Human alpha2,3-sialyltransferase (ST3Gal II) is a stage-specific embryonic antigen-4 synthase

Monosialosyl globopentaosylceramide (MSGb5), originally described as stage-specific embryonic antigen-4, is expressed in testicular germ cell tumors and in aggressive cases of human renal cell carcinoma (RCC). Clarification of the molecular mechanisms regulating synthesis of MSGb5 is very important to understand testicular carcinogenesis and the malignant progression of human RCC. For this purpose, we have investigated alpha2,3-sialyltransferase involved in the synthesis of MSGb5. We used the method of expression cloning combined with polymerase chain reaction targeted to sialylmotif to isolate a cDNA clone from RCC cell line ACHN library. The cloned cDNA was found to be identical to the previously cloned ST3Gal II in sequence. A soluble recombinant form of the protein in COS-1 cells showed an enzyme activity of alpha2,3-sialyltransferase toward globopentaosylceramide (Gb5) in addition to asialo-GM1 and GM1a. Transient transfection of COS-7 and ACHN cells with this cDNA induced an increase of MSGb5, whereas stable transfection of antisense ST3Gal II cDNA suppressed expression of MSGb5 in ACHN cells. The ST3Gal II mRNA level was increased in 7 of 8 RCC cell lines and in all six RCC tissues surgically obtained, although it was not necessarily consistent with the MSGb5 level in RCC cell lines. This study indicates that ST3Gal II is a MSGb5 (stage-specific embryonic antigen-4) synthase and that its increased expression level is closely related to renal carcinogenesis.     

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.     

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.     

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.     

An alpha2,3 sialyltransferase (ST3Gal I) is elevated in primary breast carcinomas

The MUC1 mucin is expressed on the luminal surface of most simple epithelial cells but in carcinomas, especially those of the breast and ovary, MUC1 is upregulated and aberrantly glycosylated. MUC1 contains a large amount of O-linked glycans which, in the mucin expressed by normal mammary epithelial cells, consist mainly of core 2 based structures carrying polylactosamine chains. However, the mucin expressed by breast carcinomas has shorter side-chains, often consisting of sialylated core 1 (Galbeta1-3GalNAc). in situ hybridization of primary breast tissue showed that a sialyltransferase (ST3Gal I), responsible for adding sialic acid to core 1 thereby terminating chain extension, is elevated in primary breast carcinomas when compared to normal or benign tissue. Furthermore, the level of mRNA expression encoding ST3Gal I is correlated to the intensity of staining seen with the antibody SM3, which specifically recognises underglycosylated, tumour associated MUC1. Thus, the aberrant glycosylation of MUC1 seen in breast carcinomas appears to be due, at least in part, to the elevation of ST3Gal I.     

Multiple signals are required for alpha2,6-sialyltransferase (ST6Gal I) oligomerization and Golgi localization

A single amino acid difference in the catalytic domain of two isoforms of the alpha2,6-sialyltransferase (ST6Gal I) leads to differences in their trafficking, processing, and oligomerization. The STtyr isoform is transiently localized in the Golgi and is ultimately cleaved and secreted, whereas the STcys isoform is stably localized in the Golgi and is not cleaved and secreted. The stable localization of STcys is correlated with its enhanced ability to oligomerize. To test the hypothesis that multiple signals can mediate Golgi localization and further evaluate the role of oligomerization in the localization process, we evaluated the effects of individually and simultaneously altering the cytosolic tail and transmembrane region of the STcys isoform. We found that the localization, processing, and oligomerization of STcys were not substantially changed when either the core amino acids of the cytosolic tail were deleted or the sequence and length of the transmembrane region were altered. In contrast, when these changes were made simultaneously, the STcys isoform was converted into a form that was processed, secreted, and weakly oligomerized like STtyr. We propose that STcys oligomerization is a secondary event resulting from its concentration in the Golgi via mechanisms independently mediated by its cytosolic tail and transmembrane region.     

Comparative rates of transfer of N-acetylneuraminic acid to acceptors bearing one or more Gal(beta 1-4)GlcNAc terminus by the Gal(beta 1-4)GlcNAc(NeuAc-Gal) (alpha 2-6)-sialyltransferase from embryonic chicken liver. Utilization of oligosaccharides as acceptors in sialyltransferase assays.

Using a number of branched and unbranched oligosaccharides, glycoproteins and artificial glycoproteins bearing Gal(beta 1-4)GlcNAc-R termini as acceptors (where R represents H, oligosaccharide, oligosaccharide-protein or fatty acid-protein), the comparative rates of transfer of NeuAc by the Gal(beta 1-4)GlcNAc(NeuAc-Gal) (alpha 2-6)-sialyltransferase of embryonic chicken liver were determined. Acceptor substrates were utilized at levels approximating physiological, near the Km value of the best acceptor, desialylated alpha 1 acid glycoprotein. The sialyltransferase has a marked preference for multi-branched acceptors. From the specificity data, it is concluded that the enzyme binds at least two Gal(beta 1-4)GlcNAc termini of an acceptor molecule, and that the relative orientation of the branches is an important factor determining the rate of catalysis by the enzyme. The use of oligosaccharides as acceptors to study sialyltransferase catalyses is emphasized. Results are discussed in the context of the mode of assembly of sialoside termini of known glycoprotein structures in vivo.     

Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization

Sialylation is a biosynthetic process occurring in the trans compartments of the Golgi apparatus. Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported. Here we describe generation and characterization of polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion- protein from E.coli , respectively. These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections. In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the recombinant antigen retained in the endoplasmic reticulum. However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines. Antibodies affinity purified on the beta- galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections. We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase. This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus. These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.      

The two rat alpha 2,6-sialyltransferase (ST6Gal I) isoforms: evaluation of catalytic activity and intra-Golgi localization

alpha2,6-Sialyltransferase (ST6Gal I) functions in the Golgi to terminally sialylate the N-linked oligosaccharides of glycoproteins. Interestingly, rat ST6Gal I is expressed as two isoforms, STtyr and STcys, that differ by a single amino acid in their catalytic domains. In this article, our goal was to evaluate more carefully possible differences in the catalytic activity and intra-Golgi localization of the two isoforms that had been suggested by earlier work. Using soluble recombinant STtyr and STcys enzymes and three asialoglycoprotein substrates for in vitro analysis, we found that the STcys isoform was somewhat more active than the STtyr isoform. However, we found no differences in isoform substrate choice when these proteins were expressed in Chinese hamster ovary cells, and sialylated substrates were detected by lectin blotting. Immuno-fluorescence and immunoelectron microscopy revealed differences in the relative levels of the isoforms found in the endoplasmic reticulum (ER) and Golgi of transiently expressing cells but similar intra-Golgi localization. STtyr was restricted to the Golgi in most cells, and STcys was found in both the ER and Golgi. The ER localization of STcys was especially pronounced with a C-terminal V5 epitope tag. Ultrastructural and deconvolution studies of immunostained HeLa cells expressing STtyr or STcys showed that within the Golgi both isoforms are found in medial-trans regions. The similar catalytic activities and intra-Golgi localization of the two ST6Gal I isoforms suggest that the particular isoform expressed in specific cells and tissues is not likely to have significant functional consequences.     

Detection of the Neu5 Ac (alpha 2,3) Gal (beta 1,4) GlcNAc sequence with the leukoagglutinin from Maackia amurensis: light and electron microscopic demonstration of differential tissue expression of terminal sialic acid in alpha 2,3- and alpha 2,6-linkage

The Maackia amurensis leukoagglutinin has been shown to react specifically with the Neu5Ac (alpha 2,3) Gal sequence of asparagine-linked complex type oligosaccharides. We report here the preparation of Maackia amurensis lectin-gold complexes and their application for light and electron microscopic detection of the Neu5 Ac (alpha 2,3) Gal sequence in various tissues. The use of the lectin directly gold labeled was superior to a two-step cytochemical affinity technique using a fetuin-gold complex. The Maackia amurensis lectin-gold staining was inhibited by pre-incubation of the lectin-gold complexes with 50 mM alpha 2,3 sialyllactose, whereas alpha 2,6 sialyllactose up to concentrations of 1 M had no effect, thus demonstrating the high specificity of the histochemical staining. In addition to N-glycanase-sensitive asparagine-linked oligosaccharides, beta-elimination-sensitive serine/threonine-linked oligosaccharides could be detected. Data are presented which show that cellular staining patterns obtained with Maackia amurensis lectin-gold complexes may differ from those with elderberry bark lectin-gold, which detects the Neu5 Ac (alpha 2,6) Gal/GalN Ac sequence. Electron microscopic double labeling for direct study of the differential distribution of the Neu5 Ac (alpha 2,3) Gal and Neu5 Ac (alpha 2,6) Gal sequences is reported. Therefore, the availability of two sialic acid binding lectins with different linkage specificity for histochemistry provides the first opportunity to study tissue and cell type expression of these terminal sequences of glycoproteins.     

An N-linked glycoprotein with alpha(2,3)-linked sialic acid is a receptor for BK virus

BK virus (BKV) is a common human polyomavirus infecting >80% of the population worldwide. Infection with BKV is asymptomatic, but reactivation in renal transplant recipients can lead to polyomavirus-associated nephropathy. In this report, we show that enzymatic removal of alpha(2,3)-linked sialic acid from cells inhibited BKV infection. Reconstitution of asialo cells with alpha(2,3)-specific sialyltransferase restored susceptibility to infection. Inhibition of N-linked glycosylation with tunicamycin reduced infection, but inhibition of O-linked glycosylation did not. An O-linked-specific alpha(2,3)-sialyltransferase was unable to restore infection in asialo cells. Taken together, these data indicate that an N-linked glycoprotein containing alpha(2,3)-linked sialic acid is a critical component of the cellular receptor for BKV.