Molecular cloning and expression of mouse GD1alpha/GT1aalpha/GQ1balpha synthase (ST6GalNAc VI) gene

A novel member of the mouse CMP-NeuAc:beta-N-acetylgalactosaminide alpha2,6-sialyltransferase (ST6GalNAc) subfamily, designated ST6GalNAc VI, was identified by BLAST analysis of expressed sequence tags. The sequence of the cDNA clone of ST6GalNAc VI encoded a type II membrane protein with 43 amino acids composing the cytoplasmic domain, 21 amino acids composing the transmembrane region, and 269 amino acids composing the catalytic domain. The predicted amino acid sequence showed homology to the previously cloned ST6GalNAc III, IV, and V, with common amino acid sequences in sialyl motif L and S among these four enzymes. A fusion protein with protein A and extracts from L cells transfected with ST6GalNAc VI in an expression vector showed enzyme activity of alpha2,6-sialyltransferase for GM1b, GT1b, and GD1a but not toward glycoproteins. Thin layer chromatography-immunostaining revealed that the products were GD1alpha, GQ1balpha, and GT1aalpha. Northern blotting revealed that this gene was expressed in a wide range of mouse tissues such as colon, liver, heart, spleen, and brain. It is concluded that this enzyme is a novel sialyltransferase involved in the synthesis of alpha-series gangliosides in the nervous tissues and many other tissues.     

Molecular cloning and functional expression of two members of mouse NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2,6-sialyltransferase family, ST6GalNAc III and IV

Two cDNA clones encoding NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2, 6-sialyltransferase have been isolated from mouse brain cDNA libraries. One of the cDNA clones is a homologue of previously reported rat ST6GalNAc III according to the amino acid sequence identity (94.4%) and the substrate specificity of the expressed recombinant enzyme, while the other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those of other cloned mouse sialyltransferases, although it shows the highest sequence similarity with mouse ST6GalNAc III (43.0%). The expressed soluble recombinant enzyme exhibited activity toward NeuAcalpha2, 3Galbeta1, 3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Galbeta1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the 14C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[14C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAcalpha2,6-sialyltransferase, which requires sialic acid residues linked to Galbeta1,3GalNAc residues for its activity; therefore, we designated it mouse ST6GalNAc IV. Although the substrate specificity of this enzyme is similar to that of ST6GalNAc III, ST6GalNAc IV prefers O-glycans to glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.     

Molecular cloning of a novel alpha2,3-sialyltransferase (ST3Gal VI) that sialylates type II lactosamine structures on glycoproteins and glycolipids

A novel member of the human CMP-NeuAc:beta-galactoside alpha2, 3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of alpha2, 3-sialyltransferase toward Galbeta1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity, i.e. it utilized Galbeta1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.     

Identification and expression of a sialyltransferase responsible for the synthesis of disialylgalactosylgloboside in normal and malignant kidney cells: downregulation of ST6GalNAc VI in renal cancers

Although disialyl glycosphingolipids such as GD3 and GD2 have been considered to be associated with malignant tumours, whether branched-type disialyl glycosphingolipids show such an association is not well understood. We investigated the sialyltransferases responsible for the biosynthesis of DSGG (disialylgalactosylgloboside) from MSGG (monosialylgalactosylgloboside). Among six GalNAc:alpha2,6-sialyltransferases cloned to date, we focused on ST6GalNAc III, V and VI, which utilize sialylglycolipids as substrates. In vitro enzyme analyses revealed that ST6GalNAc III and VI generated DSGG from MSGG with V(max)/K(m) values of 1.91 and 4.16 respectively. Transfection of the cDNA expression vectors for these enzymes resulted in DSGG expression in a renal cancer cell line. Although both ST6GalNAc III and VI genes were expressed in normal kidney cells, the expression profiles of ST6GalNAc VI among 20 renal cancer cell lines correlated clearly with those of DSGG, suggesting that the sialyltransferase involved in the synthesis of DSGG in the kidney is ST6GalNAc-VI. ST6GalNAc-VI and DSGG were found in proximal tubule epithelial cells in normal kidney tissues, while they were downregulated in renal cancer cell lines and cancer tissues. All these findings indicated that DSGG was suppressed during the malignant transformation of the proximal tubules as a maturation arrest of glycosylation.     

