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Glycobiology Pages 851-863  

Molecular cloning, expression and exon/intron organization of the bovine [beta]-galactoside [alpha]2,6-sialyltransferase gene
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References

Molecular cloning, expression and exon/intron organization of the bovine [beta]-galactoside [alpha]2,6-sialyltransferase gene

Molecular cloning, expression and exon/intron organization of the bovine [beta]-galactoside [alpha]2,6-sialyltransferase gene

Dominique Mercier, Anne Wierinckx, Ahmad Oulmouden, Paul F. Gallet, Monica M. Palcic1, Anne Harduin-Lepers2, Philippe Delannoy2, Jean-Michel Petit, Hubert Levéziel3 and Raymond Julien4

Institut de Biotechnologie, Faculté des Sciences, Université de Limoges, 87060 Limoges, France, 1Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada, 2Laboratoire de Chimie Biologique, UMR no. 8576 du CNRS, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France and 3Laboratoire de Génétique Biochimique et Cytogénétique, INRA-CRJ, 78350 Jouy-en-Josas, France

Received on August 27, 1998; revised on December 18, 1998; accepted on January 22, 1999

In this study, we report the first isolation and characterization of a bovine sialyltransferase gene. Bovine cDNAs prepared from different tissues contain an open-reading frame encoding a 405 amino acid sequence showing 83%, 75%, and 60% identity with human, murine, and chicken ST6Gal I ([beta]-galactoside [alpha]2,6-sialyltransferase) sequences, respectively. Whentransfected into COS-7 cells, a recombinant enzyme wasobtained which catalyzed the in vitro [alpha]2,6-sialylation of LacNAc (NeuAc[alpha]2-6Gal[beta]1-4GlcNAc) and LacdiNAc (NeuAc[alpha]2-6GalNAc[beta]1-4GlcNAc) acceptor substrates. The Km values were 2.8 and 6.9 mM, respectively. Different relative efficiencies (Vmax/Km) for the two precursors (36 for LacNAc and 4.3 for LacdiNAc) were observed. Bovine ST6Gal I gene consists of four 5[prime]-untranslated exons E(-2) to E(1), and five coding exons from E(2) to E(6). This later carries a 3[prime]-untranslated region of 2.7 kb. Gene sequence spans at least 80 kb of genomic DNA. Two processed pseudogenes have been identified. They are 94.3 and 95.6% similar to the bovine cDNA, respectively. Three families of mRNA isoforms were isolated. They differed by their 5[prime]-untranslated regions and could be generated by three tissue-specific promoters. Family 1 is made up of exons E(-2) and E(1) to E(6), family 2 of exons E(-1) to E(6), and family 3 of exons E(1) to E(6). Tissular distribution of transcript families appears noticeably different than those described in human and rat.

Key words: bovine sialyltransferase/ST6Gal I gene/LacNAc/LacdiNAc

Introduction

Sialyltransferases are a family of 12 glycosyltransferases that catalyze the transfer of sialic acid from CMP-NeuAc to glycoproteins and glycolipids (Harduin-Lepers et al., 1995). The [beta]-galactoside [alpha]2,6-sialyltransferase, recently renamed ST6Gal I (Tsuji et al., 1996), terminates sugar chains of glycoproteins with [alpha]2-6Gal linkages, and has been extensively studied. Twenty years ago, the enzyme from bovine colostrum was the first [alpha]2,6-sialyltransferase purified and enzymatically characterized (Paulson et al., 1977a,b). More recently, Nemansky and van den Eijnden (1992) have shown that this enzyme catalyzes the in vitro sialylation of both LacNAc (NeuAc[alpha]2-6Gal[beta]1-4GlcNAc) and LacdiNAc (NeuAc[alpha]2-6GalNAc[beta]1-4GlcNAc) disaccharide structures. Since no bovine sialyltransferase gene has ever been isolated or characterized, it was of interest to determine whether one or more bovine genes are involved in synthesizing the [alpha]2,6-sialyl linkage in these different trisaccharides. This clarification also relates to the recent discovery that the terminus NeuAc[alpha]2-6GalNAc[beta]1-4GlcNAc plays a role in the expression of the biological activity of prolactin/growth hormone family members during rat pregnancy (Manzella et al., 1997). The presence of NeuAc[alpha]2-6GalNAc[beta]1-4GlcNAc on only a limited number of these peptidic hormones reflects the concurrent expression of these glycoproteins with [beta]1,4-N-acetylgalactosaminyl-transferase and [alpha]2,6-sialyltransferase capable of adding sialic acid to terminal [beta]1,4 linked GalNAc. Also, the presence of [alpha]2,6-sialylated LacdiNAc on only a limited number of bovine milk glycoproteins such as lactoferrin (Coddeville et al., 1992), fat globule membrane CD36 (Nakata et al., 1992), and various milk oligosaccharides (Gyorgy et al., 1974) reflects a similar upregulated mechanism possibly related to the ability of milk glycoconjugates to protect suckling calves from pathogens (Mouricout et al., 1990).

ST6Gal I genes have been extensively studied in human (Grundmann et al., 1990; Wang et al., 1993), rat (Weinstein et al., 1987), murine (Hamamoto et al., 1993) and chicken (Kurosawa et al., 1994), notably owing to the possible roles of the [alpha]2,6-sialyl to Gal linkage (1) in tumorigenicity in cancer (Dall'Olio, 1996), (2) as ligands for CD22 in B lymphocyte activation and function (Braesch-Andersen et al., 1994), (3) as participant in hepatic inflammatory response (Kaplan et al., 1983). In these species, the level of expression of ST6Gal I varies dramatically in the different tissues and organs (Paulson et al., 1989; Kitagawa and Paulson, 1994). Regulation of the expression of ST6Gal I gene is achieved mainly at the transcriptional level through the use of different cell-specific promoters which generate transcripts differing in their 5[prime]-untranslated regions (O'Hanlon et al., 1989; Svensson et al., 1990; Wang et al., 1990; Wen et al., 1992).

