A complete {alpha}1,3-galactosyltransferase gene is present in the human genome and partially transcribed

Marion Lantéri2, Valérie Giordanengo2, Frédérique Vidal3, Patrick Gaudray4 and Jean-Claude Lefebvre1,2

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


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The synthesis of Gal{alpha}1-3Gal-terminated oligosaccharides ({alpha}-Gal) epitopes has been interrupted during the course of evolution, starting with Old World primates. Partial sequences similar to the {alpha}1,3-galactosyltransferase ({alpha}1,3GalT) gene, which governs the synthesis of {alpha}-Gal epitopes, have been detected in the human genome and were found to correspond to pseudogenes. We completed the sequence of the human {alpha}1,3GalT pseudogene present on chromosome 9 and found it to be organized like the murine {alpha}1,3GalT gene. In human cell lines and several normal and tumor tissues we detected truncated transcripts corresponding to this pseudogene. Considering these mRNAs, translation of an open reading frame containing the first four translated exons but missing the two catalytic exons could predict a truncated {alpha}1,3GalT polypeptide that should be enzymatically inactive. We show that transcription of human {alpha}1,3GalT is prematurely terminated at the level of a strong transcriptional stop signal in the middle of intron VII. We were able to reproduce this effect in vitro by subcloning the implicated DNA region upstream from a reporter cDNA. The premature transcriptional arrest of human {alpha}1,3-GalT gene leads to an ectopic splicing event and to the connection of a short intronic sequence downstream from translated exons. Finally, we show that these truncated transcripts are overexpressed in cell lines with modifications of O-glycans.

Key words: Alu element/gene expression/transcription stop/xenograft


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Most of the genes expressed in humans have counterparts in other mammals. The {alpha}1,3-galactosyltransferase ({alpha}1,3GalT) gene constitutes a noteworthy exception (Galili, 1999Go). Only partial sequences, similar to {alpha}1,3GalT genes of nonprimate mammals, have been detected in the human genome and are considered to correspond to pseudogenes because of multiple base deletions leading to premature translation stops (Joziasse et al., 1991Go; Larsen et al., 1990Go). Attempts to detect human {alpha}1,3GalT transcripts have remained unsuccessfull as of today (Galili, 1999Go).

The {alpha}1,3GalT gene is responsible for the synthesis of {alpha}-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 {alpha}-Gal (Galili et al., 1984Go).These antibodies are strongly active, in the presence of complement (Good et al., 1992Go). This explains hyperacute rejection of xenografts, and in particular, these antibodies prevent projects of transplantation of pig organs to humans (Kobayashi and Cooper, 1999Go).

The {alpha}1,3GalT gene encodes for the UDP-Gal:Galß1,4GlcNAc {alpha}1,3-galactosyltransferase enzyme that catalyzes the transfer of a Gal residue, with an {alpha}1,3 linkage, on terminal lactosaminide (Galß1,4GlcNAc-R) disaccharide beared by a glycoprotein or a glycolipid (Blanken and Van den Eijnden, 1985Go). The enzyme is not able to graft {alpha}-Gal on Fuc{alpha}1,2Galß1,3/4GlcNAc-R that is a Fuc{alpha}1,2-substituted lactosamine (i.e., blood group H structure), as does the histo-blood group B transferase (B-transferase). Therefore the {alpha}1,3GalT gene has to be clearly distinguished from the B-transferase, which is strongly expressed in blood group B individuals.

The murine {alpha}1,3GalT gene is composed of nine exonic sequences, six of which (exons 4 to 9) being translated (Joziasse, 1992Go). 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 {alpha}1,3GalT homologs have been described in the human genome. Both contained several frame-shift mutations and internal stop codons (Joziasse et al., 1991Go; Larsen et al., 1990Go). The first one is located on chromosome 12 and corresponds to a copy of an {alpha}1,3GalT cDNA, without intronic sequences (Joziasse et al., 1991Go; Larsen et al., 1990Go). This pseudogene has also been retrieved in apes and Old World monkeys (Galili and Swanson, 1991Go). The other one has been localized on chromosome 9 and could correspond to an authentic {alpha}1,3GalT gene, because intronic sequences (Joziasse et al., 1991Go) as well as one exonic sequence corresponding to the largest part of the catalytic domain of the enzyme (Larsen et al., 1990Go) have been detected in human genomic libraries. However, two frame-shift mutations present in the exonic sequence rendered this pseudogene nonfunctional (Larsen et al., 1990Go). Finally, natural transcripts of {alpha}1,3GalT genes have widely been shown in nonprimate but never in human cells (Galili, 1999Go).

