Philippe Delannoy

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.

The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach.

The animal sialyltransferases are Golgi type II transmembrane glycosyltransferases. Twenty distinct sialyltransferases have been identified in both human and murine genomes. These enzymes catalyze transfer of sialic acid from CMP-Neu5Ac to the glycan moiety of glycoconjugates. Despite low overall identities, they share four conserved peptide motifs [L (large), S (small), motif III, and motif VS (very small)] that are hallmarks for sialyltransferase identification. We have identified 155 new putative genes in 25 animal species, and we have exploited two lines of evidence: (1) sequence comparisons and (2) exon-intron organization of the genes. An ortholog to the ancestor present before the split of ST6Gal I and II subfamilies was detected in arthropods. An ortholog to the ancestor present before the split of ST6GalNAc III, IV, V, and VI subfamilies was detected in sea urchin. An ortholog to the ancestor present before the split of ST3Gal I and II subfamilies was detected in ciona, and an ortholog to the ancestor of all the ST8Sia was detected in amphioxus. Therefore, single examples of the four families (ST3Gal, ST6Gal, ST6GalNAc, and ST8Sia) have appeared in invertebrates, earlier than previously thought, whereas the four families were all detected in bony fishes, amphibians, birds, and mammals. As previously hypothesized, sequence similarities among sialyltransferases suggest a common genetic origin, by successive duplications of an ancestral gene, followed by divergent evolution. Finally, we propose predictions on these invertebrates sialyltransferase-related activities that have not previously been demonstrated and that will ultimately need to be substantiated by protein expression and enzymatic activity assays.

Tumour-associated carbohydrate antigens in breast cancer

Glycosylation changes that occur in cancer often lead to the expression of tumour-associated carbohydrate antigens. In breast cancer, these antigens are usually associated with a poor prognosis and a reduced overall survival. Cellular models have shown the implication of these antigens in cell adhesion, migration, proliferation and tumour growth. The present review summarizes our current knowledge of glycosylation changes (structures, biosynthesis and occurrence) in breast cancer cell lines and primary tumours, and the consequences on disease progression and aggressiveness. The therapeutic strategies attempted to target tumour-associated carbohydrate antigens in breast cancer are also discussed.

Current trends in the structure–activity relationships of sialyltransferases

Sialyltransferases (STs) represent an important group of enzymes that transfer N-acetylneuraminic acid (Neu5Ac) from cytidine monophosphate-Neu5Ac to various acceptor substrates. In higher animals, sialylated oligosaccharide structures play crucial roles in many biological processes but also in diseases, notably in microbial infection and cancer. Cell surface sialic acids have also been found in a few microorganisms, mainly pathogenic bacteria, and their presence is often associated with virulence. STs are distributed into five different families in the CAZy database (http://www.cazy.org/). On the basis of crystallographic data available for three ST families and fold recognition analysis for the two other families, STs can be grouped into two structural superfamilies that represent variations of the canonical glycosyltransferase (GT-A and GT-B) folds. These two superfamilies differ in the nature of their active site residues, notably the catalytic base (a histidine or an aspartate residue). The observed structural and functional differences strongly suggest that these two structural superfamilies have evolved independently.

Consequences of the expression of sialylated antigens in breast cancer.

Changes in cell surface glycosylation are common modifications that occur during oncogenesis, leading to the over-expression of tumour-associated carbohydrate antigens (TACA). Most of these antigens are sialylated and the increase of sialylation is a well-known feature of transformed cells. In breast cancer, expression of TACA such as sialyl-Lewis(x) or sialyl-Tn is usually associated with a poor prognosis and a decreased overall survival of patients. However, the specific role of these sialylated antigens in breast tumour development and aggressiveness is not clearly understood. These glycosylation changes result from the modification of the expression of genes encoding specific glycosyltransferases involved in glycan biosynthesis and the level of expression of sialyltransferase genes has been proposed to be a prognostic marker for the follow-up of breast cancer patients. Several human cellular models have been developed in order to explain the mechanisms by which carbohydrate antigens can reinforce breast cancer progression and aggressiveness. TACA expression is associated with changes in cell adhesion, migration, proliferation and tumour growth. In addition, recent data on glycolipid biosynthesis indicate an important role of G(D3) synthase expression in breast cancer progression. The aim of this review is to summarize our current knowledge of sialylation changes that occur in breast cancer and to describe the cellular models developed to analyze the consequences of these changes on disease progression and aggressiveness.