The rat ST6Gal I gene is surprisingly complex spanning over 80 kb and has at least four promoters that regulate its expression. In most rat tissues, transcription initiates from at least one upstream promoter not yet defined that drives the low level constitutive expression of a family of 4.7 kb transcripts (Wen et al., 1992). Two non coding exons E(-1) and E(0) have been described for these 4.7 kb transcripts. A downstream promoter region containing binding sites for several liver restricted transcription factors allows the very high expression of the gene in the liver (Svensson et al., 1990) giving rise to a 4.3 kb transcript lacking two exons in the 5[prime]-untranslated region. Finally, two alternative promoters would generate three short kidney specific transcripts of 3.6 kb, two of them coding for potentially truncated proteins (Wen et al., 1992).

A large heterogeneity among human ST6Gal I mRNAs in various tissues or cultured cells is also described. It results from the alternative use of several exons named E(Y) and E(Z) which are the homologues of the rat exons E(-1) and E(0) in the 4.7 kb rat transcripts (Aasheim et al., 1993; Wang et al., 1993). These observations lead to the conclusion that mRNA isoforms may arise by the usage of several alternative promoters.

As an initial step towards understanding the regulatory mechanisms for the expression of NeuAc[alpha]2-6 sialylated Gal[beta]1-4GlcNAc and GalNAc[beta]1-4GlcNAc oligosaccharides in bovine tissues, we have isolated cDNAs which encode a bovine ST6Gal I capable of catalyzing the in vitro [alpha]2,6-sialylation of both LacNAc and LacdiNAc disaccharide structures. Using a PCR approach, three families of transcripts have been identified which differed in their 5[prime]-untranslated exons. Gene organization has been deciphered and two distinct retrotransposed pseudogenes highly homologous to the coding sequence of the functional ST6Gal I gene have been also found.

Nucleotide sequences reported in this paper have been submitted to the GenBank/EBI Data Bank with accession number Y15111 for the coding sequence, AJ006830 for E(-2), AJ006831 for E(-1), AJ006832 for E(0), and AJ006833 for E(1) extension.

Results

Isolation and analysis of coding sequence

A PCR based approach was used assuming that the bovine gene sequence is similar to the human gene. A first PCR experiment was performed using bovine brain cDNA as a template with human p10 and p20 primers (Figure 1). A unique PCR amplification product of 740 bp was obtained, which has been subcloned and sequenced (see Materials and methods). Several bovine primers were then designed (Figure 1) and the complete cDNA sequence was determined using rapid amplification of 5[prime] and 3[prime] cDNA ends (RACE) of brain, heart, kidney, liver, lung, spleen, and testis.


Figure 1. Primers used in this study. (A) Schematic representation of primer positions on exonic sequences. (B) Primer sequences, origin and nucleotide position.

5[prime] RACE products resulting from a first round of amplification with AP1 and p14 primers, were used as templates for a second round of PCR with AP2 and p12 primers (Materials and methods and Figure 1). A similar nested-PCR approach, first using AP1 and p15 primers and then AP2 and p19, primers was carried out for the 3[prime] coding region (Materials and methods and Figure 1). All PCR products were analyzed on agarose gels, and then subcloned and sequenced. Finally, in order to clone the complete sequence the bovine primer pair p7 and p23 was designed to obtain a PCR product which was cloned and sequenced (Figure 2).


Figure 2. Sequence of bovine ST6Gal I gene. Nucleotide sequence found in all bovine ST6Gal I cDNAs and containing the 1218 nt coding region. The adenine residue of the putative initiation codon is assigned as residue 1. Dashed lines below the bST6Gal I sequence denote DNA identity of the putative P1 and P2 pseudogenes. Gaps in the nucleotide sequence alignments are indicated by dots and are not taken into account for nucleotide numbering. Untranslated sequences are written with lowercase letters. Exon limits are indicated by thick lines above the five 4 nt sequences dividing the six exons present in cDNAs. ATG, TGA, TAG, TAA present in 5[prime]-untranslated E(1) exon were pointed out by a thin line topped by a solid or open circle to indicate the putative initiation and in frame stop codons, respectively. Partial sequences of putative ST6Gal I P1 (P1) and P2 (P2) pseudogenes were aligned with homologous cDNA sequence. Primers p7 and p23 are defined by horizontal arrows. Limits of probes used for Southern blot analysis are indicated by vertical solid (probe I) or open (probe II) arrows.

The bovine ST6Gal I nucleotide sequence (Figure 2) shown a coding region of 1218 nucleotides beginning with ATG codon within a sequence context consistent with Kozak's consensus rules for mammalian translation initiation (Kozak, 1987). Three additional ATGs were found in the 5[prime]-untranslated region at position -181, -156, and -118 with in frame stop codons at -132, -97, -64, and -60. The coding sequence shown 85%, 79%, 78%, and 64% homologies to human, rat, mouse, and chicken ST6Gal I, respectively. As expected, a lower homology of 67%, 55%, 53%, and 42% was found between the bovine ST6Gal I 3[prime]-untranslated region and the corresponding 3[prime]-untranslated regions of human, rat, mouse, and chicken, respectively.

A single open reading frame encoding a protein of 405 residues with a theoretical molecular weight of 46250 Da (Figure 3) was observed in cDNA clones. Identities (similarities) of 83.1% (86.8%), 75% (79%), and 60.4% (67.4%) were found compared to human, murine, and chicken ST6Gal I polypeptide sequences, respectively. Analysis of the hydropathy profile revealed one potential transmembrane domain located 10 residues from NH2 terminus. This transmembrane domain consists of 18 hydrophobic amino acids flanked on either end by three or four lysines. Interestingly, we found a 44 amino acid hypervariable domain located in the stem region at the amino acid positions 46-89 which presented high species specificity, the bovine enzyme showing a deletion of four amino acids in this region. The three conserved sequence called sialylmotifs L, S, and VS were identified like in all other sialyltransferases (Drickamer, 1993; Livingston and Paulson, 1993; Geremia et al., 1997). Nine cysteine residues (including the three residues of the transmembrane domain) were common to bovine and human ST6Gal I enzyme, but only six residues located into the catalytic domain were conserved with other species. At the COOH end, the bovine enzyme had two additional amino acid residues (Gly and Ala) while all other ST6Gal I enzymes ended with a cysteine residue. Two potential consensus sites for asparagine-linked glycosylation (bovine amino acids 146 and 158) are common to the bovine enzyme and other mammalian and chicken enzymes (Figure 3). These structural features suggest that the bovine ST6Gal I protein has a type II membrane topology characteristic of all other glycosyltransferases cloned to date (Paulson and Colley, 1989).


