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.     

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.     

Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells

Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies.     

High-level expression of human glycosyltransferases in insect cells as biochemically active form

cDNAs, encoding human beta1,4-galactosyltransferase (hGalT I, EC 2.4.1.22), human Galbeta1,3(4)-GlcNAc alpha2,3-sialyltransferase (hST3GalIII, EC 2.4.99), and human Galbeta1,4-GlcNAc alpha2,6-sialyltransferase (hST6Gal I, EC 2.4.99.1), were cloned from human cell lines. In order to express these glycosyltransferases as secreted form in insect cells, cDNAs were inserted into a novel baculovirus transfer vector equipped with the mouse IgM signal peptide and IgG binding domain of the Staphylococcus aureus protein A as an N-terminal fusion partner. About 14 mg hGalT I, 8 mg hST3GalIII, and 6.4 mg hST6Gal I were purified from 1 liter of recombinant baculovirus infected insect cell culture media. The specific activities of recombinant hGalT I and hST6Gal I were determined as 0.65 and 1.6 U/mg protein, respectively. These results indicated that the recombinant hGalT I and hST6Gal I retained enzyme activities at similar level to those of the authentic one although they were fused with the IgG binding domain at the N-terminus. Taken together, the mouse IgM signal peptide and IgG binding domain of the protein A could be efficiently used as an N-terminus fusion partner for the over-expression of heterologous proteins in insect cells.     

Recognition of cell surface acceptors by two human alpha-2,6-sialyltransferases produced in CHO cells

The action of sialyltransferases (STs) on cell surface glycoconjugates is a key process in shaping cell phenotype in a variety of cells mostly involved in migratory and adhesive pathways. The factors determining cell-specific pattern of glycosylation are so far poorly understood. Most STs are resident proteins of the Golgi apparatus, where acceptors are sialylated while they are in transit to the cell surface. To identify putative structural features that may account for their acceptor preference, we analyzed 53 cloned animal and human STs. We could identify conserved regions and peptide motifs representative of ST subfamilies, located at the C-terminal end of the hypervariable region upstream from the L-sialyl motif. Residues 93-100 in human ST6Gal I (hST6Gal I) were shown to be crucial for enzymatic activity when deleted and expressed in CHO cells. The Delta100 hST6Gal I mutant protein was fully recognized by polyclonal anti-hST6Gal I antibodies and followed the intracellular secretory pathway. This indicated that the conserved QVWxKDS sequence is essential for the whole catalytic domain to acquire a biologically active conformation. When full-length epitope-tagged hST6Gal I and hST6GalNAc I constructs were transfected in CHO cells, the alpha-2,6 sialylated glycotope was found to be largely restricted to intracellular resident acceptors and enzymatic activity based on fluorescent lectin staining. In contrast, both enzymes deprived of their membrane anchor and part of the hypervariable region but still possessing the conserved domains exhibited a very efficient transfer of sialic acid to cell surface glycoconjugates. Colocalization of the ST6Gal I mutant proteins with early and late Golgi markers such as giantin or rab6 proteins confirmed that soluble STs migrate forward in these subcompartments where they can act upon newly synthesized acceptors and follow the secretory pathway. It is thus concluded that downstream from the transmembrane domain, native STs possess peptide sequences that allow them to sialylate glycoprotein acceptors selectively along their transit within Golgi stacks.     

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.     

Combinatorial PCR approach to homology-based cloning: Cloning and expression of mouse and human GM3-synthase

GM3-synthase, also known as sialyltransferase I (ST-I), catalyzes the transfer of a sialic acid residue from CMP-sialic acid onto lactosylceramide to form ganglioside GM3. In order to clone this enzyme, as well as other sialyltransferases, we developed an approach that we termed combinatorial PCR. In this approach, degenerate primers were designed on the basis of conserved sequence motifs of the ST3 family of sialyltransferases (STs). The nucleotide sequence of the primers was varied to cover all amino acid variations occurring in each motif. In addition, in some primers the sequence was varied to cover possible homologous substitutions that are absent in the available motifs. A panel of cDNA from 12 mouse and 8 human tissues was used to enable cloning of tissue- and stage-specific sialyltransferases. Using this approach, the fragments of 11 new putative sialyltransferases were isolated and sequenced so far. Analysis of the expression pattern of a particular sialyltransferase across the panel of cDNA from the different tissues provided information about the tissue specificity of ST expression. We chose two new ubiquitously expressed human and mouse STs to clone full-length copies and to assay for GM3-synthase activity. One of the STs, which exhibited the highest homology to ST3 Gal III, showed activity toward lactosylceramide (LacCer) and was termed ST3 Gal V according to the suggested nomenclature [1]. The other ubiquitously expressed sialyltransferase was termed ST3Gal VI. All isolated sialyltransferases were screened for alternatively spliced forms (ASF). Such forms were found for both human ST3Gal V and ST3Gal VI in human fetal brain cDNA library. The detailed cloning strategy, functional assay, and full length cDNA and protein sequences of GM3 synthase (ST3Gal V, or ST-I) are presented.     

