Multiplex reverse transcription polymerase chain reaction assessment of sialyltransferase expression in human breast cancer.

Increased sialylation, especially involving the Sialyl-Lewisa and Sialyl-Lewisx determinants, has been reported in breast cancer. A multiplex reverse transcription-PCR method was used here to determine the expression of five sialyltransferases (ST3Gal III, ST6Gal I, ST3Gal IV, ST3Gal I, and ST3Gal II) in 49 patients surgically treated for locoregional breast cancer. We assessed the relationship between these expressions and clinical, pathological, and biological features. The most expressed sialyltransferase was ST3Gal 1II, which is involved in Sialyl-Lewisa synthesis. ST3Gal III expression was positively correlated to ST6Gal I and ST3Gal IV expressions, to tumor size, and to the number of involved axillary nodes. Patients with high ST3Gal III expression had a shorter overall survival. High ST6Gal I expression was associated with histoprognostic grade III. ST6Gal I expression was negatively correlated to expression of progesterone receptor. In conclusion, high ST3Gal III and ST6Gal I expressions in human breast tumors are associated with poor prognosis markers.

Expression of glypican-1, syndecan-1 and syndecan-4 mRNAs protein kinase C-regulated in rat immature Sertoli cells by semi-quantitative RT-PCR analysis.

In seminiferous tubules, Sertoli cells provide structural and nutritional support for the developing germinal cells. Cell to cell signalization and cell adhesion require proteoglycans expressed at the cell membrane. A preliminary biochemical and structural approach indicated that cell surface proteoglycans are mostly heparan sulfate (HSPG) in immature rat Sertoli cells. The present study focused on the qualitative and quantitative expression of three membrane HSPG, syndecan-1, syndecan-4 and glypican-1 in Sertoli cells of 20-day-old rat. A semi-quantitative multiplex RT-PCR strategy was developed to appreciate the effect of PKC activation on the mRNA expression of the three HSPG. Our data show that the syndecan-1 and glypican-1 mRNA expression is increased by the phorbol myristate acetate (PMA) suggesting a regulation of their expression by the phosphatidyl inositol pathway, as previously hypothesized (Fagen et al., Biochim. Biophys. Acta, 1472 (1999) 250-261). In addition, a physiological effector of the PKC as ATP gave similar effects. Thus, this over-expression could be related with paracrine factors secreted by germ cells.

Molecular cloning and functional expression of human ST6GalNAc II. Molecular expression in various human cultured cells.

A cDNA clone encoding a human Galbeta1-3GalNAc alpha2, 6-sialyltransferase (designated hST6GalNAc II) was identified employing the PCR with degenerated primers to the sialylmotifs, followed by BLAST analysis of databanks. This sialyltransferase sequence is similar to that of previously cloned ST6GalNAc II (chicken and mouse) and shows the sialylmotifs that are present in all eukaryotic members of the sialyltransferase gene family. The predicted amino acid sequence encodes a putative type II transmembrane protein as found for other eukaryotic sialyltransferases and shows significant similarity to chicken (56. 8% identity) and mouse (74.6% identity) enzymes. Expression of a secreted form of hST6GalNAc II in COS-7 cells showed that the gene product had Galbeta1-3GalNAc (sialyl to GalNAc) alpha2, 6-sialyltransferase activity. In vitro analysis of substrate specificity revealed that the enzyme required a peptide aglycone fraction to be active and used both Galbeta1-3GalNAc and Neu5Acalpha2-3Galbeta1-3GalNAc as acceptor substrates. Northern analysis revealed a restricted expression pattern of two hST6GalNAc II transcripts, a 2.0 kb mRNA found mainly in skeletal muscle, heart and kidney and a 1.8 kb mRNA found in placenta, lung and leukocytes. No transcriptional expression was detected in brain, thymus or spleen. Transcriptional expression of the ST6GalNAc II gene was followed in various human cell lines and found to be expressed in almost all cell types with notable exceptions for several myeloid and lymphoid cell lines.

