2 INSERM U526, IFR50, Faculté de Médecine, 06107 Nice Cedex 2, France; 3 INSERM U470, Centre de Biochimie, Parc Valrose, 06108 Nice Cedex 2, France; and 4 CNRS-UNSA-UMR 6549, IFR50, Faculté de Médecine, 06107 Nice Cedex 2, France
Received on December 20, 2001; revised on May 29, 2002; accepted on July 2, 2002
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Abstract |
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Key words: Alu element/gene expression/transcription stop/xenograft
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Introduction |
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The 1,3GalT gene is responsible for the synthesis of
-Gal epitopes in all mammals, except Old World primates. The expression of this gene has been completely lost in Old World monkeys, apes, and humans, thus enabling the production of significant amounts of natural antibodies to
-Gal (Galili et al., 1984
).These antibodies are strongly active, in the presence of complement (Good et al., 1992
). This explains hyperacute rejection of xenografts, and in particular, these antibodies prevent projects of transplantation of pig organs to humans (Kobayashi and Cooper, 1999
).
The 1,3GalT gene encodes for the UDP-Gal:Galß1,4GlcNAc
1,3-galactosyltransferase enzyme that catalyzes the transfer of a Gal residue, with an
1,3 linkage, on terminal lactosaminide (Galß1,4GlcNAc-R) disaccharide beared by a glycoprotein or a glycolipid (Blanken and Van den Eijnden, 1985
). The enzyme is not able to graft
-Gal on Fuc
1,2Galß1,3/4GlcNAc-R that is a Fuc
1,2-substituted lactosamine (i.e., blood group H structure), as does the histo-blood group B transferase (B-transferase). Therefore the
1,3GalT gene has to be clearly distinguished from the B-transferase, which is strongly expressed in blood group B individuals.
The murine 1,3GalT gene is composed of nine exonic sequences, six of which (exons 4 to 9) being translated (Joziasse, 1992
). Its structure diverges from that of the blood group transferase genes that make up seven translated exons with extensive similarities between the two last exons that correspond to the catalytic domain of this group.
Two 1,3GalT homologs have been described in the human genome. Both contained several frame-shift mutations and internal stop codons (Joziasse et al., 1991
; Larsen et al., 1990
). The first one is located on chromosome 12 and corresponds to a copy of an
1,3GalT cDNA, without intronic sequences (Joziasse et al., 1991
; Larsen et al., 1990
). This pseudogene has also been retrieved in apes and Old World monkeys (Galili and Swanson, 1991
). The other one has been localized on chromosome 9 and could correspond to an authentic
1,3GalT gene, because intronic sequences (Joziasse et al., 1991
) as well as one exonic sequence corresponding to the largest part of the catalytic domain of the enzyme (Larsen et al., 1990
) have been detected in human genomic libraries. However, two frame-shift mutations present in the exonic sequence rendered this pseudogene nonfunctional (Larsen et al., 1990
). Finally, natural transcripts of
1,3GalT genes have widely been shown in nonprimate but never in human cells (Galili, 1999
).
In the course of our study of the alterations of glycosylation patterns (hyposialylation and poly-N-acetyllactosaminyl extensions) in human HIV-1-infected lymphoblasts (Lefebvre et al., 1994a,b), we have found transcripts similar to some of the coding exons of marmoset
1,3GalT, a New World monkey gene, overexpressed in latently HIV-1-infected lymphoblasts and also retrieved in human Expressed Sequences Tag (EST) databases. In the present article, we report the characterization of these transcripts and of the genomic sequences corresponding to these messengers. Moreover, we propose that Alu-like sequences may act as transcriptional terminator in this gene, accounting for part of the regulation of
1,3GalT gene expression in humans.
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Results and discussion |
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Among various mechanisms, it was conceivable that an 1,3GalT enzymatic activity could be responsible for these changes. Actually, it has been demonstrated that expression of a transgene
1,3GalT could compete with endogenous
2,3-sialyltransferases without limiting the elongation of poly-N-acetyllactosamine chains (Smith et al., 1990
).