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., 1994aGo,b), we have found transcripts similar to some of the coding exons of marmoset {alpha}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 {alpha}1,3GalT gene expression in humans.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We have previously reported modifications of O-glycans that appear on major lymphocytic surface glycoproteins CD45 and CD43 after HIV-1 infection. The modified O-glycans side chains, harbored by the latently HIV-1-infected CEMLAI/NP cells, are hyposialylated and elongated with poly-N-acetyllactosamine chains (Lefebvre et al., 1994aGo,b).

Among various mechanisms, it was conceivable that an {alpha}1,3GalT enzymatic activity could be responsible for these changes. Actually, it has been demonstrated that expression of a transgene {alpha}1,3GalT could compete with endogenous {alpha}2,3-sialyltransferases without limiting the elongation of poly-N-acetyllactosamine chains (Smith et al., 1990Go).

HIV-1 latently infected CEM cells are reactive with the plant lectin Griffonia simplicifolia specific for {alpha}-Gal epitopes
Although the hypothesis of expression of {alpha}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 {alpha}-Gal (Murphy and Goldstein, 1977Go) (data not shown). This surprising result has already been reported by others (Castronovo et al., 1989Go; Galili et al., 1985Go; Kagawa et al., 1988Go).

Detection of human {alpha}1,3GalT mRNA
To validate this observation, we decided to search for {alpha}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 {alpha}1,3GalT of marmoset (Henion et al., 1994Go), ox (Joziasse et al., 1989Go), and mouse (Larsen et al., 1989Go). 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 {alpha}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 {alpha}1,3GalT transcripts (3.6–3.9 kb) detected in mammals with a functional {alpha}1,3GalT gene (Joziasse et al., 1989Go; Smith et al., 1990Go). Hence, the expression of truncated mRNA similar to {alpha}1,3GalT seemed to be correlated with changes in glycosylation in CEMLAI/NP cells.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 1. Northern blot analysis of human {alpha}1,3GalT-FRAG mRNA. Poly(A)+ RNA were electrophoresed (5 µg/lane), transferred to a nylon membrane, and then probed with {alpha}-32P random-labeled amplicons Galfive, corresponding to the 5' coding region of human {alpha}1,3GalT (exons IV–VI). Lane 1, CEMLAI/NP; lane 2, parental CEM cells.

 
An entire human {alpha}1,3GalT-inactivated gene (pseudogene) was retrieved in the GenBank database
Cloning the entire human {alpha}1,3GalT cDNA was performed by the screening of a cDNA library synthesized by retrotranscription of poly(A)-mRNA isolated from CEMLAI/NP cells, using a 5'-end 32P-radiolabeled oligonucleotide designed on the basis of the sequence of Galfive PCR fragments (see Materials and methods). Three positive clones were isolated and the longest ({alpha}1,3GalT-FRAG, 1.1 kb) was sequenced. An unique open reading frame of only 303 nt was identified, corresponding to the first four translated exons 4–7 of the marmoset {alpha}1,3GalT cDNA (Figure 2A), linked to an unknown untranslated region of 0.8 kb (unk3') (Figure 2C). The unk3' region is followed by a poly-A sequence. The first coding exon of this truncated cDNA matched exactly with the clone HGT-10 (Figure 2B) described by Joziasse et al. (1991)Go). The size discrepancy between the transcripts seen after northern blot (1.6 kb) and revealed after cloning (1.1 kb) can be explained by an unusually long 5'-untranslated region (UTR) of these truncated {alpha}1,3GalT as it has already been reported for the bovine {alpha}1,3GalT mRNA with a quite long 5' UTR, totaling 468 bp (Joziasse et al., 1989Go). It is also known that the three exons composing the {alpha}1,3GalT 5' UTR are differentially spliced depending on the tissues among various vertebrates, very likely leading to differential expression (Galili, 1999Go).



