown); a comparable value for this parameter had been

obtained for purified mouse (5.5 AM) and porcine (11 AM)

hydroxylases [40,41]. However, a substrate concentration of

2.1 AM, which allowed the reliable determination of

[14C]Neu5Gc, was used to compare hydroxylase activities

of the various CHO clones. This CMP-Neu5Ac concentra-

tion was significantly lower than KM. Nevertheless, sub-

strate consumption was always less than 15% (Fig. 5C–F)

and increased linearly with the incubation time (Fig. 5C and

D) suggesting that the above results allow a quantitative

comparison of the enzyme activity of the various clones

presented in Fig. 6. To confirm this, the activity of the

Fig. 6. Specific activity of the CMP-Neu5Ac hydroxylase in different CHO-clones,

four separate measurements is shown. Standard deviations are denoted with error ba

8 measurements.

hydroxylase in these cell lines was also measured with

substrate concentrations of 12 and 18 AM, both of which

are greater than KM (data not shown). The less accurate data

obtained for the substrate concentrations above the KM

revealed the same tendency as in Fig. 6 (data not shown).

A reduction of the hydroxylase activity in comparison

with parental UH cells was observed in seven out of the

eight clones studied (Fig. 6). The greatest reduction of

hydroxylase activity was observed in the cell line 199m2,

in which a 78% reduction of the activity measured in the

parental CHO-UH cells was detected (Figs. 4E and 5). This

antisense cell line was therefore chosen for further inves-

tigations and renamed CHO-AsUH2.

3.5. mRNA expression profile

The mRNA expression pattern of CMP-Neu5Ac hydrox-

ylase was studied by RT-PCR analysis on both CHO-UH

parental and the selected CHO-AsUH2 cells (Fig. 7). Total

RNA was isolated from both cell lines and reversed tran-

scribed using random nonamers. Two different 35 cycle-

PCR reactions were then performed with two sets of

primers, using the same quantity of the respective template

cDNA. The first PCR served to amplify a portion of the

hydroxylase cDNA, whereas the second was aimed at the

amplification of the 199 nt antisense RNA. As shown in Fig.

7B, the presence of the 199 nt antisense RNA fragment was

confirmed in the CHO-AsUH2 cell line, and as expected it

was absent in the parental cell line. In order to compare the

level of hydroxylase mRNA expression between both cell

lines, the relative amounts of the amplified RT-PCR prod-

ucts were quantified using three different gels, by densito-

metric analysis on each (Fig. 7A). Although the band

determined using 2.1 AM CMP-[14C]Neu5Ac. The mean specific activity of

rs. For the definition of clones, see legend to Fig. 4. (*) Data for 12 and (**)

Fig. 8. Sialic acid composition of glycoconjugates from CHO-UH or CHO-

AsUH2 cells. DMB-modified sialic acids were separated by reversed phase

HPLC coupled to fluorimetric detection. Upper panel: sialic acids extracted

from glycoconjugates of CHO-UH cells. Lower panel: sialic acids from

glycoconjugates of CHO-AsUH2 cells.


corresponding to the hydroxylase mRNA was present in

both cell lines, an 80% (F 6%) decrease in its intensity was

observed in the CHO-AsUH2 cells. As a control for RNA

quality and to assess RT-PCR conditions, single-stranded

cDNA was used as a template for the amplification of h-actin with specific forward and reverse primers. In all

experiments, the h-actin specific primers amplified the

expected 550 bp product (data not shown). The formation

of the hydroxylase PCR-product in CHO-UH cells had not

reached a plateau, since a 40 cycle amplification yielded a

band of higher intensity.

3.6. Analysis of the sialic acids in CHO-UH and CHO-

AsUH2 cell lines

For the sialic acid analyses, both parental and CHO-

AsUH2 cells were cultured for 32 passages in medium

supplemented with 4% human serum, which is devoid of

Neu5Gc residues. This culture medium containing human

serum (not relevant from a biotechnological point of view

for the production of a recombinant protein) was selected

with the aim of removing all traces of Neu5Gc, which could

appear in the cells as the result of the uptake and utilization

of the Neu5Gc-containing glycoconjugates from FCS. How-

ever, the type of serum added to the culture medium had no

influence on the activity of the hydroxylase, i.e. the specific

enzyme activities were 190F 25 fmol/min/mg protein for

CHO-UH cells and 42F 10 fmol/min/mg protein for CHO-

AsUH2 cells grown in presence of calf or human serum.