Cell surface n-acetylneuraminic acid alpha2,3-galactoside-dependent intercellular adhesion of human colon cancer cells

Sialoglycans on the cell surface of human colon cancer (HCC) cells have been implicated in cellular adhesion and metastasis. To clarify the role of N-acetylneuraminic acid (NeuAc) linked alpha2,3 to galactose (Gal) on the surface of HCC cells, we studied the intercellular adhesion of HCC cell lines expressing increasing NeuAcalpha2,3Gal-R. Our model system consisted of the HCC SW48 cell line, which inherently possesses low levels of cell surface alpha2,3 and alpha2,6 sialoglycans. To generate SW48 clonal variants with elevated cell surface NeuAcalpha2,3Gal-R linkages, we transfected the expression vector, pcDNA3, containing either rat liver cDNA encoding Galbeta1,3(4)GlcNAc alpha2,3 sialyltransferase (ST3Gal III) or human placental cDNA encoding Galbeta1,3GalNAc/Galbeta1,4GlcNAc alpha2,3 sialyltransferase (ST3Gal IV) into SW48 cells. Selection of neomycin-resistant clones (600 microgram G418/ml) having a higher percentage of cells expressing NeuAcalpha2,3Gal-R (up to 85% positive Maackia amurenis agglutinin staining compared with 30% for wild type cells) was performed. These ST3Gal III and ST3Gal IV clonal variants demonstrated increased adherence to IL-1beta-activated human umbilical vein endothelial cells (HUVEC) (up to 90% adherent cells compared with 63% for wild type cells). Interestingly, ST3Gal III and ST3Gal IV clonal variants also bound non-activated HUVEC up to 4-fold more effectively than wild type cells. Cell surface NeuAcalpha2,3Gal-R expression within the various SW48 clonal variants correlated directly with increased adhesion to HUVEC (r=0.84). Using HCC HT-29 cells, which express high levels of surface NeuAcalpha2,3Gal-R, addition of synthetic sialyl, sulfo or GalNAc Lewis X structures were found to specifically inhibit intercellular adhesion. At 1.0mM, NeuAcalpha2,3Galbeta1,3(Fucalpha1, 4)GlcNAc-OH and Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,6(SE-6Galbeta1++ +, 3)GalNAcalpha1-O-methyl inhibited HT-29 cell adhesion to IL-1beta-stimulated HUVEC by 100% and 68%, respectively. GalNAcbeta1, 4(Fucalpha1,3)GlcNAcbeta1-O-methyl and GalNAcbeta1,4(Fucalpha1, 3)GlcNAcbeta1,6Manalpha1,6Manbeta1-0-C30H61, however, did not possess inhibitory activity. In conclusion, these studies demonstrated that cell surface NeuAcalpha2,3Gal-R expression is involved in HCC cellular adhesion to HUVEC. These specific carbohydrate-mediated intercellular adhesive events may play an important role in tumor angiogenesis, metastasis and growth control.     

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.     

Computational characterisation of the interactions between human ST6Gal I and transition‐state analogue inhibitors: insights for inhibitor design

Human β‐galactoside α‐2,6‐sialyltransferase I (hST6Gal I) catalyses the synthesis of sialylated glycoconjugates involved in cell–cell interactions. Overexpression of hST6Gal I is observed in many different types of cancers, where it promotes metastasis through altered cell surface sialylation. A wide range of sialyltransferase (ST) inhibitors have been developed based on the natural donor, cytidine 5′‐monophosphate N‐acetylneuraminic acid (CMP‐Neu5Ac). Of these, analogues that are structurally similar to the transition state exhibit the highest inhibitory activity. In order to design inhibitors that are readily accessible synthetically and with favourable pharmacokinetic properties, an investigation of the replacement of the charged phosphodiester‐linker, present in many ST inhibitors, with a potential neutral isostere such as a carbamate or a 1,2,3‐triazole has been undertaken. To investigate this, molecular docking and molecular dynamics simulations were performed. These simulations provided an insight into the binding mode of previously reported phosphodiester‐linked ST inhibitors and demonstrated that targeting the proposed sialyl acceptor site is a viable option for producing selective inhibitors. The potential for a carbamate‐ or triazole‐linker as an isosteric replacement for the phosphodiester in transition‐state analogue ST inhibitors was established using molecular docking. Molecular dynamics simulations of carbamate‐ and phosphodiester‐linked compounds revealed that both classes exhibit consistent interactions with hST6Gal I. Overall, the results obtained from this study provide a rationale for synthetic and biological evaluation of triazole‐ and carbamate‐linked transition‐state analogue ST inhibitors as potential new antimetastatic agents. Copyright © 2015 John Wiley & Sons, Ltd.     

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.