Figure 3. Comparison of protein sequences of bovine ST6 Gal I with human, mouse, rat, and chicken homologous enzymes. Methionine 1 corresponded to the adenine containing ATG (Figure 2). Amino acid sequence identities are indicated by dashed lines and gaps by dots. The double underlined amino acids corresponded to the putative transmembrane domain. Solid circles represented common cysteine residues. Solid triangles represented potential asparagine-linked glycosylation sites. Sialylmotifs L (178-225), S (318-340) and VS (367-372) were boxed. (§) indicated an amino acid position totally conserved in the alignment and (*) a position that is only well conserved among all sialyltransferases already described. b, Bovine (this study); h, human (X17247); m, mouse (D16106);r, rat (M18769); c, chicken (X75558).

Identification of three transcript families

5[prime]-Untranslated exons were mapped by 5[prime] RACE analysis (see Materials and methods) using cDNAs from various tissues as targets and p14 and p12 (Figure 1) as specific primers. Three 5[prime]-untranslated exon families were identified.

3[prime]-Untranslated region mapped by a similar nested PCR procedure with the p22/AP2 second primer pair (Figure 1) revealed a single RACE fragment of 2.4 kb in size corresponding to a 2.7 kb 3[prime]-untranslated region whatever cDNAs examined.

The examination of 3[prime] and 5[prime]-untranslated regions and coding sequence, allowed us to determine that ST6Gal I gene is expressed in three mRNA transcript families having 4.1-4.5 kb in size. Relative level of expression, calculated using [beta]-actin hybridization signal as reference, indicated that kidney contains the highest level of ST6Gal I mRNA (Figure 4). Comparatively to kidney, levels of transcripts in spleen, heart, brain, or lung were 0.41, 0.15, and 0.18, respectively.


Figure 4. Expression level of ST6Gal I gene. Blot containing mRNA from five bovine tissues was probed with a bovine ST6Gal I cDNA spanning the sialylmotif L (228 bp), and after stripping and assessing for complete removal of signal, with bovine [beta]-actin probe (308 bp). Experimental conditions were described in Materials and methods. A relative quantification of ST6Gal I mRNA isoforms was carried out owing to [beta]-actin signal normalization based on a densitometric determination (ImageQuant software from Molecular Dynamics, Inc.). B, Brain; H, heart; K, kidney;L, lung; S, spleen.

The first transcript family, characterized by an association of 5[prime]-untranslated E(-2) and E(1) exons (Figure 5), was present in brain and kidney. The second cDNA family, including transcripts with 5[prime]-untranslated exons E(-1), E(0), E(1) (Figure 5), was present in lung and spleen. The third cDNA family present in heart, kidney, liver, spleen, and testis possesses a 19 bp sequence upstream the untranslated E(1) exon (Figure 5).


Figure 5. Bovine exons from 5[prime]-untranslated regions of cDNA families. Comparison with human and rat corresponding sequences. 5[prime]-untranslated exons were obtained by sequencing for each tissues several clones of RACE products. (A) cDNA family 1 (brain, kidney); (B) cDNA family 2 (lung and spleen); (C) cDNA family 3 (heart, kidney, liver, spleen, and testis). Dashed lines below bovine sequences denote DNA identity. Gaps are indicated by dots. Exon limits are indicated by (/) at the level of conserved splice sites. Italic letters indicated the beginning of E(1) exon for each cDNA family. Nucleotides are numbered from adenine 1 containing ATG (Figure 2). Accession numbers: for bovine 5[prime]-untranslated E(-2), E(-1), E(0) exons AJ006830, AJ006831, AJ006832 respectively, and for 19 bp extension of E(1) exon AJ006833; for human 5[prime]-untranslated E(Y) and E(Z) exons X17147 and for E(X) X62822; for rat 5[prime]-untranslated E(-1) and E(0) exons M83142.

Expression of ST6Gal I gene in COS-7 cells and kinetic parameters of the enzyme

In order to check the ability of bovine cDNA sequence (Figure 2) to promote enzyme activity, the entire open reading frame of the bovine ST6Gal I cDNA was inserted into the mammalian expression vector pcDNAI/Amp and transiently transfected into COS-7 cells. The [alpha]2,6-sialyltransferase was detected by immunoblot analysis (Figure 6) using an affinity purified anti ST6Gal I antibody. The strong immunoreactive band corresponding to the ST6Gal I enzyme found in transfected COS-7 cells migrated at the same molecular mass than bovine liver and kidney Golgi enzymes (about 55 kDa); 110 and 220 kDa immunoreactive protein bands may represent a disulfide-bonded dimer or tetramer of the [alpha]2,6-sialyltransferase (Ma and Colley, 1996). A discrete immunoreactive band was observed at a molecular mass lower than 55 kDa in untransfected COS-7 cells. Prior to determine kinetic parameters of bovine ST6Gal I, we assessed endogenous COS-7 sialyltransferase activities using asialofetuin as acceptor substrate (terminal galactose concentration: 80 µM). Compared to enzyme activity of transfected cells, the relative rate of [14C] sialic acid incorporated was about 1.5%.


Figure 6. Immunodetection of bovine ST6Gal I protein. Untransfected (lane 1) and transfected COS-7 cells with bovine ST6Gal I gene (lane 2) were lyzed and centrifuged. Bovine liver (lane 3) and kidney (lane 4) Golgi membranes were isolated as described under Materials and methods. In all cases, 60 µg of protein were used for Western blotting. Prior to SDS-polyacrylamide gel electrophoresis, the samples were prepared with 5% (v/v) [beta]-mercaptoethanol and with heating for 3 min at 100°C. Following electrophoresis, ST6Gal I proteins were detected by immunoblotting as described under Materials and methods. m, Monomer; d, dimer; t, tetramer.