View larger version (49K):
[in this window]
[in a new window]
 
Fig. 2. Sequence and localization of {alpha}1,3GalT-FRAG cDNA. (A) The four potentially translated exons IV–VII of {alpha}1,3GalT-FRAG and the corresponding sequence of marmoset {alpha}1,3GalT were aligned; only divergent bases were denoted. The position of the base 240 deletion is indicated by a vertical arrow, and the poly(A) signal is underscored. (B) The sequence of exon IV, underscored on panel A, matched exactly with the exon described in the clone HGT-10 flanked by intronic sequences (Joziasse et al., 1991Go). (C) The unknown 3'UTR sequence (unk3') of {alpha}1,3GalT-FRAG cDNA is localized on the intron VII of the human {alpha}1,3GalT gene. The sequence of this gene was extracted from clone RP11-162D16 (Genbank accession no. AL359644). The unk3' sequence is linked to the potentially translated exon VII after an intermediate splicing event that excises a segment of 4361 bases after a premature trancriptional arrest in the middle of intron VII. The positions of stopGal and AluGal sequences are denoted by hooks. Splice donor and acceptors are framed. The splice acceptor at position 7920 corresponds to the expected end of intron VII in the case of a complete trancription of the human {alpha}1,3GalT gene.

 
The recent availability of the human genome allowed us to retrieve a 172-kb-long clone (GenBank accession no. AL359644) from chromosome 9 containing all the six translated exons that matched almost exactly with those of marmoset (Figure 3). The size of each coding exon (89, 36, 66, 116, 138, 692 nt for exons IV–IX, respectively) was remarkably similar to the corresponding exon of mouse {alpha}1,3GalT (Joziasse et al., 1992Go), apart from exon VII (116 versus 102 nt). (Roman numerals are used to number human {alpha}1,3GalT exons by analogy with mouse exons [Arabic numerals], to take into account that the first three exons of human gene are hypothetical.) It is also noteworthy that the mouse coding exons IV–VI and VIII–IX are highly similar (82%) to the corresponding potentially coding human exons, whereas exons VII are <50% similar. This gene contains the partial {alpha}1,3GalT genomic sequences previously described as clones HGT-10 (Joziasse et al., 1991Go) and HGT-1 (Larsen et al., 1990Go) that correspond to exons IV and IX, respectively.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3. Schematic and comparative representation of mouse and human {alpha}1,3GalT genomic organization. Corresponding human transcripts are also shown. Roman numerals are used to number human {alpha}1,3GalT exons by analogy with mouse exons (Arabic numerals) to take into account that the first three exons of human gene are hypothetical. The sizes of exons (thick bars) are indicated in parentheses. Human exons 1–3 are not known. Border sequences of human introns (thin bars) are displayed. The positions of base deletions are circled. The point of transcriptional stop signal is shown with a thick arrow.

 
These potentially coding sequences are interrupted by five successive introns of 4.6, 2, 1, 7.9, and 4.5 kb (Figure 3). The unknown 3' region (unk3') of 0.8 kb of the {alpha}1,3GalT-FRAG cDNA was retrieved in the middle of intron VII (Figure 2C).

Using the BLAST program, we searched for alignments between {alpha}1,3GalT-FRAG and Expressed Sequence Tag databases. This search allowed the discovery of several clones that matched almost exactly (95–100% identities) with the 3' end of {alpha}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 {alpha}1,3GalT-FRAG and allowed us to confirm the presence in human cells of a particular transcript of {alpha}1,3GalT containing the first four translated exons elongated with the unk3' sequence of 0.8 kb.