Comparison of the enzymatic properties of mouse beta-galactoside alpha2,6-sialyltransferases,ST6Gal I and II

The cDNA encoding a second type of mouse beta-galactoside alpha2,6-sialyltransferase (ST6Gal II) was cloned and characterized. The sequence of mouse ST6Gal II encoded a protein of 524 amino acids and showed 77.1% amino acid sequence identity with human ST6Gal II. Recombinant ST6Gal II exhibited alpha2,6-sialyltransferase activity toward oligosaccharides that have the Galbeta1,4GlcNAc sequence at the nonreducing end of their carbohydrate groups, but it exhibited relatively low and no activity toward some glycoproteins and glycolipids, respectively. On the other hand, ST6Gal I, which has been known as the sole member of the ST6Gal-family for more than ten years, exhibited broad substrate specificity toward oligosaccharides, glycoproteins, and a glycolipid, paragloboside. The ST6Gal II gene was mainly expressed in brain and embryo, whereas the ST6Gal I gene was ubiquitously expressed, and its expression levels were higher than those of the ST6Gal II gene. The ST6Gal II gene is located on chromosome 17 and spans over 70 kb of mouse genomic DNA consisting of at least 6 exons. The ST6Gal II gene has a similar genomic structure to the ST6Gal I gene. In this paper, we have shown that ST6Gal II is a counterpart of ST6Gal I.     

Cloning and expression of the Gal beta 1, 3GalNAc alpha 2,3-sialyltransferase.

Sequence information obtained by NH2-terminal sequence analysis of two molecular weight forms (45 and 48 kDa) of the porcine Gal beta 1,3GalNAc alpha 2,3-sialyltransferase was used to clone a full-length cDNA of the enzyme. The cDNA sequence revealed an open reading frame coding for 343 amino acids and a putative domain structure consisting of a short NH2-terminal cytoplasmic domain, a signal-anchor sequence, and a large COOH-terminal catalytic domain. This domain structure was confirmed by construction of a recombinant sialyltransferase in which the cytoplasmic domain and signal-anchor sequence of the enzyme was replaced with the cDNA of insulin signal sequence. Expression of the resulting construct in COS-1 cells produced an active sialyltransferase which was secreted into the medium in soluble form. Comparison of the cDNA sequence of the sialyltransferase with GenBank produced no significant homologies except with the previously described Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. Although the cDNA sequences of these two enzymes were largely nonhomologous, there was a 45-amino acid sequence which exhibited 65% identity. This observation suggests that the two sialyltransferases were derived, in part, from a common gene.     

ST6GalNAc I expression in MDA-MB-231 breast cancer cells greatly modifies their O-glycosylation pattern and enhances their tumourigenicity

Sialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers, including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac:GalNAcalpha2,6-sialyltransferase: CMP-Neu5Ac: R-GalNAcalpha1-O-Ser/Thr alpha2,6-sialyltransferase (EC 2.4.99.3) (ST6GalNAc I), which transfers a sialic acid residue in alpha2,6-linkage to the GalNAcalpha1-O-Ser/Thr structure. However, established breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn. We have previously shown that stable transfection of MDA-MB-231, a human breast cancer cell line, with ST6GalNAc I cDNA induces sialyl-Tn antigen (STn) expression. We report here the modifications of the O-glycosylation pattern of a MUC1-related recombinant protein secreted by MDA-MB-231 sialyl-Tn positive cells. We also show that sialyl-Tn expression and concomitant changes in the overall O-glycan profiles induce a decrease of adhesion and an increase of migration of MDA-MB-231. Moreover, STn positive clones exhibit an increased tumour growth in severe combined immunodeficiency (SCID) mice. These observations suggest that modification of the O-glycosylation pattern induced by ST6GalNAc I expression are sufficient to enhance the tumourigenicity of MDA-MB-231 breast cancer cells.     