The total amount of sialic acid was practically the same

in both cell lines grown in FCS-free medium (CHO-UH

cells: 1.8 Ag sialic acid/mg protein; CHO-AsUH2 cells: 1.5

Ag sialic acid/mg protein). Significantly, the amount of

Fig. 7. RT-PCR analysis of CHO-UH and CHO-AsUH2 cell lines. The PCR

products were loaded on a 1.5% agarose gel. Panel A: CMP-Neu5Ac

hydroxylase mRNA amplification in CHO-UH and CHO-AsUH2 cell lines,

respectively. Panel B: 199m-antisense RNA amplification in CHO-UH and

CHO-AsUH2 cell lines, respectively. For both panels, MW=DNA

molecular weight marker.

Neu5Gc as a percentage of total sialic acids was consider-

ably decreased in the CHO-AsUH2 cells (0.62F 0.05%)

compared with the CHO-UH cells (3.7F 0.3%) (Fig. 8).

4. Discussion

The aim of this work was to reduce CMP-Neu5Ac

hydroxylase activity in a CHO cell line, and thus generate

a stable cell line performing a glycosylation as similar as

possible to that present in humans. This involved obtaining

cells which produce a2,6-linked sialic acids and lacked

Neu5Gc. To achieve this, a genetically modified ‘‘universal

host’’ cell line CHO-UH [2], stably expressing a2,6 sialyl-

transferase, was used in an antisense RNA approach to

inhibit the translation of CMP-Neu5Ac hydroxylase.

Despite a growing body of literature on the use of

antisense nucleic acids in modifying gene expression

[22,23,35–39], no general rules for the selection of an

efficient antisense RNA have yet emerged. In order to

design an optimal antisense RNA fragment, partial nucleo-

tide sequence of the hamster CMP-Neu5Ac hydroxylase

was cloned by RT-PCR amplification. The deduced nucle-

otide sequence of the hamster hydroxylase (Fig. 1) was

highly homologous to that of the mouse enzyme and the

hydroxylase from other mammals [5,34].

Various antisense RNAs of 714, 403, and 199 nucleo-

tides, derived from the mouse and hamster sequences, and a

full-length antisense RNA from the mouse cDNA (Fig. 3)


were preliminary tested in hydroxylase translation inhibition

in vitro tests with different amounts of antisense and sense

RNAs. All antisense RNAs, independent of the animal

source, were able to inhibit the translation of the hydroxy-

lase in vitro, though the 714 nt fragment was markedly less

effective, probably due to the formation of stable secondary

structures. The shorter antisense RNAs, which appeared to

be most efficient, were therefore used to generate stable

transfectants of the CHO-UH cells.

Several approaches were tested to analyse which of the

cell lines expressing different antisense fragments were most

efficient at suppressing hydroxylase production. However,

due to the very low level of enzyme in the parental CHO-

UH cells, the sensitivity of the detection methods was

generally insufficient. For example, the screening of a

CHO cDNA library using the full-length mouse CMP-

Neu5Ac hydroxylase cDNA as a probe, failed to detect

any hydroxylase-specific clones (data not shown). Accord-

ingly, a RT-PCR analysis showed that mRNA coding for

CMP-Neu5Ac hydroxylase is expressed at a very low level

in CHO-UH cells (Fig. 7A). Furthermore, a sensitive

Western blot analysis with a CMP-Neu5Ac hydroxylase-

specific antibody, at a detection limit of about 20 ng

enzyme, failed to reveal the hydroxylase in 90 Ag of total

protein from high-speed supernatants of CHO-UH cells

resolved by SDS-PAGE (data not shown). Determining

Neu5Gc was also a misleading measure of cellular hydrox-

ylase, due to the small amount of this sialic acid (3.7% of

total sialic acid) in the parental CHO-UH cell line. This

latter analysis is further complicated by the fact that cells in

culture incorporate Neu5Gc from FCS present in the medi-

um [42,43]. For this reason, screening the clones by sialic

acid analysis was only meaningful after cultivating cells for

a long period of time in FCS-free medium. Despite the fact

that sub-KM CMP-[14C]Neu5Ac concentrations had to be

used, the sensitive radio-TLC-based assay provided a quan-

titative measure of the relative hydroxylase content of the

various cell lines.