View this table:
[in this window]
[in a new window]
 
Table I. Human ESTs similar to truncated {alpha}1,3GalT mRNA
 
The hypothetical human {alpha}1,3GalT coding entire cDNA (1127 nt) was further deduced by comparison between the marmoset {alpha}1,3GalT cDNA and the human gene (Figure 3). The difference of three bases in length between the human and marmoset {alpha}1,3GalT cDNAs corresponds to three base deletions in the human gene, which are located, if numbered from 1 on marmoset ATG, at positions 240 (exon VII), 767, and 846 (exon IX). Deletions 767 and 846 correspond to those found by Larsen et al. (1990)Go) in the clone HGT-1 and would lead to the inactivation of the catalytic domain of the {alpha}1,3GalT enzyme in the case of a complete transcription of the gene. In contrast, the deletion 240 that we identified induces a frame shift that leads to a premature termination by 75 bases downstream, just after the end of exon VII (Figure 2A). Nevertheless, the {alpha}1,3GalT-FRAG transcript we have isolated could be translated as a short peptide of 100 amino acids containing the signal peptide and the major part of the stem region of the theoretical human {alpha}1,3GalT enzyme.

The unk3' region is polyadenylated downstream from the noncanonical poly(A) signal AAATAAAAA. This one is retrieved in the human ESTs similar to {alpha}1,3GalT found in databases (Table I), and which have been primed with a poly(T) oligonucleotide.

Thus it appeared that the {alpha}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., 1978Go; Nevins and Darnell, 1978Go), 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., 1995Go) and (2) the receptor for the complement C3b/C4b called CD35 (Hourcade et al., 1988Go). 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 {alpha}1,3GalT-FRAG cDNA and {alpha}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 {alpha}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 {alpha}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., 1982Go). 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 {alpha}1,3GalT-FRAG. By computer analysis, we found significant similarities between this sequence and AluSx sequences belonging to the Alu family (Batzer et al., 1996Go), (AluGal, Figures 2C and 4).



View larger version (62K):
[in this window]
[in a new window]
 
Fig. 4. Aligment of Alu sequence found in {alpha}1,3GalT gene and AluSx consensus (Rowold and Herrera, 2000Go). The dots represent the same nucleotide. Deletions are indicated by dashes.

 
The Alu sequence corresponds to an human family of repetitive sequences related to 7SL RNA. These sequences were composed of a tandem duplication of 130 bp, with an unrelated insertion of a 31-bp sequence in the right half of the dimer. The individual members of the Alu family are rather related than identical (Rowold and Herrera, 2000Go). Some of them appeared to be transcribed in vivo by the RNA polymerase III, thus leading to transcriptional interferences. A variety of properties have been found for this family, and its ubiquity has led to many suggestions concerning its function. In fact, repeated sequences seem to be modular components of regulatory elements whose effects depend on the influence of flanking sequences. One of the proposed functions is that due to their ability to form hairpin structures, some of these sequences could act as transcriptional terminators and in that way could be involved in the control of gene expression (Maraia and Sarrowa, 1995Go). To test this hypothesis in the case of {alpha}1,3GalT gene, we cloned the genomic fragment containing the Alu-like sequence (stopGal) in a reporter plasmid between the ß-galactosidase gene and its promoter.

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).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5. Transcriptional stop activity of the stopGal sequence. A sequence of 865 nucleotides downstream from the poly(A) signal located in the intron VII of human {alpha}1,3GalT was constructed into the plasmid pCMVß between the promoter CMV IE and the ß-galactosidase reporter gene in a sense orientation (Stop-sense) in the place of the SV40 intron, and in an antisense orientation (Stop-antisense) with conservation of the intron. Each construct and control plasmid (pCMVbeta and pCMVbeta-deltaSV40) was cotransfected in HEK-293 cells, on six-well plates with the expression vector pGL2-promoter containing the luciferase gene. Data are the ratios of ß-galactosidase versus luciferase activities, arbitrarily set as a 100% for pCMVbeta. Results are shown as means of at least three experiments done with triplicates on 6 wells each time (A), after normalization according to the amount of protein evaluated in each sample at 560 nm (B).