The specificity of [alpha]2,6 neuraminic acid transfer to Gal has been examined using 4-deoxy-[beta]-d-Gal[beta]1-4GlcNAc-R and 4-O-methyl-[beta]-d-Gal[beta]1-4GlcNAc-R (Table I). Modifications at C4 of galactose, especially the sterically conservative substitution of the 4-hydroxyl by hydrogen were well accepted by [alpha]2,6-sialyltransferases and not by [alpha]2,3-sialyltransferases (Van Dorst et al., 1996). With the recombinant bovine enzyme, a significant incorporation of Neu5Ac was observed using this acceptor (38.6% compared to [beta]-d-Gal[beta]1-4GlcNAc-R in the same experimental conditions). On the other hand, substitution of the 4-hydroxyl by an O-methyl was well accepted by [alpha]2,3-sialyltransferases and not by [alpha]2,6-sialyltransferases (Van Dorst et al., 1996). With the 4-O-methyl-[beta]-d-Gal[beta]1-4GlcNAc-R, an incorporation of only 5% was observed compared to [beta]-d-Gal[beta]1-4GlcNAc-R.

Kinetic parameters of the recombinant bovine [alpha]2,6-sialyltransferase were determined using asialofetuin and synthetic LacNAc and LacdiNAc as substrates. Gal[beta]1-4GlcNAc-R termini appear to be 10-fold more efficient as acceptor than its LacdiNAc analogue, GalNAc[beta]1-4GlcNAc-R (Table II). By analyzing the rate of formation of [alpha]2,6-sialylated fetuin at various concentrations of CMP-NeuAc, the Km and Vmax for this donor substrate were determined (Table II). By comparison with all the substrates tested, the sugar nucleotide exhibited the higher enzyme affinity.

Table I. Transfer specificity of neuraminic acid to galactose by recombinant bovine sialyltransferase expressed in transfected COS-7 cells
Acceptors Untransfected Mock transfected Transfected Preferential linkage detection
LacNAc-R 360 300 18,900 [alpha](2,3) and [alpha](2,6)
4-Deoxy-LacNAc-R ud ud 7300 [alpha](2,6)
4-O-Methyl-LacNAc-R ud ud 960 [alpha](2,3)
ud, Undetected. Values expressed in cpm are obtained as described in Materials and methods. Acceptor concentration was 0.2 mM.

Table II. Kinetic parameters of bovine [alpha]2,6-sialyltransferase calculated for several substrates
Substrate Km (µM) 104 Vmax (nmol/min per mg protein) Relative efficiency (Vmax/Km)
CMP-NeuAca 8.1 2.8  
Asialofetuinb 81 5.8 720
Gal[beta]1-4GlcNAc-R 2.8 103 1.0 36
GalNAc[beta]1-4GlcNAc-R 6.9 103 0.3 4.3
R= O(CH2)8COOCH3.
aKinetic parameters have been determined with asialofetuin (concentration of terminal galactose: 80 µM) as acceptor substrate.
bFor determination of the Km value for this desialylated bovine protein, we take into account the eight terminal galactoses per asialofetuin molecule. Km and Vmax were calculated from Lineweaver-Burk plots.

Bovine ST6Gal I gene organization

Putative intron/exon boundary domains emphasized the high conservation of splice sites between bovine, human and rat genes. They define six exons E(1) to E(6) ranging in size from 96 bp (E(3) exon) to 2.9 kb (E(6) exon). A set of PCR experiments and Southern blot analysis was carried out in order to determine the complete bovine ST6Gal I gene organization. In a first round of PCR, a 4.1 kb amplification product was obtained using the p15/p21 primer pair (lane 1, Figure 7). Assuming a bovine exon/intron organization comparable to that of human and rat genes, this fragment should correspond to the bovine E(4)/i4/E(5)/i5/E(6) sequence. According to the data obtained from coding and non coding exons analysis, bovine intron size were determined owing to a set of primers (Figure 1) designed with the aim to establish gene structure by long PCR experiments. The intron lengths range from 1.2 kb to more than 20 kb (Figure 8). In order to clarify the localization of E(-2) exon, a set of PCR experiments has been carried out by means of different primer pairs, each composed of a specific primer of E(-2) exon (p1 or p2) combined with a specific primer of each untranslated E(-1) (p3 or p4), E(0) (p5 or p6), and E(1) (p7 or p8) exons. Whatever the primer pair or PCR conditions retained, negative results led us to the conclusion that E(-2) exon was the most upstream untranslated exon of bovine gene. Gene organization summarized in Figure 9, consist of five coding and four 5[prime] upstream untranslated exons spanned up to 80 kb of genomic DNA.


Figure 7. Comparative PCR analysis of genomic and cDNA sequences corresponding to catalytic domain of ST6Gal I. Genomic DNA (lane 1) and cDNA from bovine brain (lane 2) were used as target. The primers were p15 and p21 (Figure 1), and PCR procedure was made as described under Materials and methods. Diagram summarizes gene organization between E(4) and E(6) exons.


Figure 8. Long PCR analysis of intron sizes. Genomic DNA was used as target for long PCR experiments performed using p3/p6 primers (lane 1), p5/p8 (lane 2), p7/p12 (lane 3), p11/p14 (lane 4), p15/p18 (lane 5), and p17/p21 (lane 6). The largest i-2 and i3 introns have not been amplified. Intron sizes were determined using two DNA ladders as reference.


Figure 9. Genomic organization of the bovine ST6Gal I gene. (A) Schematic gene structure. Untranslated exons (striped areas) are labeled E(-2) to E(1) and coding exons (black boxes) E(2) to E(6). Intron (i) sizes in kilobases are indicated above the gene structure. Promoter regions are named Pr1, Pr2, and Pr3. Protein domains are schematized under the coding sequence (CytD, cytoplasmic domain; TMD, transmembrane domain; CD, catalytic domain). (B) Putative differential splicing of primary transcripts result in three families of mature mRNAs.