Sialyltransferase isoforms are phosphorylated in the cis-medial Golgi on serine and threonine residues in their luminal sequences

ST6Gal-I (alpha2,6-sialyltransferase) is expressed as two isoforms, STTyr and STCys, which exhibit differences in catalytic activity, trafficking through the secretory pathway, and proteolytic processing and secretion. We have found that the ST6Gal-I isoforms are phosphorylated on luminal Ser and Thr residues. Immunoprecipitation of 35S- and 32P-labeled proteins expressed in COS-1 cells suggests that the STTyr isoform is phosphorylated to a greater extent than the STCys isoform. Analysis of domain deletion mutants revealed that STTyr is phosphorylated on stem and catalytic domain amino acids, whereas STCys is phosphorylated on catalytic domain amino acids. An endoplasmic reticulum retained/retrieved chimeric Iip33-ST protein demonstrates drastically lower phosphorylation than does the wild type STTyr isoform. This suggests that the bulk of the ST6Gal-I phosphorylation is occurring in the Golgi. Treatment of cells with the ionophore monensin does not significantly block phosphorylation of the STTyr isoform, suggesting that phosphorylation is occurring in the cis-medial Golgi prior to the monensin block. This study demonstrates the presence of kinase activities in the cis-medial Golgi and the substantial phosphorylation of the luminal sequences of a glycosyltransferase.     

Construction and characterization of stably transfected BHK-21 cells with human-type sialylation characteristic

The human Golgi enzyme CMP-NeuAc:Gal(β1–4)GlcNAc-R α2,6-sialyltransferase (ST6N) was stably coexpressed with human erythropoietin (EPO) from a BHK-21A cell line. The cell line was characterized with respect to the expression and in vitro activity of the ST6N and the endogenous α2,3-sialyltransferase. Detailed structural analysis of the N-linked carbohydrates of the rhuEPO expressed from the new cell line was performed by HPAE-PAD-mapping, MALDI/TOF-MS and methylation analysis after purification of the recombinant protein by immunoaffinity chromatography. This is the first report describing that the human α2,6-sialyltransferase is capable of sialylating, apart from Gal(β1–4)GlcNAc-R, also GalNAc(β1–4)GlcNAc-R motifs in vivo, which is not the case for the endogenous BHK-cell α2,3-sialyltransferase. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of mouse sialyltransferase genes: their evolution and diversity

Sialic acids are negatively charged acidic sugars, and sialylglycoconjugates often play important roles in various biological phenomena. Sialyltransferases are involved in the synthesis of sialylglycoconjugates, and 20 members of the mammalian sialyltransferase family have been identified to date. These sialyltransferases are grouped into four families according to the carbohydrate linkages they synthesize: beta-galactoside alpha2,3-sialyltransferases (ST3Gal I-VI), beta-galactoside alpha2,6-sialyltransferases (ST6Gal I and II), GalNAc alpha2,6-sialyltransferases (ST6GalNAc I-VI), and alpha2,8-sialyltransferases (ST8Sia I-VI). Analysis of the amino acid sequence similarities, substrate specificities, and gene structures of mouse sialyltransferases has revealed that they can be further divided into seven subfamilies. The genomic structural resemblance of members of the same subfamily suggests that they arose from a common ancestral gene through gene duplication events. These multiple sialyltransferase genes are needed for fine control of the expression of sialylglycoconjugates, resulting in a variety of developmental stage- and tissue-specific glycosylation patterns.     

Exploring the acceptor substrate recognition of the human beta-galactoside alpha 2,6-sialyltransferase