 
These results could be explained by the orientation of the Alu sequence inserted in the constructs. In the sense construction, in addition to secondary hairpin structures, transcriptional interferences could arise due to the presence of potential RNA polymerase III promoter retrieved in Alu sequence. This observation is reminiscent of what is found in natural transcriptional stop of some viruses (Lee et al., 1981Go).

An important question arising in view of the widespread tissue distribution of {alpha}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., 1997Go).

Even if any glycosyltransferase activity cannot be expected from {alpha}1,3GalT-FRAG, a short protein (100 amino acids) containing the signal peptide and the almost entire stem region of {alpha}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., 1998Go; Klumperman, 2000Go). On this basis, it could be hypothesized that {alpha}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 {alpha}1,3GalT could compete with endogenous {alpha}2,3-sialyltransferases (Smith et al., 1990Go). 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 {alpha}1,3GalT-FRAG peptides.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cells and cultures
The cell line HEK-293 (ATCC CRL-1573) was maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Rockville, MD) containing 7% (v/v) fetal calf serum (FCS) (Whittaker Bioproducts, Walkersville, MD), and transfected with the Transfection MBS Mammalian Transfection Kit (Stratagene, La Jolla, CA). The HIV-1 latently infected CEMLAI/NP cell line bearing hyposialylated surface glycoproteins was described elsewhere (Lefebvre et al., 1994aGo). The parental CCRF-CEM (ATCC CCL-119) and CEMLAI/NP cell lines were propagated in RPMI 1640 medium (Life Technologies) containing 5% (v/v) FCS.

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:

Galfive: 5'-ATGAATGTCAARGGAAAAGTRAT and 5'-CTTSTTGATAATYGTGGRTCCC (R = A + G; S = G + C; Y = C + T) for detection of {alpha}1,3GalT sequences (Genbank S71333, nt 1–23 and nt 181–202, respectively) by RT-PCR; and stopGal: 5'-CTAAAAGCACCATGTAAGCTACTAA and 5'-CTCATTTAGTCTCTGCAACAGC for the cloning of the transcriptional stop sequence from total genomic DNA (Genbank AL359644, nt 102,539–102,560 and 103,380–103,404, respectively).

Cloning of truncated human {alpha}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 manufacturer’s recommendations. About 4 x 106 colonies of Escherichia coli DH5{alpha} 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 [{gamma}-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 Denhardt’s, 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 {alpha}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 I–stopGal–Sma 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., 1997Go). 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 [{alpha}-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)Go. 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.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by institutional grants from the Institut Nationalde la Santé et de la Recherche Médicale (INSERM) and the Association pour le Développement du Diagnostic des Maladies Virales (ADDMV). The sequence reported in this article has been deposited in the GenBank database (accession no. AF305838)


    Abbreviations
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
EST, Expressed Sequence Tag; FCS, fetal calf serum; HEK, human embryonic kidney; ONPG, ortho-nitrophenyl ß-D-galactopyranoside; RT-PCR, reverse transcription polymerase chain reaction; SDS, sodium dodecyl sulfate; UTR, untranslated region.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: lefebvre@unice.fr Back


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Ancian, P., Lambeau, G., Mattei, M.G., and Lazdunski, M. (1995) The human 180-kDa receptor for secretory phospholipases A2. Molecular cloning, identification of a secreted soluble form, expression, and chromosomal localization. J. Biol. Chem., 270, 8963–8970.[Abstract/Free Full Text]

Bannykh, S.I., Nishimura, N., and Balch, W.E. (1998) Getting into the Golgi. Trends Cell Biol., 8, 21–25.[CrossRef][ISI][Medline]

Batzer, M.A., Deininger, P.L., Hellmann-Blumberg, U., Jurka, J., Labuda, D., Rubin, C.M., Schmid, C.W., Zietkiewicz, E., and Zuckerkandl, E. (1996) Standardized nomenclature for Alu repeats. J. Mol. Evol., 42, 3–6.[ISI][Medline]