During the time course of this PCR amplification of bovine genomic DNA using the p15/p21 primer pair, a shorter PCR product of ~450 bp was observed (lane 1, Figure 7). Subcloning and sequencing of this 450 bp PCR product revealed two distinct sequences of 461 and 449 bp highly homologous (94.3% and 95.6%, respectively) to the 464 bp sequence of the bovine ST6Gal I cDNA (Figure 2 and lane 2 of Figure 7). These two genomic sequences correspond probably to distinct pseudogenes. Confirmation of a genomic organization including one ST6Gal I gene and two pseudogenes was obtained by a Southern blot analysis performed under high stringency conditions (see Materials and methods) with probes I and II (Figure 2). Whatever the restriction enzyme used (Figure 10), probe I revealed three distinct bands (Figure 10A) while probe II hybridized mainly DNA containing the full length E(2) exon (Figure 10B). The two discrete 3.2 kb and 3.5 kb bands (Figure 10B, lanes 1 and 2) corresponded to the pseudogene having a sequence domain homologous to E(2) exon.


Figure 10. Southern blot analysis of genomic DNA. DNA was digested by PstI (lane 1) and HindIII (lane 2) and hybridized (A) with 228 bp probe I(SmL region) and (B) with a 432 bp probe II (upstream domain of E(2) exon) under high stringency conditions (Materials and methods). P1 and P2 referred to pseudogenes ST6Gal I P1 and P2.

Discussion

Until today, only five bovine glycosyltransferase genes have been cloned and characterized (Shaper et al., 1986; Joziasse et al., 1989; Homa et al., 1993; Oulmouden et al., 1997) compared to approximately 30 human and murine genes. We report here the first bovine sialyltransferase gene to be isolated and extensively characterized. It encodes a transmembrane type 2 glycoprotein which presents the ability to form the [alpha]2,6-sialyl to Gal or to GalNAc linkages. Nevertheless, the relative substrate efficiency (Vmax/Km) of the sialylation was clearly different for both acceptor substrates, 36 for LacNAc and 4.3 for LacdiNAc. Interestingly, in a previous study (Nemansky and van den Eijnden, 1992) ST6Gal I purified from bovine colostrum presents a relative efficiency very close to each other (1.16 for LacNAc and 0.45 for LacdiNAc). In the first study of the bovine enzyme, Paulson et al., (1977a) have observed two soluble forms in colostrum. Equally, for the rat liver enzyme (Weinstein et al., 1987; O'Hanlon and Lau, 1992) soluble forms are generated by proteolytic cleavage between the NH2 terminal signal anchor and the catalytic domain. The membrane-bound and soluble forms in rat liver are encoded by two different RNAs that differ by a single nucleotide (Ma et al., 1997). One form which presents the highest activity is rapidly cleaved and secreted. Hence, the bovine enzyme might also exist as soluble (Nemansky and van den Eijnden, 1992) or membrane-bound (this study) forms that could differ in its ability to perform the [alpha]2,6-sialylation of LacNAc and LacdiNAc. The soluble forms could have lost their specificity for LacNAc or might have increased their ability to transfer sialic acid on LacdiNAc. Such a possibility would be of interest to understand the sialylation of therapeutical transgenic glycoproteins secreted into cow milk (Cumming, 1991; Pietro et al., 1995).

The bovine ST6Gal I is a Golgi apparatus protein of 405 amino acids which possesses the main structural characteristics of previously described glycosyltransferases (Weinstein et al., 1987; Gross et al., 1989; Sticher et al., 1991; Kurosawa et al., 1994), including a significantly divergent region in the stem domain (amino acids 46-89, Figure 3). It exhibits a higher sequence homology with human enzyme than with rat, mouse, or chicken. The three sialylmotifs (L, S, and VS) are present in the catalytic domain (Drickamer, 1993; Livingston and Paulson, 1993; Geremia et al., 1997). For the rat liver enzyme, only two of the three present potential N-glycosylation sites are effectively modified and seem to play an important role for activity (Fast et al., 1993). The two sites carried by the bovine enzyme are homo-logous to those that are effectively glycosylated in rat. All the six Cys residues in the catalytic domain are conserved; the sialylmotif L, which plays an important role in the donor substrate binding (Datta and Paulson, 1995), contains Cys181. As was demonstrated for rat liver ST6Gal I, one or more of these residues should be implicated in disulfide-bonded dimer formation recently described for bovine liver enzyme (Ma and Colley, 1996). Dimerization leads to a loss of catalytic activity arising from a decrease of enzyme affinity for CMP-NeuAc, and to the emergence of a galactose-specific lectin property. Thus, the control of the enzyme conversion to one or other of these forms could be a mechanism for a downregulation of [alpha]2,6-sialylation (Ma and Colley, 1996).

Bovine ST6Gal I gene organization seems to follow the same rules of the four other ST6Gal I genes already cloned (Svensson et al., 1990; Wang et al., 1990, 1993; Wen et al., 1992). The coding sequence is made up of five exons from E(2) to E(6), this latter contains also a 3[prime]-untranslated region of 2.7 kb. The 5[prime]-untranslated sequences are divided in four exons. All together, these nine exons span up to 80 kb of genomic DNA (Figure 9).

In vivo, the bovine ST6Gal I results in the translation of three main families of transcripts (Figure 9), differing only by their 5[prime]-untranslated sequences (they possess in common five coding exons and a large 3[prime]-untranslated region).

Transcript family 1 which is 4.2 kb in size was mainly encountered in brain and kidney and specified E(-2) and E(1) untranslated exons. It was probably synthesized from the promoter region 1 (Pr1) since E(-2) exon was located as the most 5[prime] upstream untranslated exon. This family was homologous to the human form 2 transcript (Figure 5) which contains 5[prime]-untranslated E(1) and E(X) exons (Lo and Lau, 1996b). Form 2 mRNA is expressed in B-lymphoblastoid cells, spleen and kidney, but has not yet described in murine species.

Transcript family 2 which is 4.5 kb in size, contains three untranslated exons (E(-1), E(0), and E(1) exons). This mRNA family is specific to lung and spleen and probably transcribed from Pr2 promoter (Figure 9). The human (form 3, containing E(Y), E(Z), and E(1) 5[prime]-untranslated exons) and rat (E(-1), E(0) and E(1) exons) counterparts, unlike in bovine species, are constitutively expressed in most tissues (Wen et al., 1992; Wang et al., 1993).