Delineation of the minimal catalytic domain of human Galbeta1-3GalNAc alpha2,3-sialyltransferase (hST3Gal I).

The CMP-Neu5Ac:Galbeta1-3GalNAc alpha2,3-sialyltransferase (ST3Gal I, EC 2.4.99.4) is a Golgi membrane-bound type II glycoprotein that catalyses the transfer of sialic acid residues to Galbeta1-3GalNAc disaccharide structures found on O-glycans and glycolipids. In order to gain further insight into the structure/function of this sialyltransferase, we studied protein expression, N-glycan processing and enzymatic activity upon transient expression in the COS-7 cell line of various constructs deleted in the N-terminal portion of the protein sequence. The expressed soluble polypeptides were detected within the cell and in the cell culture media using a specific hST3Gal I monoclonal antibody. The soluble forms of the protein consisting of amino acids 26-340 (hST3-Delta25) and 57-340 (hST3-Delta56) were efficiently secreted and active. In contrast, further deletion of the N-terminal region leading to hST3-Delta76 and hST3-Delta105 gave also rise to various polypeptides that were not active within the transfected cells and not secreted in the cell culture media. The kinetic parameters of the active secreted forms were determined and shown to be in close agreement with those of the recombinant enzyme already described (H. Kitagawa, J.C. Paulson, J. Biol. Chem. 269 (1994)). In addition, the present study demonstrates that the recombinant hST3Gal I polypeptides transiently expressed in COS-7 cells are glycosylated with complex and high mannose type glycans on each of the five potential N-glycosylation sites.

The human sialyltransferase family.

The human genome encodes probably more than 20 different sialyltransferases involved in the biosynthesis of sialylated glycoproteins and glycolipids but to date only 15 different human sialyltransferase cDNAs have been cloned and characterized. Each of the sialyltransferase genes is differentially expressed in a tissue-, cell type-, and stage-specific manner to regulate the sialylation pattern of cells. These enzymes differ in their substrate specificity, tissue distribution and various biochemical parameters. However, enzymatic analysis conducted in vitro with recombinant enzyme revealed that one linkage can be synthesized by multiple enzymes. We present here an overview of these human genes and enzymes, the regulation of their occurrence and their involvement in several physiological and pathological processes.

Prognostic value of tumoral sialyltransferase expression and circulating E-selectin concentrations in node-negative breast cancer patients.

AIMS AND BACKGROUND: A crucial step in the metastatic process is the interaction between the endothelial molecule E-selectin and its tumoral ligands sialyl-Lewis- and sialyl-Lewis. Sialyltranferases are involved in the biosynthesis of these ligands. The aim of this study was to assess the prognostic value of tumoral sialyltransferase expression and of circulating soluble E-selectin (sE-selectin) in node-negative breast cancer patients. METHODS: Using a multiplex RT-PCR method, we measured the expression of five sialyltransferases (ST3Gal III, ST6Gal I, ST3Gal IV, ST3Gal I and ST3Gal II) in tumors of 135 surgically treated node-negative breast cancer patients. Circulating sE-selectin concentrations were measured by an ELISA method prior to surgery. We also analyzed tumor size, histoprognostic grading and steroid hormone receptor status. RESULTS: The median follow-up was 7.5 years. Expression of estrogen receptors was associated with a good prognosis for relapse-free survival in univariate analysis. A high ST3Gal III/ST6Gal I ratio and a high sE-selectin concentration were associated with a bad prognosis for relapse-free survival and overall survival in univariate and multivariate analysis. CONCLUSION: In the present study, tumoral sialyltransferase expression and circulating sE-selectin concentrations had prognostic value in patients with node-negative breast cancer. This result provides further evidence for the important role of these agents in the metastatic process.

Differential expression of alpha-2,3-sialyltransferases and alpha-1,3/4-fucosyltransferases regulates the levels of sialyl Lewis a and sialyl Lewis x in gastrointestinal carcinoma cells.