Blanken, W.M. and Van den Eijnden, D.H. (1985) Biosynthesis of terminal Gal alpha 1,3Gal beta 1,4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1,3-galactosyltransferase from calf thymus. J. Biol. Chem., 260, 12927–12934.[Abstract/Free Full Text]

Brasier, A.R., Tate, J.E., and Habener, J.F. (1989) Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques, 7, 1116–1122.[ISI][Medline]

Castronovo, V., Colin, C., Parent, B., Foidart, J.M., Lambotte, R., and Mahieu, P. (1989) Possible role of human natural anti-Gal antibodies in the natural antitumor defense system. J. Natl Cancer Inst., 81, 212–216.[Abstract]

Galili, U. (1999) Evolution of {alpha}1, 3galactosyltransferase and of the {alpha}-Gal epitope. In Galili, U. and Avila, J.L. (Eds.), Alpha-Gal and Anti-Gal: alpha-1, 3-galactosyltransferase, alpha-Gal epitopes, and the natural anti-Gal antibody. Kluwer Academic/Plenum, New York, NY, pp. 1–18.

Galili, U. and Swanson, K. (1991) Gene sequences suggest inactivation of alpha-1, 3-galactosyltransferase in catarrhines after the divergence of apes from monkeys. Proc. Natl Acad. Sci. USA, 88, 7401–7404.[Abstract]

Galili, U., Rachmilewitz, E.A., Peleg, A., and Flechner, I. (1984) A unique natural human IgG antibody with anti-alpha-galactosyl specificity. J. Exp. Med., 160, 1519–1531.[Abstract]

Galili, U., Macher, B.A., Buehler, J., and Shohet, S.B. (1985) Human natural anti-alpha-galactosyl IgG. II. The specific recognition of alpha (1, 3)-linked galactose residues. J. Exp. Med., 162, 573–582.[Abstract]

Giordanengo, V., Bannwarth, S., Laffont, C., Van Miegem, V., Harduin-Lepers, A., Delannoy, P., and Lefebvre, J.C. (1997) Cloning and expression of cDNA for a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase from the CEM T-cell line. Eur. J. Biochem., 247, 558–566.[Abstract]

Good, A.H., Cooper, D.K., Malcolm, A.J., Ippolito, R.M., Koren, E., Neethling, F.A., Ye, Y., Zuhdi, N., and Lamontagne, L.R. (1992) Identification of carbohydrate structures that bind human antiporcine antibodies: implications for discordant xenografting in humans. Transplant Proc., 24, 559–562.[ISI][Medline]

Henion, T.R., Macher, B.A., Anaraki, F., and Galili, U. (1994) Defining the minimal size of catalytically active primate alpha 1, 3 galactosyltransferase: structure-function studies on the recombinant truncated enzyme. Glycobiology, 4, 193–201.[Abstract]

Hourcade, D., Miesner, D.R., Atkinson, J.P., and Holers, V.M. (1988) Identification of an alternative polyadenylation site in the human C3b/C4b receptor (complement receptor type 1) transcriptional unit and prediction of a secreted form of complement receptor type 1. J. Exp. Med., 168, 1255–1270.[Abstract]

Joziasse, D.H. (1992) Mammalian glycosyltransferases: genomic organization and protein structure. Glycobiology, 2, 271–277.[Abstract]

Joziasse, D.H., Shaper, J.H., Van den Eijnden, D.H., Van Tunen, A.J., and Shaper, N.L. (1989) Bovine alpha 1, 3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA. J. Biol. Chem., 264, 14290–14297.[Abstract/Free Full Text]

Joziasse, D.H., Shaper, J.H., Jabs, E.W., and Shaper, N.L. (1991) Characterization of an alpha 1, 3-galactosyltransferase homologue on human chromosome 12 that is organized as a processed pseudogene. J. Biol. Chem., 266, 6991–6998.[Abstract/Free Full Text]

Joziasse, D.H., Shaper, N.L., Kim, D., Van den Eijnden, D.H., and Shaper, J.H. (1992) Murine alpha 1, 3-galactosyltransferase. A single gene locus specifies four isoforms of the enzyme by alternative splicing. J. Biol. Chem., 267, 5534–5541.[Abstract/Free Full Text]

