Impact of sialyltransferase ST6GAL1 overexpression on different colon cancer cell types.

Cancer-associated glycan structures can be both tumor markers and engines of disease progression. The structure Siaα2,6Galß1,4GlcNAc (Sia6LacNAc), synthesized by sialyltransferase ST6GAL1, is a cancer-associated glycan. Although ST6GAL1/Sia6LacNAc are often overexpressed in colorectal cancer (CRC), their biological and clinical significance remains unclear. To get insights into the clinical relevance of ST6GAL1 expression in CRC, we interrogated The Cancer Genome Atlas with mRNA expression data of hundreds of clinically characterized CRC and normal samples. We found an association of low ST6GAL1 expression with microsatellite instability (MSI), BRAF mutations and mucinous phenotype but not with stage, response to therapy and survival. To investigate the impact of ST6GAL1 expression in experimental systems, we analyzed the transcriptome and the phenotype of the CRC cell lines SW948 and SW48 after retroviral transduction with ST6GAL1 cDNA. The two cell lines display the two main pathways of CRC transformation: chromosomal instability and MSI, respectively. Constitutive ST6GAL1 expression induced much deeper transcriptomic changes in SW948 than in SW48 and affected different genes in the two cell lines. ST6GAL1 expression affected differentially the tyrosine phosphorylation induced by hepatocyte growth factor, the ability to grow in soft agar, to heal a scratch wound and to invade Matrigel in the two cell lines. These results indicate that the altered expression of a cancer-associated glycosyltransferase impacts the gene expression profile, as well as the phenotype, although in a cancer subtype-specific manner.

Oxidative damage and response to Bacillus Calmette-Guérin in bladder cancer cells expressing sialyltransferase ST3GAL1.

BACKGROUND: Treatment with Bacillus Calmette-Guérin (BCG) is the gold standard adjuvant immunotherapy of non-muscle invasive bladder cancer (NMIBC), although it fails in one third of the patients. NMIBC expresses two tumor-associated O-linked carbohydrates: the disaccharide (Galß1,3GalNAc) Thomsen-Friedenreich (T) antigen, and its sialylated counterpart (Siaα2,3Galß1,3GalNAc) sialyl-T (sT), synthesized by sialyltransferase ST3GAL1, whose roles in BCG response are unknown. METHODS: The human bladder cancer (BC) cell line HT1376 strongly expressing the T antigen, was retrovirally transduced with the ST3GAL1 cDNA or with an empty vector, yielding the cell lines HT1376sT and HT1376T, that express, respectively, either the sT or the T antigens. Cells were in vitro challenged with BCG. Whole gene expression was studied by microarray technology, cytokine secretion was measured by multiplex immune-beads assay. Human macrophages derived from blood monocytes were challenged with the secretome of BCG-challenged BC cells. RESULTS: The secretome from BCG-challenged HT1376sT cells induced a stronger macrophage secretion of IL-6, IL-1ß, TNFα and IL-10 than that of HT1376T cells. Transcriptomic analysis revealed that ST3GAL1 overexpression and T/sT replacement modulated hundreds of genes. Several genes preserving genomic stability were down-regulated in HT1376sT cells which, as a consequence, displayed increased sensitivity to oxidative damage. After BCG challenge, the transcriptome of HT1376sT cells showed higher susceptibility to BCG modulation than that of HT1376T cells. CONCLUSIONS: High ST3GAL1 expression and T/sT replacement in BCG challenged-BC cancer cells induce a stronger macrophage response and alter the gene expression towards genomic instability, indicating a potential impact on BC biology and patient's response to BCG.

Glycosylation as a Main Regulator of Growth and Death Factor Receptors Signaling.

Glycosylation is a very frequent and functionally important post-translational protein modification that undergoes profound changes in cancer. Growth and death factor receptors and plasma membrane glycoproteins, which upon activation by extracellular ligands trigger a signal transduction cascade, are targets of several molecular anti-cancer drugs. In this review, we provide a thorough picture of the mechanisms bywhich glycosylation affects the activity of growth and death factor receptors in normal and pathological conditions. Glycosylation affects receptor activity through three non-mutually exclusive basic mechanisms: (1) by directly regulating intracellular transport, ligand binding, oligomerization and signaling of receptors; (2) through the binding of receptor carbohydrate structures to galectins, forming a lattice thatregulates receptor turnover on the plasma membrane; and (3) by receptor interaction with gangliosides inside membrane microdomains. Some carbohydrate chains, for example core fucose and ß1,6-branching, exert a stimulatory effect on all receptors, while other structures exert opposite effects on different receptors or in different cellular contexts. In light of the crucial role played by glycosylation in the regulation of receptor activity, the development of next-generation drugs targeting glyco-epitopes of growth factor receptors should be considered a therapeutically interesting goal.