Sialyl Lewis x and sialyl Lewis a expression depends on sialyltransferases and fucosyltransferases. In this study, we screened for major variations of sialyltransferases and fucosyltransferases involved in the synthesis and regulation of sialyl Lewis x and sialyl Lewis a epitopes in gastrointestinal carcinoma cells. Our results show that expression of ST3Gal IV in several gastrointestinal cell lines is correlated with the expression of sialyl Lewis x at the cell surface. ST3Gal IV overexpressed in the gastric MKN45 cell line, showed exclusive enzymatic activity towards glycoproteins containing terminal Galbeta1-4GlcNAc structure. On the other hand, when ST3Gal III was overexpressed in MKN45, an increase in the expression levels of both sialyl Lewis epitopes was observed. ST3Gal III and ST3Gal IV lead to de novo synthesis of sialyl Lewis x determinant on different molecular weight glycoproteins of MKN45 cells suggesting that each enzyme used different substrates within the available glycoproteome. The final glycosylation step in sialyl Lewis x and sialyl Lewis a biosynthesis in MKN45 cell line was shown to be associated to FUT5, which efficiently fucosylated sialyl Lewis precursors on glycoproteins. Moreover we demonstrate that the expression of sialyl Lewis epitopes in the MKN45 was induced by cell confluence, which can be regarded as a model to study altered glycosylation during tumour progression. This increase was observed together with an increase in mRNA levels of ST3GAL3, FUT5 and FUT6, and a decrease in FUT4 transcript levels in MKN45 confluent cells, suggesting a possible control at the transcriptional level.

Characterization of two cis-regulatory regions in the murine beta 1,4-galactosyltransferase gene. Evidence for a negative regulatory element that controls initiation at the proximal site.

The beta 1,4-galactosyltransferase (beta 1,4-GT) gene is unusual in that it specifies two mRNAs in somatic cells of 3.9 and 4.1 kilobases (kb). These two transcripts arise as a consequence of initiation at two different sets of start sites that are separated by approximately 200 base pairs. Translation of each mRNA results in the predicted synthesis of two related protein isoforms that differ only in the length of their NH2-terminal cytoplasmic domain (Russo, R.N., Shaper, N. L., and Shaper, J.H. (1990) J. Biol. Chem. 265, 3324-3331). In this study we show that the cellular requirements for beta 1,4-GT correlate with the transcriptional start site used. In cells and tissues that express low transcript levels, the 4.1-kb transcriptional start site is apparently used exclusively. Increased transcription from the 4.1-kb start site plus low levels of transcription from the 3.9-kb start site result in the intermediate beta 1,4-GT transcript levels that are found in almost all somatic cell types. However, in mid- to late pregnant and lactating mammary glands very high transcript levels are observed, which correlate with the predominant use of the 3.9-kb transcriptional start site. To identify the cis-acting elements that regulate the use of the two transcriptional start sites, we constructed a series of beta 1,4-GT/CAT hybrids and carried out transient transfection assays using mouse L cells and Drosophila SL2 cells. These studies have delineated both a distal and proximal regulatory region just upstream of the 4.1- and 3.9-kb transcriptional start sites, respectively. In addition, a negative cis-acting regulatory region was identified that represses transcription from the proximal site. These results suggest a model of transcriptional regulation in which the distal region functions as a housekeeping promoter while the proximal region functions as a mammary cell-specific promoter. Differential initiation from the two promoters is a mechanism for regulation of beta 1,4-GT enzyme levels. The predictions from this model are consistent with the conclusion that both protein isoforms are functionally equivalent resident trans-Golgi membrane-bound enzymes.

Male germ cell expression of murine beta 4-galactosyltransferase. A 796-base pair genomic region, containing two cAMP-responsive element (CRE)-like elements, mediates male germ cell-specific expression in transgenic mice.