Human beta1,4-galactoside alpha2,6-sialyltransferase I (ST6GalI) recognition of glycoprotein acceptors has been investigated using various soluble forms of the enzyme deleted to a variable extent in the N-terminal half of the polypeptide. Full-length and truncated forms of the enzyme have been investigated with respect to their specificity for a variety of desialylated glycoproteins of known complex glycans as well as related proteins with different carbohydrate chains. Differences in transfer efficiency have been observed between membrane and soluble enzymatic forms, indicating that deletion of the transmembrane fragment induces loss of acceptor preference. No difference in substrate recognition could be observed when soluble enzymes of similar peptide sequence were produced in yeast or mammalian cells, confirming that removal of the membrane anchor and heterologous expression do not alter enzyme folding and activity. When tested on free oligosaccharides, soluble ST6GalI displayed full ability to sialylate free N-glycans as well as various N-acetyllactosaminyl substrates. Progressive truncation of the N terminus demonstrated that the catalytic domain can proceed with sialic acid transfer with increased efficiency until 80 amino acids are deleted. Fusion of the ST6GalI catalytic domain to the N-terminal half of an unrelated transferase (core 2 beta1,6-N-acetylglucosaminyltransferase) further showed that a chimeric form of broad acceptor specificity and high activity could also be engineered in vivo. These findings therefore delineate a peptide region of approximately 50 amino acids within the ST6GalI stem region that governs both the preference for glycoprotein acceptors and catalytic activity, thereby suggesting that it may exert a steric control on the catalytic domain.     

Endothelial alpha 2,6-linked sialic acid inhibits VCAM-1-dependent adhesion under flow conditions.

We have previously shown that costimulation of endothelial cells with IL-1 + IL-4 markedly inhibits VCAM-1-dependent adhesion under flow conditions. We hypothesized that sialic acids on the costimulated cell surfaces may contribute to the inhibition. Northern blot analyses showed that Gal beta 1-4GlcNAc alpha 2, 6-sialyltransferase (ST6N) mRNA was up-regulated in cultured HUVEC by IL-1 or IL-4 alone, but that the expression was enhanced by costimulation, whereas the level of Gal beta 1-4GlcNAc/Gal beta 1-3GalNAc alpha2,3-sialyltransferase (ST3ON) mRNA was unchanged. Removing both alpha 2,6- and alpha 2,3-linked sialic acids from IL-1 + IL-4-costimulated HUVEC by sialidase significantly increased VCAM-1-dependent adhesion, whereas removing alpha 2,3-linked sialic acid alone had no effect; adenovirus-mediated overexpression of ST6N with costimulation almost abolished the adhesion, which was reversible by sialidase. The same treatments of IL-1-stimulated HUVEC had no effect. Lectin blotting showed that VCAM-1 is decorated with alpha 2,6- but not alpha 2,3-linked sialic acids. However, overexpression of alpha 2,6-sialyltransferase did not increase alpha 2,6-linked sialic acid on VCAM-1 but did increase alpha 2,6-linked sialic acids on other proteins that remain to be identified. These results suggest that alpha 2,6-linked sialic acids on a molecule(s) inducible by costimulation with IL-1 + IL-4 but not IL-1 alone down-regulates VCAM-1-dependent adhesion under flow conditions.     

Photobacterium sp. JT-ISH-224 produces two sialyltransferases, alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase

A novel bacterium, Photobacterium sp. JT-ISH-224, that produces alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase, was isolated from the gut of a Japanese barracuda. The genes that encode the enzymes were cloned from the genomic library of the bacterium using the genes encoding alpha-/beta-galactoside alpha2,3-sialyltransferase from P. phosphoreum and beta-galactoside alpha2,6-sialyltransferase from P. damselae as probes. The nucleotide sequences were determined, and open reading frames of 1,230 and 1,545 bp for encoding an alpha2,3-sialyltransferase and an alpha2,6-sialyltransferase of 409- and 514-amino acid residues, respectively, were identified. The alpha2,3-sialyltransferase had 92% amino acid sequence identity with the P. phosphoreum alpha2,3-sialyltransferase, whereas the alpha2,6-sialyltransferase had 54% amino acid sequence identity with the P. damselae alpha2,6-sialyltransferase. For both enzymes, the DNA fragments that encoded the full-length protein and its truncated form lacking the putative signal peptide sequence were amplified by a polymerase chain reaction and cloned into an expression vector. Each gene was expressed in Escherichia coli, and the lysate from each strain had enzymatic activity. The alpha2,3-sialyltransferase catalysed the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to lactose, alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside with low apparent K(m) and the alpha2,6-sialyltransferase catalysed the transfer of NeuAc from CMP-NeuAc to lactose with low apparent K(m).