HIV-1 latently infected CEM cells are reactive with the plant lectin Griffonia simplicifolia specific for -Gal epitopes
Although the hypothesis of expression of 1,3GalT in human cells was unlikely, we were led to explore it after the discovery of a weak differential reactivity of CEMLAI/NP versus parental CEM cells with Griffonia simplicifolia GS-I isolectin B4 specific for terminal
-Gal (Murphy and Goldstein, 1977
) (data not shown). This surprising result has already been reported by others (Castronovo et al., 1989
; Galili et al., 1985
; Kagawa et al., 1988
).
Detection of human 1,3GalT mRNA
To validate this observation, we decided to search for 1,3GalT mRNA by reverse transcription polymerase chain reaction (RT-PCR) on CEMLAI/NP cells lysates. Several pairs of degenerate primers were designed on the basis of conserved sequences between
1,3GalT of marmoset (Henion et al., 1994
), ox (Joziasse et al., 1989
), and mouse (Larsen et al., 1989
). One of them, Galfive, allowed the production of a 200-bp fragment. The sequence of this fragment was nearly identical (94.5%) to the 5' region of marmoset
1,3GalT cDNA. The Galfive amplified DNA fragment was then used to probe a northern blot carried out with poly(A)-rich RNA isolated from parental CEM and CEMLAI/NP cells. As shown in Figure 1, a ~1.6-kb species was detected in both cell lines but was much stronger in CEMLAI/NP cells. It is noteworthy that the size of these mRNA is much smaller than the size of
1,3GalT transcripts (3.63.9 kb) detected in mammals with a functional
1,3GalT gene (Joziasse et al., 1989
; Smith et al., 1990
). Hence, the expression of truncated mRNA similar to
1,3GalT seemed to be correlated with changes in glycosylation in CEMLAI/NP cells.
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Using the BLAST program, we searched for alignments between 1,3GalT-FRAG and Expressed Sequence Tag databases. This search allowed the discovery of several clones that matched almost exactly (95100% identities) with the 3' end of
1,3GalT-FRAG (Table I). Through the IMAGE Consortium, it has been possible to receive one of these clones (Genbank accession no. R24770), originated from an infant brain cDNA library constructed by Bento Soares and M. Fatima Bonaldo (University of Iowa). The insert sequence (1.2 kb) matched exactly (100% identities) with our
1,3GalT-FRAG and allowed us to confirm the presence in human cells of a particular transcript of
1,3GalT containing the first four translated exons elongated with the unk3' sequence of 0.8 kb.
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The unk3' region is polyadenylated downstream from the noncanonical poly(A) signal AAATAAAAA. This one is retrieved in the human ESTs similar to 1,3GalT found in databases (Table I), and which have been primed with a poly(T) oligonucleotide.
Thus it appeared that the 1,3GalT-FRAG cDNA was terminated at an intra-intronic poly(A) signal present within intron VII and that the unk3' fragment was attached to the exonVII after intermediate splicing of this intron (Figure 2C). Because it has been shown that the addition of a poly(A) tail to the 3' ends of nuclear RNA occurs more rapidly than RNA splicing (Lai et al., 1978
; Nevins and Darnell, 1978
), the most probable chronology of events that lead to the production of truncated transcripts polyadenylated at an intra-intronic site could be as follows: First, arrest of transcription at an intra-intronic site followed by addition of poly(A) tail and finally splicing of the transcript. There are at least two other examples of this mechanism, the alternative production of the membrane-anchored and secreted forms of (1) the phospholipase A2 receptor (Ancian et al., 1995
) and (2) the receptor for the complement C3b/C4b called CD35 (Hourcade et al., 1988
). In these two cases, an intra-intronic polyadenylation event leads to the exclusion of a 3' end exon that encodes for a transmembrane domain, and thus to the production of a functional secreted form of the receptor.
The DNA structure downstream from the intronic poly(A) signal is propitious to a transcriptional stop
Comparison between human 1,3GalT-FRAG cDNA and
1,3GalT gene organization led to the conclusion that the splicing of exon VII with the unk3' sequence in intron VII is the result of the excision of a 4.3-kb intron. However, according to the splicing events that generate a complete
1,3GalT cDNA in nonprimate mammals, the size of the corresponding intron might be 7.8 kb. No obvious mutations within the donor or the acceptor consensus sites of splicing could be revealed in the genomic sequence in these regions (Figures 2C and 3). Therefore, one way to explain this observation was that the transcription of
1,3GalT gene was terminated using a transcriptional-stop signal and/or a polyadenylation signal present within intron VII.