In murine somatic cells, transcription of the single gene encoding beta 4-galactosyltransferase results in two transcripts of 4.1 and 3.9 kilobases (kb), as a consequence of the use of two transcriptional start sites that are located on exon one separated by 200 base pairs (bp). In early male germ cell development, spermatogonia use only the 4.1-kb start site to yield a transcript that is identical to its somatic cell counterpart. As these cells enter meiosis, there is a switch from the use of this somatic cell start site to the exclusive use, beginning in pachytene spermatocytes, of a male germ cell-specific start site. Germ cell-specific transcripts are distinguished from their somatic counterparts by an additional approximately 560 nucleotides of 5'-untranslated sequence that is located immediately upstream and contiguous with the transcriptional start site defined for the 4.1-kb mRNA (Harduin-Lepers, A., Shaper, N.L., Mahoney, J.A., and Shaper, J.H. (1992) Glycobiology 2, 361-368). This observation predicts the use of a different upstream male germ cell-specific promoter. In this study we show that a 796-bp fragment containing 543 bp of genomic sequence upstream of the germ cell specific transcriptional start site and 253 bp of flanking downstream sequence, directs expression of the reporter gene, beta-galactosidase, exclusively to the pachytene spermatocytes and round spermatids of transgenic mice. This pattern of cell type-specific expression of the transgene is comparable with that of the endogenous beta 4-galactosyltransferase gene.

Multiplex RT-PCR method for the analysis of the expression of human sialyltransferases: application to breast cancer cells.

In many cases of human cancer, the appearance of hypersialylated glycan structures is related to a precise stage of the disease; this may depend on altered regulation of one or more sialyltransferases genes. Since several distinct sialyltransferase enzymes arising from different unique genes transfer sialic acid residues in the same linkage onto the same acceptor, it is impossible to precisely determine which enzyme is involved in the observed phenotype based on enzymatic assays. We have developed a very sensitive and highly reproducible multiplex reverse transcriptase-polymerase chain reaction technique in order to monitor the expression of four human sialyltransferases genes ST6Gal I, ST3Gal I, ST3Gal III and ST3Gal IV in small cell samples. Multiplex PCR amplification using specific primers for each sialyltransferase and detection of amplification products by polyacrylamide gel electrophoresis is a method that is fast and easy to handle and has proven to be useful for establishing sialyltransferase patterns of expression in breast immortalized cell line HBL100 as well as in breast cancer cell lines MCF-7/6, MCF-7/AZ and MDA.

Murine beta 1,4-galactosyltransferase: round spermatid transcripts are characterized by an extended 5'-untranslated region.

We have previously shown that the expression of the gene encoding murine beta 1,4-galactosyltransferase (beta 1,4-GT, UDP-galactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D galactosyltransferase, EC 2.4.1.38) is fundamentally different between somatic and male germ cells (Shaper et al., 1990b). In somatic cells, two transcripts of 3.9 kb and 4.1 kb are produced. In contrast, in spermatogonia only the 4.1 kb transcript is expressed. Maturation of spermatogonia to pachytene spermatocytes is accompanied by reduced expression of the 4.1 kb transcript to barely detectable levels. Continued differentiation to haploid round spermatids is coincident with renewed expression in which the 4.1 kb transcript is replaced by two truncated transcripts of 2.9 and 3.1 kb. In this study, we report the characterization of a full-length beta 1,4-GT cDNA clone from a murine round spermatid library that corresponds to the 2.9 kb transcript. This transcript encodes the same open reading frame as the 4.1 kb transcript, but utilizes alternative poly(A) signals embedded within the long 3'-untranslated region of the somatic transcript. Based on sequence analysis, together with primer extension and S1 nuclease protection experiments, both the 2.9 and the 3.1 kb round spermatid beta 1,4-GT transcripts are distinguished by the presence of an additional 5'-untranslated sequence of approximately 560 bp that is absent in premeiotic germ cells and somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of sialyl-Tn antigen in breast cancer cells transfected with the human CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase (ST6GalNac I) cDNA.