The family 3 of transcripts is the main form encountered in bovine tissues (heart, kidney, liver, spleen, and testis). This 4.1 kb mRNA form presents a short additional sequence 19 bp upstream E(1) exon. In human and rat, the homologous form has a similar structure but this form is only expressed in liver (Wen et al., 1992; Wang et al., 1993; Aas-Eng et al., 1995) and intestinal epithelial cells (Vertino-Bell et al., 1994). Mouse liver mRNA form is divergent from that observed in other species, due to the presence of an upstream 5[prime] exon (exon H), but this form is still liver-specific (Hu et al., 1997).

In lung, a minor 2.3 kb in size mRNA form was also detected by 3[prime] RACE experiment (data not shown). The possibility that shortened transcripts bearing in their 3[prime] ends a weak secondary polyadenylation site cannot be excluded, as this has been observed in chicken (Kurosawa et al., 1994).

Recently, for human B-lymphoblastoid cell line, a novel heterogeneity has been described in the 5[prime]-untranslated region. Three new families were depicted which could be generated from other promoter regions (Lo and Lau, 1996a).

All in all, bovine gene organization and expression seem to be both different and similar to human (Wang et al., 1993) and murine (Svensson et al., 1990; Wang et al., 1990; Wen et al., 1992; Stanley, 1998). They were similar due to the presence of three main transcript families. Whereas human and rat transcript families are present in homologous tissues, bovine ST6Gal I transcripts are distributed according to a specific pattern (Table III).

Some features of ST6Gal I P1 and P2 pseudogenes (the lack of intervening sequences and the extent of homology) strongly suggest that these sequences derive from mature mRNAs (Vanin, 1985). This could signify an integration mechanism initiated from RNA transcripts devoid of their 5[prime] ends. These truncated RNAs would correspond to a loss of NH2 terminus of enzyme, a characteristic which points out the possible ancestral presence of soluble truncated enzymes in bovine species.

Materials and methods

Nomenclature

The gene described represents the first bovine sialyltransferase gene. It is designated bST6Gal I with reference to the Neu5Ac[alpha]2-6 Gal linkage formed with Gal[beta]1-4GlcNAc precursor termini. In this paper, we show that bST6Gal I encodes an enzyme which also exhibits the property to synthesize a Neu5Ac[alpha]2-6 GalNAc linkage with GalNAc[beta]1-4GlcNAc disaccharide precursor termini.

Materials

The oligonucleotides used for various experiments in this manuscript are listed in Figure 1. They were obtained from Eurogentec, France. Before sequencing, all PCR products were cloned into the pMOSBlue T-Vector (Amersham). Transfections of COS-7 cells were carried out using pcDNAI/Amp expression vector (Invitrogen). Bovine brain, heart, kidney, liver, lung, and spleen mRNAs were purchased from CLONTECH. Testis mRNAs were prepared using TRIZOL Reagent (Life Technologies) and Oligo(dT) Cellulose Column (Life Technologies) according to the manufacturer protocols. Autoradiograms were scanned, sized, grouped, and labeled using Adobe Photoshop 4.0 (Mountain View, CA).

Table III. Tissue distributions of ST6Gal I transcript families found in human, rat, and bovine
  Brain Heart Kidney Liver Lung Spleen Testis
Family 1 B   B, H     H  
Family 2 H, R H, R H, R   B, H, R B, H, R H, R
Family 3   B B B, H, R   B B
B, Bovine; H, human; R, rat. Tissue distribution of transcript families found in rat and human are described by Wen et al., (1992) and Lo and Lau, (1996b), respectively. Each bovine transcript family is characterized by its specific 5[prime]-untranslated region (see also Figure 9).

Rapid amplification of 5[prime] and 3[prime] cDNA ends (RACE)

The Marathon cDNA Amplification Kit (CLONTECH) was used to obtain a library of adaptor-ligated double-stranded cDNA from bovine brain, heart, kidney, liver, lung, spleen, and testis tissues; 1 µg of poly(A+) RNA was used as a template for the first strand synthesis, with the 52-mer CDS primer (5[prime]-TTCTAGAATTCAGCGGCCGC (T)30 (G/A/C) (G/A/T/C)-3[prime]) and 100 units of the MMLV reverse transcriptase in a total volume of 10 µl. Synthesis was carried out at 42°C for 1 h. Next, the second strand was synthesized at 16°C for 90 min in a total volume of 80 µl containing the enzyme mixture (RNase H, Escherichia coli DNA polymerase I, and E.coli DNA ligase), the second strand buffer, the dNTP mix, and the first strand reaction. cDNA ends were then made blunt by adding 10 U of T4 DNA polymerase and incubating at 16°C for 45 min. The double-stranded cDNA was phenol/chloroform extracted, ethanol precipitated, and resuspended in 10 µl of water. Half of this volume was used to ligate the adaptor to the cDNA ends (adaptor sequence: 5[prime]-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3[prime]) in a total volume of 10 µl using 1 U of T4 DNA ligase. The ligation reaction was incubated 16 h at 16°C. The resulting cDNA library was diluted to a final concentration of 0.2 µg/ml.

The 5[prime] end of bST6Gal I was PCR-amplified using 2 µl of the library as a template with the sense oligonucleotide AP1, and the antisense oligonucleotide p14 (Figure 1). The 25 µl PCR reaction mixture contained 2.5 nmol of each dNTP, 10 pmol of each primer, 25 nmol of MgCl2, and 1.25 U of Taq DNA polymerase (Promega). After one pre-PCR cycle (2 min at 94°C, 15 s at 68°C, 5 min at 72°C) 35 cycles were done at the following conditions: 10 s at 94°C, 15 s at 68°C, and 1 min at 72°C, incremented with 1 s at each cycle, followed by a final extension at 72°C for 7 min. The resulting PCR product (0.01 µl) was reamplified under same conditions using the nested oligonucleotides AP2 and p12 (nested to AP1 and p14, respectively; Figure 1). Final PCR products were analyzed on a 1.2% agarose gel, and RACE fragments were gel-extracted (PCR pure-bind kit, CLONTECH), subcloned, and sequenced.

For the determination of the 3[prime] end of bST6Gal I, the same procedure was carried out except that the 3[prime] end specific sense primers were p15 and p19 (Figure 1) for the first and second PCR amplification, respectively.