Little is known about either the signals for termination or the process involved for most eukaryotic RNA polymerases. However, experiments performed in Xenopus oocytes suggest that formation of secondary structures is more important in that process than the exact sequence (Nishikura et al., 1982). To check for the presence of a sequence that could be involved in forming secondary structures, we looked at the genomic sequence located downstream from the 3' sequence of
1,3GalT-FRAG. By computer analysis, we found significant similarities between this sequence and AluSx sequences belonging to the Alu family (Batzer et al., 1996
), (AluGal, Figures 2C and 4).
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The 865-bp sequence arround the poly(A) signal of intron VII functions as a transcriptional stop
A fragment of 865 bp (stopGal) was synthesized from total DNA extracted from CEM cells using PCR and a pair of primers flanking the AAATAAAAA signal from the 83 to the +782 nucleotide (Figure 2C). The stopGal fragment was then ligated into the pCMVß plasmid between the promoter CMV IE and the ß-galactosidase reporter gene (see Materials and methods). Stop-sense and Stop-antisense constructs were obtained. These recombinants and control plasmids were transfected in human embryonic kidney (HEK)293 cells. As shown in Figure 5, the activity of ß-galactosidase was quantified from lysates of transfected cells, using ortho-nitrophenyl ß-D-galactopyranoside (ONPG) substrate. When compared to corresponding controls, the stopGal sequence was able to block almost completely the activity of the reporter gene when it was constructed in the sense orientation. Moreover, although significant, its blocking efficiency was much more moderate when it was constructed in the antisense orientation (Figure 5).
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An important question arising in view of the widespread tissue distribution of 1,3GalT-FRAG transcripts concerns the role that these mRNA could play, especially in the case of their overexpression discovered in CEMLAI/NP cells (Figure 1). Indeed, the CD43/CD45 molecules of CEMLAI/NP cells are severely hyposialylated and this status is associated with a (apparently paradoxical) normal expression of concerned sialyltransferases (Giordanengo et al., 1997
).
Even if any glycosyltransferase activity cannot be expected from 1,3GalT-FRAG, a short protein (100 amino acids) containing the signal peptide and the almost entire stem region of
1,3GalT can be translated from the corresponding mRNA. This putative protein could thus be addressed to the Golgi apparatus just like an authentic glycosyltransferase. It is generally admitted that the concentration of these enzymes is well regulated, implicating sorting, anterograde and retrograde transport vesicles (Bannykh et al., 1998
; Klumperman, 2000
). On this basis, it could be hypothesized that
1,3GalT-FRAG proteins interfere in the trans-Golgi network compartment with functional enzymes, such as sialyltransferases, by way of their conserved stem domain in the manner of negative transdominants. This could account for results indicating that expression of a transgene
1,3GalT could compete with endogenous
2,3-sialyltransferases (Smith et al., 1990
). To pursue the hypothesis, the hyposialylation status of CD43/CD45 molecules of CEMLAI/NP cells could be explained by the overexpression of nonfunctional but competitive
1,3GalT-FRAG peptides.
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Materials and methods |
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PCR
All the PCR reactions were carried out in buffer added with 10% (v/v) dimethyl sulfoxide, and the amplicons were subcloned into the plasmid pCR II-TOPO (TOPO TA Cloning Kit, Invitrogen, Faraday, CA). PCR primers pairs were:
Cloning of truncated human 1,3GalT cDNA
A CEMLAI/NP cell line cDNA library was constructed using pcDNA3 vector plasmid and a cDNA synthesis kit (both from Invitrogen) with an oligo(dT) (NotI) primer, according to the manufacturers recommendations. About 4 x 106 colonies of Escherichia coli DH5 transfected with this library were isolated by using the antisense oligonucleotide probe 5'-CCCAAAACACAATGATCACAGTTGAGACAACCA designed on the basis of the sequence of PCR fragments obtained with the primers pair Galfive.