Sialyl-Tn antigen (STn) is a cancer associated carbohydrate antigen over-expressed in several cancers including breast cancer, and currently associated with more aggressive diseases and poor prognosis. However, the commonly used breast cancer cell lines (MDA-MB-231, T47-D and MCF7) do not express STn antigen. The key step in the biosynthesis of STn is the transfer of a sialic acid residue in alpha2,6-linkage to GalNAc alpha-O-Ser/Thr. This reaction is mainly catalyzed by a CMP-Neu5Ac GalNAc alpha2,6-sialyltransferase: ST6GalNAc I. In order to generate STn-positive breast cancer cells, we have cloned a cDNA encoding the full-length human ST6GalNAc I from HT-29-MTX cells. The stable transfection of MDA-MB-231 with an expression vector encoding ST6GalNAc I induces the expression of STn antigen at the cell surface. The expression of STn short cuts the initial O-glycosylation pattern of these cell lines, by competing with the Core-1 beta1,3-galactosyltransferase, the first enzyme involved in the elongation of O-glycan chains. Moreover, we show that STn expression is associated with morphological changes, decreased growth and increased migration of MDA-MB-231 cells.

Cloning and expression of cDNA for a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase from the CEM T-cell line.

Complementary DNA encoding a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase type II (hST3Gal II) was cloned from a CEM T-cell cDNA library using a 23-base oligonucleotide probe. The sequence of this probe was established on the basis of a slightly divergent sialylmotif L that was obtained by polymerase chain reaction with degenerate oligonucleotide primers based on the conserved sialylmotif L of mammalian Gal(beta1-3)GalNAc alpha2,3-sialyltransferases. It was thus confirmed that a short oligonucleotide probe may be sensitive and highly specific. The nucleotide and amino acid sequences of hST3Gal II show, respectively, 56.3% and 49.3% similarity to hST3Gal I [Kitagawa, H. & Paulson, J. C. (1994) J. Biol. Chem. 269, 17872-17878] and 88.1% and 93.7% similarity to murine ST3Gal II [Lee, Y. C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hamamoto, T. & Tsuji, S. (1994) J. Biol. Chem. 269, 10028-10033]. hST3Gal II mRNA was highly expressed in heart, liver, skeletal muscle and various lymphoid tissues but not in brain and kidney. A soluble form of hST3Gal II expressed in COS-7 cells was tested in vitro for substrate specificity and kinetic properties. Asialofetuin and asialo-bovine submaxillary mucin appeared better substrates for hST3Gal II than for its murine counterpart as previously reported [Kojima, N., Lee, Y.-C., Hamamoto, T., Kurosawa, N. & Tsuji, S. (1994) Biochemistry 33, 5772-5776]. In previous studies, we have shown hyposialylation of O-glycans attached to two major lymphocyte CD43 and CD45 cell surface molecules in human-immunodeficiency-virus-1(HIV-1)-infected T-cell lines. Since comparable levels of hST3Gal I and hST3Gal II mRNA and enzymatic activity were observed in parental and HIV-1-infected CEM T-cell lysates, the sialylation defect associated with HIV infection of this cell line is probably due to a mechanism different from a simple altered catalytic activity of these sialyltransferases.

Molecular cloning, expression and exon/intron organization of the bovine beta-galactoside alpha2,6-sialyltransferase gene.

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 alpha2,6-sialyltransferase) sequences, respectively. When transfected into COS-7 cells, a recombinant enzyme was obtained which catalyzed the in vitro alpha2, 6-sialylation of LacNAc (NeuAcalpha2-6Galbeta1-4GlcNAc) and LacdiNAc (NeuAcalpha2-6GalNAcbeta1-4GlcNAc) acceptor substrates. The K (m) 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'-untranslated exons E(-2) to E(1), and five coding exons from E(2) to E(6). This later carries a 3'-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'-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.

Microheterogeneity of the oligosaccharides carried by the recombinant bovine lactoferrin expressed in Mamestra brassicae cells.