Kagawa, Y., Takasaki, S., Utsumi, J., Hosoi, K., Shimizu, H., Kochibe, N., and Kobata, A. (1988) Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells. J. Biol. Chem., 263, 17508–17515.[Abstract/Free Full Text]

Klumperman, J. (2000) Transport between ER and Golgi. Curr. Opin. Cell Biol., 12, 445–449.[CrossRef][ISI][Medline]

Kobayashi, T. and Cooper, D. (1999) Anti-Gal, alpha-Gal epitopes, and xenotransplantation. In Galili, U. and Avila, J.L. (Eds.), Alpha-Gal and Anti-Gal: alpha-1, 3-galactosyltransferase, alpha-Gal epitopes, and the natural anti-Gal antibody. Kluwer Academic/Plenum, New York, NY, pp. 229–257.

Lai, C.J., Dhar, R., and Khoury, G. (1978) Mapping the spliced and unspliced late lytic SV40 RNAs. Cell, 14, 971–982.[ISI][Medline]

Larsen, R.D., Rajan, V.P., Ruff, M.M., Kukowska-Latallo, J., Cummings, R.D., and Lowe, J.B. (1989) Isolation of a cDNA encoding a murine UDPgalactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase: expression cloning by gene transfer. Proc. Natl Acad. Sci. USA, 86, 8227–8231.[Abstract]

Larsen, R.D., Rivera-Marrero, C.A., Ernst, L.K., Cummings, R.D., and Lowe, J.B. (1990) Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA. J. Biol. Chem., 265, 7055–7061.[Abstract/Free Full Text]

Lee, F., Mulligan, R., Berg, P., and Ringold, G. (1981) Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids. Nature, 294, 228–232.[ISI][Medline]

Lefebvre, J.C., Giordanengo, V., Doglio, A., Cagnon, L., Breittmayer, J.P., Peyron, J.F., and Lesimple, J. (1994a) Altered sialylation of CD45 in HIV-1-infected T lymphocytes. Virology, 199, 265–274.[CrossRef][ISI][Medline]

Lefebvre, J.C., Giordanengo, V., Limouse, M., Doglio, A., Cucchiarini, M., Monpoux, F., Mariani, R., and Peyron, J.F. (1994b) Altered glycosylation of leukosialin, CD43, in HIV-1-infected cells of the CEM line. J. Exp. Med., 180, 1609–1617.[Abstract]

Maraia, R.J. and Sarrowa, J. (1995) Alu-family SINE RNA: interacting proteins and pathways of expression. In Maraia, R.J. and Sarrowa, J. (Eds.), The impact of short interspersed elements (SINEs) on the host genome. R.G. Landes, Austin, TX, pp. 163–196.

Murphy, L.A. and Goldstein, I.J. (1977) Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea simplicifolia seeds. J. Biol. Chem., 252, 4739–4742.[Abstract]

Nevins, J.R. and Darnell, J.E. Jr. (1978) Steps in the processing of Ad2 mRNA: poly(A)+ nuclear sequences are conserved and poly(A) addition precedes splicing. Cell, 15, 1477–1493.[ISI][Medline]

Nishikura, K., Kurjan, J., Hall, B.D., and De Robertis, E.M. (1982) Genetic analysis of the processing of a spliced tRNA. EMBO J., 1, 263–268.[ISI][Medline]

Rowold, D.J. and Herrera, R.J. (2000) Alu elements and the human genome. Genetica, 108, 57–72.[CrossRef][ISI][Medline]

Smith, D.F., Larsen, R.D., Mattox, S., Lowe, J.B., and Cummings, R.D. (1990) Transfer and expression of a murine UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase gene in transfected Chinese hamster ovary cells. Competition reactions between the alpha 1,3-galactosyltransferase and the endogenous alpha 2,3-sialyltransferase. J. Biol. Chem., 265, 6225–6234.[Abstract/Free Full Text]