PCR amplification of gene and pseudogene fragments

PCR amplification of a 740 bp bovine ST6Gal I fragment was carried out using p10/p20 primer pair designed in the corresponding human ST6Gal I sequence (Figure 1). Amplification conditions were as above except that MgCl2 was 2 mM, annealing temperature was 60°C, and elongation time was 45 s with bovine brain cDNA as template. After cloning and sequencing, two bovine specific primers were designed (p14 and p15, Figure 1) and used in combination with p11 and p21 (bovine primers homologous to human primers p10 and p20) to probe bovine genomic DNA.

Gene and pseudogene fragments were amplified using the Expand Long Template PCR System (Boehringer Mannheim). The 50 µl reaction contained 15 pmol of each primer (sense p15 and antisense p21 shown in Figure 1), 2.6 U of DNA polymerase, 25 nmol of each dNTP, 500 ng of genomic bovine DNA, and 2.25 mM of MgCl2. Amplifications were performed using the following cycling parameters: one cycle of denaturation at 94°C for 2 min followed by 10 cycles (10 s at 94°C, 5 s at 60°C, and 5 min at 68°C), then followed by 20 cycles (10 s of denaturation at 94°C, 5 s of annealing at 60°C, and 5 min of extension at 68°C, incremented with 20 s at each cycle). The final polymerization step was extended to 7 min at 68°C.

DNA sequence analysis

Sequencing was carried out using T7 and U-21 sequencing primers and a dye terminal labeling chemistry (kit PRISM Ready Reaction Ampli Taq FS) and the ABI PRISM 310 Genetic Analyzer (Perkin Elmer).

Southern blot analysis of genomic DNA

Bovine genomic DNA was digested with restriction endonucleases PstI and HindIII, fractionated through 0.8% agarose gel and subjected to Southern transfer. Blots were hybridized with [32P] radioactive probe I (228 bp) and probe II (432 bp) both generated by PCR. Probe I and probe II were synthesized from bovine brain cDNA using p11/p14 and p9/p12 primer pairs (Figure 1), respectively. Probe I corresponds to sialylmotif L (228 bp). Probe II corresponds to the upstream part of E(2) exon (-51 to 381, Figure 2), the PCR product being deleted of its 3[prime] downstream part by SmaI cleavage (Figure 2). After electrophoresis on a 2% agarose gel and gel-extraction, 25 ng of each probe were labeled with [32P]dCTP (Amersham) by random priming (Random Primers DNA Labeling System, Life Technologies) and purified to avoid unincorporated isotope (QIAquick Nucleotide Removal Kit, QIAGEN) at a specific activity of 0.5 109 c.p.m./mg or higher. High stringent hybridizations were performed for at least 12 h at 65°C in a buffer containing 0.26 M of Na2HPO4, 0.7% (w/v) SDS, 5% (w/v) dextran sulfate, 1% (w/v) bovine serum albumin, and 0.2 mg/ml denatured salmon sperm DNA. Blots were rinsed three times for 20 min each at 65°C in 0.2× SSC and then subjected to autoradiography.

Northern blot analysis

Total RNA was isolated from 100 mg of tissue using TRIZOL Reagent from Life Technologies. Poly(A+) RNA was prepared using oligo(dT)-cellulose chromatography, according to manufacturer recommendations (Life Technologies). Purified messenger RNAs were also purchased from CLONTECH. Poly(A+) RNA (1 µg) was denatured and fractionated with 0.8% formaldehyde agarose gel electrophoresis (Wen et al., 1992) and transferred to Hybond-N+ membranes (Amersham). The blots were prehybridized for 4 h at 42°C in formamide buffer (50% deionized formamide (v/v), 1% SDS (w/v), 5× Denhardt's, 5× SSC, 1 mg/ml salmon sperm DNA). Probe I, labeled as described above, was used as gene-specific probe. Hybridization to [beta]-actin messenger was used as control (Svensson et al., 1990). Bovine [beta]-actin probe was synthesized by PCR using cDNAs and primers corresponding to the most conserved domain of various bovine tissue actin (sense primer : 5[prime]-TTTACAACGAGCTGCGTGTGGCC-3[prime]; antisense primer : 5[prime]-GATCTTCATGAGGTAGTCTGTCAGG-3[prime]). Hybridization was carried out at 42°C for 16-20 h. The final wash was carried out at 65°C with 1× SSC for 20 min. Blots were analyzed using a PhosphorImager 445 SI (Molecular Dynamics). Autoradiography was performed with intensifying screens at -80°C for 10 days.

SDS-polyacrylamide gel electrophoresis and immunoblot analysis

COS-7 untransfected and transfected cells were solubilized in 200 µl of breaking buffer containing 10 mM sodium cacodylate, 20% (v/v) glycerol, 1 mM DTT, 1% (v/v) Triton X-100, pH 6, for 30 min at 4°C. The suspension was centrifuged for 5 min at 14,000 × g and 4°C. To the supernatant, 100 µl of a protease inhibitor cocktail (Sigma) was then added.

Golgi membranes were prepared from bovine liver and kidney as previously described by Fleischer and Kervina, (1974). Golgi membranes were stored in buffer containing 0.25 M sucrose, 10 mM HEPES, pH 7.5, and the protease inhibitor cocktail. In all cases, the protein content was determined by the Bradford assay (Bio-Rad) using bovine serum albumin as a standard (Bradford, 1976).

COS-7 supernatants and Golgi membrane preparations (60 µg proteins) were mixed with 6× SDS/sampling buffer (Sambrook et al., 1989), containing 5% [beta]-mercaptoethanol (v/v). Samples were heated to 100°C for 3 min before loading. Electrophoresis was carried out on SDS-PAGE using a 10% acrylamide Tris-Tricine gel (Schägger and VonJagow, 1987). Following electrophoresis, proteins were electrophoretically transferred to nitrocellulose membrane and processed for immunoblotting according to the Boehringer protocol (BM Chemiluminescence Blotting Substrate (POD), Boehringer). Primary antibody (rabbit anti-rat liver [alpha]2,6-sialyltransferase antibody) was diluted to 1:500 and second antibody, mouse anti-rabbit IgG conjugated to horseradish peroxidase, was diluted to 1:1000. Immunoblot was developed according to the Boehringer method. Molecular mass for standards were 200 kDa for myosin, 97.4 kDa for phosphorylase B, 68 kDa for bovine serum albumin, 43 kDa for ovalbumin, 29 kDa for carbonic anhydrase, and 18.4 kDa for [beta]-lactoglobulin (GibcoBRL).