The development of therapeutic glycoprotein production using the baculovirus expression system depends on the ability of insect cell lines to reproduce site specific mammalian-like N-glycans. A combination of 1H-NMR and mass spectrometry techniques (MALD-MS, ES-MS, and CID-MS-MS) allowed us to elucidate the N-linked oligosaccharides microheterogeneity on three different N-glycosylation sites, Asn233, Asn476, and Asn545, of a baculovirus-expressed recombinant bovine lactoferrin produced in Mamestra brassicae. Two families of N-glycan structures have been found: first, oligomannosidic glycans (Man[9-5]GlcNAc2) and secondly, short truncated partially fucosylated glycans (Man(3-2)[Fuc(0-1)]GlcNAc2). These results indicate that Mamestra brassicae cell line is not able to synthesize complex N-glycans, even if an alpha1,6-linked fucose residue is frequently present on the asparagine-bound N-acetylglucosamine residue of short truncated structures. Nevertheless, we have shown that Mamestra brassicae ensures the same N-glycosylation pattern as found on natural bovine lactoferrin showing the same distribution between complex and high-mannose type glycans on the different glycosylation sites. Sites which are naturally occupied by high-mannose glycans (Asn233 and Asn545) are substituted essentially by the same type of N-glycans in the recombinant counterpart, and the site Asn476,which carries sialylated complex type chains in the natural glycoprotein, is substituted by short, truncated, partially fucosylated chains in Mamestra brassicae-expressed bovine lactoferrin. These various results lead us to the conclusion that bovine lactoferrin is an interesting model to determine the potential of glycosylation of the baculovirus/insect cell expression systems.

Regulation of sialyltransferase expression by estradiol and 4-OH-tamoxifen in the human breast cancer cell MCF-7.

We have addressed the effects of estradiol and 4-OH-tamoxifen on the expression of five sialyltransferases in the hormono-dependent MCF-7 cell line using a Multiplex RT-PCR approach. Estradiol induced a statistically significant increase in ST3Gal III and a decrease in ST6Gal I, whereas the two other enzymes, ST3Gal IV and ST3Gal I, are not modified and expression of the fifth enzyme, ST3Gal II, was very low or not detectable. Estradiol effects were dose dependent and completely antagonized by 4OH-tamoxifen. In addition, there is no direct relation between cellular proliferation and sialyltransferase expression. This suggests that ST3Gal III and ST6Gal I could be used as supplementary markers of hormono-sensitivity in breast cancer.

Cloning, expression and gene organization of a human Neu5Ac alpha 2-3Gal beta 1-3GalNAc alpha 2,6-sialyltransferase: hST6GalNAcIV.

On the basis of the detection of expressed sequence tag ('EST') similar to the rat N-acetylgalactosamine alpha2,6-sialyltransferase (ST6GalNAc) III cDNA, we have identified a novel member of the human ST6GalNAc family. We have isolated a cDNA clone containing an open reading frame that codes for a type II membrane protein of 302 amino acids with a seven-amino-acid cytoplasmic domain, an 18-amino-acid transmembrane domain and the smallest described catalytic domain of 277 amino acids. This predicted sialyltransferase sequence is similar to the rat ST6GalNAc III (46.6%), but was found to be even more similar to the recently reported mouse ST6GalNAc IV (88.1%) on the basis of amino acid sequence identity. Northern-blot analysis showed that the newly identified gene is expressed constitutively in various adult human tissues as a 2.2kb transcript, but was also found to be expressed at lower levels in brain, heart and skeletal muscle as a 2.5kb transcript. Expression of the hST6GalNAc IV gene was investigated by reverse transcription PCR in various human cancer cells, and was found to be present in the majority of cell types with the exception of the carcinoma cell line T47D and pro-monocyte THP cells. The transient expression in COS-7 cells of the full-length cDNA led to the production of an active enzyme sharing the acceptor specificity of the ST6GalNAc family towards Neu5Ac alpha 2-3Gal beta 1-3GalNAc alpha-O-R (where 'R' denotes H, benzyl, or a peptidic chain). Detailed analysis in vitro of substrate specificity revealed that the enzyme required the trisaccharide Neu5Ac alpha 2-3Gal beta1-3GalNAc found on O-glycans and arylglycosides. In addition, we have clarified the genomic organization of ST6GalNAc IV gene.