Stable expression of sialyl-Tn antigen in T47-D cells induces a decrease of cell adhesion and an increase of cell migration

SummarySialyl-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: GalNAc α2,6-sialyltransferase: ST6GalNAc I, which catalyzes the transfer of a sialic acid residue in α2,6-linkage to the GalNAcα1-O-Ser/Thr structure. The resulting disaccharide (Neu5Acα2-6GalNAcα1-O-Ser/Thr) cannot be further elongated and sialyl-Tn expression results therefore in a shortening of the O-glycan chains. However, usual breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn antigen. We have previously shown that stable transfection of MDA-MB-231 cells with the hST6GalNAc I cDNA induces the sialyl-Tn antigen expression at the cell surface and leads to a decreased cell growth and an increased cell migration. We describe herein the generation of new T47-D clones expressing sialyl-Tn antigen after hST6GalNAc I cDNA stable transfection. sialyl-Tn antigen is carried by several high molecular weight membrane bound O-glycoproteins, including MUC1. We show that sialyl-Tn expression induces a decrease of cell growth and adhesion, and an increase of cell migration in sialyl-Tn positive clones compared to mock transfected cells. These observations show that the alteration of the O-glycans pattern is sufficient to modify the biological features of cancer cells. These T47-D sialyl-Tn expressing clones might allow further in vivo investigation to determine precisely the impact of such O-glycosylation modifications on breast cancer development.

Involvement of glycosylation in the intracellular trafficking of glycoproteins in polarized epithelial cells

The surface of epithelial cells is composed of apical and basolateral domains with distinct structure and function. This polarity is maintained by specific sorting mechanisms occurring in the Trans-Golgi Network. Peptidic signals are responsible for the trafficking via clathrin-coated vesicles by means of an interaction with an adaptor complex (AP). The basolateral targeting is mediated by AP-1B, which is specifically expressed in epithelial cells. In contrast, the apical targeting is proposed to occur via apical raft carriers. It is thought that apically targeted glycoproteins contain glycan signals that would be responsible for their association with rafts and for apical targeting. However, the difficulty in terms of acting specifically on a single step of glycosylation did not allow one to identify such a specific signal. The complete inhibition of the processing of N-glycans by tunicamycin often results in an intracellular accumulation of unfolded proteins in the Golgi. Similarly, inhibition of O-glycosylation can be obtained by competitive substrates which gave a complex pattern of inhibition. Therefore, it is still unknown if glycosylation acts in an indirect manner, i.e. by modifying the folding of the protein, or in a specific manner, such as an association with specific lectins.

IL-6 and IL-8 increase the expression of glycosyltransferases and sulfotransferases involved in the biosynthesis of sialylated and/or sulfated Lewisx epitopes in the human bronchial mucosa

Bronchial mucins from patients suffering from CF (cystic fibrosis) exhibit glycosylation alterations, especially increased amounts of the sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc-R) and 6-sulfo-sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3][SO(3)H-6]GlcNAc-R) terminal structures. These epitopes are preferential receptors for Pseudomonas aeruginosa, the bacteria responsible for the chronicity of airway infection and involved in the morbidity and early death of CF patients. However, these glycosylation changes cannot be directly linked to defects in CFTR (CF transmembrane conductance regulator) gene expression since cells that secrete airway mucins express no or very low amounts of the protein. Several studies have shown that inflammation may affect glycosylation and sulfation of various glycoproteins, including mucins. In the present study, we show that incubation of macroscopically healthy fragments of human bronchial mucosa with IL-6 (interleukin-6) or IL-8 results in a significant increase in the expression of alpha1,3/4-fucosyltransferases [FUT11 (fucosyltransferase 11 gene) and FUT3], alpha2-6- and alpha2,3-sialyltransferases [ST3GAL6 (alpha2,3-sialyltransferase 6 gene) and ST6GAL2 (alpha2,6-sialyltransferase 2 gene)] and GlcNAc-6-O-sulfotransferases [CHST4 (carbohydrate sulfotransferase 4 gene) and CHST6] mRNA. In parallel, the amounts of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes at the periphery of high-molecular-mass proteins, including MUC4, were also increased. In conclusion, our results indicate that IL-6 and -8 may contribute to the increased levels of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes on human airway mucins from patients with CF.

Expression of Sialyl-Tn antigen in breast cancer cells transfected with the human CMP-Neu5Ac: GalNAc α2,6-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 α2,6-linkage to GalNAcα-O-Ser/Thr. This reaction is mainly catalyzed by a CMP-Neu5Ac GalNAc α2,6-sialyltransferase: ST6GalNAc I. In order to generate STn-positive breast cancer cells, we have cloned a cDNA encoding the full-lenght 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 β1,3-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.

Sialyltransferases functions in cancers.

Abnormally elevated levels of sialylated tumor associated carbohydrate antigens are frequently described at the surface of cancer cells and/or secreted in biological fluids. It is now well established that this over-expression may result from deregulation in sialyltransferases enzymatic activity involved in their biosynthesis, but the precise molecular mechanisms remain unknown. Twenty different human sialyltransferases preside to the sialylation of glycoconjugates, either glycolipids or glycoproteins. This review summarizes the current knowledge on human sialyltransferases implicated in the altered expression of sialylated tumor associated antigens, the molecular basis of their regulated expression in cancer cells and the various tools developed by researchers and clinicians for their study in pathological samples.