This probe was 5'-end radiolabeled using T4 Polynucleotide Kinase (Clontech Laboratories, Palo Alto, CA) and [-32P]ATP (ICN Biomedicals, Costa Mesa, CA). Membrane prehybridization was carried out at 60°C for 3 h in 20 mM phosphate buffer, pH 7.5, containing 5x NaCL/Cit (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 7% (w/v) sodium dodecyl sulfate (SDS), 5x Denhardts, and 1% salmon sperm DNA. Hybridization was achieved overnight at 60°C. Then membranes were washed twice in 2x NaCl/Cit, 2% (w/v) SDS for 20 min at 20°C, once in 0.1x NaCl/Cit, 0.1% SDS 20 min at 20°C, and once in 0.1x NaCl/Cit, 0.1% SDS, 30 min at 48°C and thereafter exposed to XAR5 Kodak film for 3 days at 80°C. Three positive clones were isolated and sequenced.
Insertion of a plasmid containing the transcriptional stop fragment between the coding sequence of the reporter gene ß-galactosidase and its promoter and transient transfections
To test the ability of the stopGal sequence (865 nt) to end the transcription of human 1,3GalT gene, the stopGal fragment was inserted into the plasmid pCMVß (Clontech) between the ß-galactosidase coding sequence and the CMV IE promoter. To conserve the SV40 intron located between the promoter and the ß-galactosidase gene of pCMVß, we first attempted to subclone directly blunt-ended stopGal into the Sma I unique site of pCMVß downstream from the SV40 intron. All the constructs obtained by these means were in an antisense orientation. These constructs (called STOP-antisense) were nevertheless tested as indicated.
To insert stopGal in sense orientation, the SV40 intron of pCMVß flanked by the unique restriction sites Xho I and Sma I was excised and replaced by appropriated stopGal. For that purpose, a Sma I site was first created between the Hind III and Bam HI sites located in the multiple cloning site of the intermediate stopGal/pCR II-TOPO construct, by insertion of a double strand oligonucleotide generated by hybridization of the 5'-AGCTTGGTACCCGGGAGCTCG and 5'-GATCCGAGCTCCCGGGTACCA (the Sma I linker underscored) oligonucleotides. It was thereafter possible to ligate the Xho IstopGalSma I fragments into pCMVß in the orientation expected to obtain the constructs called STOP-sense. The control pCMVß without the SV40 intron (pCMVß delta SV40) was also prepared.
HEK-293 cells were transiently transfected, on six-well plates using MBS mammalian transfection kit (Stratagene). To control transfection efficiency, the expression vector GL2-promoter (Promega) containing the luciferase gene was cotransfected (3 µg/well) with each of the ß-galactosidase constructs (3 µg/well).
Northern analysis
Total RNA were extracted according to Guanidine Isothiocyanate Technic, and Poly(A)-rich RNA were selected as described elsewhere (Giordanengo et al., 1997). Poly(A)-rich RNA were electrophoresed on denaturing 1.2% agarose gel and transferred in 20x NaCl/Cit to a hybond N nylon (Amersham Corp). The probe was [
-32P]-random-labeled Galfive amplicons. The detection of the glyceraldehyde 3-phosphate-dehydrogenase mRNA were used as internal control.
ß-Galactosidase and luciferase assays
Colorimetric assays were conducted with soluble ONPG (Sigma) on HEK-293 cells transfected in 24-well plates (5 x 104 cells/well) transfected with STOP-sense, STOP-antisense, and control plasmids (2 µg/well). After a 48-h period of culture, cells were disrupted in 100 µl lysis Tris buffer (0.25 M, pH 7.8; 0.5% Nonidet P-40; 1% Triton) for 15 min. From supernatant of each well, a 5-µl aliquot was taken off to protein quantification, and 70 µl were transferred in a new plate. Then, successively and in the same order, to each well was added 1 ml of 2-ME buffer (0.1 M NaHPO4, pH 7.5; 10 mM KCl; 1 mM MgSO4; 0.1% 2-ME) for 15 s, 200 µl ONPG solution (0.1 M NaHPO4, pH 7.5; 4 mg/ml ONPG) for 15 s, and finally 500 µl 1 M Na2CO3 to stop the reaction. Two days after transfection, ß-galactosidase activity was evaluated by measuring the release of o-nitrophenol at 420 nm. Luciferase assay was performed according to Brasier et al. (1989). Colorimetric and luminescent points were normalized according to the amount of protein evaluated in each sample with the BCA Protein Assay Reagent (Pierce, Rockford, IL). All transfections were performed in triplicate for each point and repeated at least three times.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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