Using this enzyme assay, it was first shown that most of

the tested clones displayed a reduced CMP-Neu5Ac hy-

droxylase activity; in fact, this is probably due to the use of

the combined in vitro/in vivo amplification procedures

yielding a high rate of strong producer clones. Second, the

efficiency of the antisense fragments in vivo was indepen-

dent of the animal source, and the 199 nt fragments were

more effective than the 403 nt antisense RNAs at suppress-

ing hydroxylase expression (Fig. 6). The cell line trans-

fected with the 199 nt fragment derived from the cDNA of

the mouse enzyme exhibited the greatest (78%) reduction in

the hydroxylase activity and was termed CHO-AsUH2.

Although the latter antisense fragment differed from the

corresponding hamster fragment by 15 nt, both were effi-

cient at inhibiting the translation of the hydroxylase mRNA

in vivo. In fact, these sequence differences could be advan-

tageous, since natural antisense RNAs do not exhibit com-

plete complementary to the target mRNA and thus are less

prone to intracellular RNases [44,45]. Two different mech-

anisms could account for the inhibition of translation by

antisense fragments [37]: (i) steric blocking of translation

initiation, and (ii) degradation of the antisense RNA-mRNA

duplex by specific RNases. However, since none of the 199

nt antisense fragments was located upstream the AUG

initiator codon, the reduction of CMP-Neu5Ac hydroxylase

activity in CHO-AsUH2 is probably mainly due to the

degradation of mRNAs. Indeed, the semi-quantitative esti-

mation of the amounts of mRNA by RT-PCR showed a

significant reduction (approximately 80%) in CMP-Neu5Ac

hydroxylase mRNA in the CHO-AsUH2 cell line as com-

pared to CHO-UH cells (Fig. 7A). This result is also in a

good agreement with previous findings demonstrating that

(antisense RNA-sense mRNA) hybrids are rapidly degraded

by RNases specific for RNA duplexes, leading to a decrease

in the endogenous message levels of a targeted gene [46,47].

An analysis of Neu5Gc, the product of the hydroxylase

reaction, in total cellular glycoconjugates produced by cells

cultivated for 32 passages in the presence of human serum,

revealed an 80% decrease in the amount of Neu5Gc in the

CHO-AsUH2 cell line relative to the parental cells (Fig. 8).

Since a reduction in CMP-Neu5Ac hydroxylase activity of

about 80% led to the same decrease in the amount of

Neu5Gc residues present on the glycoconjugates in the

new CHO-AsUH2 cell line, the amount of hydroxylase

protein appears to be governing the incorporation of

Neu5Gc into glycoconjugates. This is in agreement with

previous studies on various mammalian tissues [33,41,48] in

which the amount of Neu5Gc was found to be regulated by

the level of hydroxylase activity. This, in turn, generally

correlates with the quantity of hydroxylase protein. More-

over, the estimated 80% reduction in the levels of hydrox-

ylase mRNA found in CHO-AsUH2 cells suggests that in

this cell line, the observed decrease in the CMP-Neu5Ac

hydroxylase activity and the amount of Neu5Gc are due to

the presence of antisense RNA.

Although we were able to significantly reduce the CMP-

Neu5Ac hydroxylase activity in CHO-UH cells, the anti-

sense RNA approach does not yield a complete suppression

of the enzyme activity. This agrees with previous reports on

the inhibition of sialidase activity in CHO cells [23]. Despite

the fact that the CMP-Neu5Ac hydroxylase mRNA only

seems to be present in a low copy number, and the antisense

fragment was amplified by means of DHFR-MTX selection

and in vitro concatenamer amplification systems, the CHO-

AsUH2 cells still exhibited 20% of the parental enzyme

activity. Nevertheless, compared to previous studies [21,23,

38], the approach described here enabled the generation of a

stable cell line which retained a significant reduction of the

target enzyme activity for more than 30 passages. Though

the overproduction of a recombinant protein by this cell line

might require the use of a selection/amplification marker

different from DHFR/MTX (for instance glutamine syn-

thase/methionine sulfoximine) [49], these new CHO-

AsUH2 cells are suitable for industrial purposes and further


studies will focus on the assessment of the reduction of

Neu5Gc levels in recombinant glycoproteins produced by

these cells.

Acknowledgements

This work was partially supported by the European

community, grant No. 960767. The authors thank Dr.

Kawano for providing the pBSCNAH vector containing the

mouse CMP-Neu5Ac hydroxylase cDNA.

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