Determination of gene organization

Comparison of bovine cDNA sequences with human and rat counterparts (Grundmann et al., 1990; Weinstein et al., 1987) allowed us to design exonic primers (Figure 1) within each exon for determination of gene organization. Intron sizes result of different PCR experiments based on primer pair and carried out as follows. introns were amplified with flanking specific primers using the Expand[trade] Long Template PCR System using the same conditions as above except for elongation time. According to human and rat intron sizes, elongation time was adjusted as mentioned (5 min for sizes from 1 to 4 kb, 10 min for sizes less than 10 kb, and 20 min for the longest introns). PCR products were analyzed on 0.8% (w/v) agarose gel and intron sizes were estimated using 1 kb (Life Technologies) and [lambda]/HindIII (CLONTECH) ladders as references.

Transfection and expression of the bovine ST6Gal I gene

A DNA fragment including the entire coding sequence was amplified from bovine brain cDNA with p7/p23 primer pair (Figures 1 and 2). PCR conditions were as described in the RACE experiments except that the elongation time was 75 s. The amplified fragment was cloned into pMOSBlue T-vector and sequenced. A 1790 bp fragment was subcloned between EcoRI and HindIII sites into the mammalian expression plasmid pcDNA I/Amp. COS-7 cells were transfected by the DEAE-dextran method (Davis et al., 1986) with the bovine ST6Gal I construct. After 48 h, transfected cells were trypsinized and washed twice with PBS.

Sialyltransferase enzyme assay

The assays with oligosaccharides and asialofetuin as acceptors (final volume of 60 µl) were carried out in 50 mM sodium cacodylate, pH 6.5, 0.25% (v/v) of Triton X-100 and 25 µg of total protein extracted from transfected COS-7 cells (1 h at 4°C in extraction buffer consisting in 10 mM sodium cacodylate, pH 6.5, 0.5% (v/v) of Triton X-100, 20% (v/v) of glycerol, 0.5 mM DTT and 5 mM MnCl2). Kinetic data for CMP-NeuAc were obtained with a concentration of asialofetuin fixed at 80 mM in termsof galactose sites, while [14C]-CMP-NeuAc (243 mCi/mmol,Amersham) was varied from 0.8 to 32 mM. For all other assays, the final concentration of [14C]-CMP-NeuAc was 1.6 mM.

Kinetic data for the different synthetic 8-methoxycarbonyloctyl disaccharide acceptors were obtained over a range of concentrations, from 0.5-4 mM for LacNAc and 1-7.7 mM for LacdiNAc. Mixtures were incubated for 30 min (LacNAc) or 2 h (LacdiNAc) at 37°C, and the reaction was stopped by addition of 3 ml of water, centrifuged, and the supernatant applied to a conditioned Sep-Pak C18 reverse phase chromatography cartridge (Waters, Milford). CMP-[14C]-NeuAc and its hydrolysis products were washed out with 10 ml of H2O, and the radiolabeled reaction products were eluted with two 5 ml portions of methanol collected directly into scintillation vials and counted with 1 volume of Instagel (Packard, IL) in a liquid scintillation beta counter.

Kinetic data for bovine asialofetuin were obtained with concentrations of asialofetuin ranging from 10 to 480 mM in terms of galactose sites (from 3 to 144 mg). After 5 min, the reaction was stopped by addition of 1 ml of an ice-cold solution containing 5% (w/v) phosphotungstic acid. Precipitated protein was collected by suction filtration on Whatman GF/A glass fiber filters followed by three washes with 1 ml of 5% (w/v) phosphotungstic acid, 1 ml of 5% (w/v) trichloracetic acid and 1 ml of ethanol. The filter loaded with the precipitated [14C]-NeuAc-fetuin was dried and counted with 10 ml of Instagel (Packard, IL) in a liquid scintillation beta counter.

Synthetic oligosaccharide acceptors

[beta]1,4-Galactosyltransferase was isolated from bovine milk (Barker et al., 1972), UDP-GalNAc was from Calbiochem, and calf intestine alkaline phosphatase (Molecular Biology grade, 1 U/ml) was obtained from Boehringer Mannheim. Gal[beta]1-4GlcNAc[beta]-R (R=O(CH2)8COOCH3) and GlcNAc[beta]-R were synthesized by established procedures (Palcic and Hindsgaul, 1991). GalNAc[beta]1-4GlcNAc[beta]-R was synthesized by adding 5.6 mg GlcNAc[beta]-R and 10.5 mg UDP-GalNAc to 1 U of [beta]1,4-Galactosyltransferase and 2 U of alkaline phosphatase in 1.5 ml 25 mM sodium cacodylate, pH 7.1, containing 25 mM MnCl2. Reaction was carried out at ambient temperature for 5 days with the addition of 1.1 mg of UDP-GalNAc to the mixture after 3 days of reaction. The product was isolated by applying the mixture onto two Sep-Pak C18reverse phase cartridges in tandem. The cartridges were washed with 100 ml of water, 3× 10 ml of 15% (v/v) methanol/H2O and then the product was eluted with 30 ml of HPLC grade methanol. The methanol eluent was evaporated to dryness and the residue was lyophilized from water after passing through a 0.22 mM Millex-GV filter (Millipore). The structure of isolated product (7.0 mg, 82% yield) was confirmed by [1H]-NMR spectroscopy (Palcic and Hindsgaul, 1991).

Acknowledgments

This work was supported in part by a grant from Conseil Régional du Limousin. We thank Dr. K.J.Colley for providing the rabbit anti-ST6Gal I antibody.

Abbreviations

ST6Gal I, [beta]-galactoside [alpha]2,6-sialyltransferase (E.C. 2.4.99.1); LacNAc, Gal[beta]1-4GlcNAc; LacdiNAc, GalNAc[beta]1-4GlcNAc.

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4To whom correspondence should be addressed at: Institut de Biotechnologie, 123 Avenue Albert Thomas, F-87060 Limoges Cedex, France

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