Expression of sialyl-Tn sugar antigen in bladder cancer cells affects response to Bacillus Calmette Guérin (BCG) and to oxidative damage.

The sialyl-Tn (sTn) antigen is an O-linked carbohydrate chain aberrantly expressed in bladder cancer (BC), whose biosynthesis is mainly controlled by the sialyltransferase ST6GALNAC1. Treatment with Bacillus Calmette-Guérin (BCG) is the most effective adjuvant immunotherapy for superficial BC but one third of the patients fail to respond. A poorly understood correlation between the expression of sTn and BC patient's response to BCG was previously observed. By analyzing tumor tissues, we showed that patients with high ST6GALNAC1 and IL-6 mRNA expression were BCG responders. To investigate the role of sTn in BC cell biology and BCG response, we established the cell lines MCRsTn and MCRNc by retroviral transduction of the BC cell line MCR with the ST6GALNAC1 cDNA or with an empty vector, respectively. Compared with MCRNc, BCG-stimulated MCRsTn secreted higher levels of IL-6 and IL-8 and their secretome induced a stronger IL-6, IL-1ß, and TNFα secretion by macrophages, suggesting the induction of a stronger inflammatory response. Transcriptomic analysis of MCRNc and MCRsTn revealed that ST6GALNAC1/sTn expression modulates hundreds of genes towards a putative more malignant phenotype and down-regulates several genes maintaining genomic stability. Consistently, MCRsTn cells displayed higher H2O2 sensitivity. In MCRsTn,, BCG challenge induced an increased expression of several regulatory non coding RNA genes. These results indicate that the expression of ST6GALNAC1/sTn improves the response to BCG therapy by inducing a stronger macrophage response and alters gene expression towards malignancy and genomic instability, increasing the sensitivity of BC cells to the oxidizing agents released by BCG.

Identification of novel plasma glycosylation-associated markers of aging.

The pro- or anti-inflammatory activities of immunoglobulins G (IgGs) are controlled by the structure of the glycan N-linked to Asn297 of their heavy chain. The age-associated low grade inflammation (inflammaging) is associated with increased plasmatic levels of agalactosylated IgGs terminating with N-acetylglucosamine (IgG-G0) whose biogenesis has not been fully explained. Although the biosynthesis of glycans is in general mediated by glycosyltransferases associated with internal cell membranes, the extracellular glycosylation of circulating glycoproteins mediated by plasmatic glycosyltransferases has been recently demonstrated. In this study we have investigated the relationship between plasmatic glycosyltransferases, IgG glycosylation and inflammatory and aging markers. In cohorts of individuals ranging from infancy to centenarians we determined the activity of plasmatic ß4 galactosyltransferase(s) (B4GALTs) and of α2,6-sialyltransferase ST6GAL1, the glycosylation of IgG, the GlycoAge test (a glycosylation-based marker of aging) and the plasma level of inflammatory and liver damage markers. Our results show that: 1) plasmatic B4GALTs activity is a new marker of aging, showing a linear increase throughout the whole age range. 2) plasmatic ST6GAL1 was high only in children and in people above 80, showing a quadratic relationship with age. 3) Neither plasmatic glycosyltransferase correlated with markers of liver damage. 4) plasmatic ST6GAL1 showed a positive association with acute phase proteins in offspring of short lived parents, but not in centenarians or in their offspring. 5) Although the glycosylation of IgGs was not correlated with the level of the two plasmatic glycosyltransferases, it showed progressive age-associated changes consistent with a shift toward a pro-inflammatory glycotype.

Sialosignaling: sialyltransferases as engines of self-fueling loops in cancer progression.

BACKGROUND: Glycosylation is increasingly recognized as one of the most relevant postranslational modifications. Sialic acids are negatively charged sugars which frequently terminate the carbohydrate chains of glycoproteins and glycolipids. The addition of sialic acids is mediated by sialyltransferases, a family of glycosyltransferases with a crucial role in cancer progression. SCOPE OF THE REVIEW: To describe the phenotypic and clinical implications of altered expression of sialyltransferases and of their cognate sialylated structures in cancer. To propose a unifying model of the role of sialyltransferases and sialylated structures on cancer progression. MAJOR CONCLUSIONS: We first discuss the biosynthesis and the role played by the major cancer-associated sialylated structures, including Thomsen-Friedenreich-associated antigens, sialyl Lewis antigens, α2,6-sialylated lactosamine, polysialic acid and gangliosides. Then, we show that altered sialyltransferase expression in cancer, consequence of genetic and epigenetic alterations, generates a flow of information toward the membrane through the biosynthesis of aberrantly sialylated molecules (inside-out signaling). In turn, the presence of aberrantly sialylated structures on cell membrane receptors generates a flow of information toward the nucleus, which can exacerbate the neoplastic phenotype (outside-in signaling). We provide examples of self-fueling loops generated by these flows of information. GENERAL SIGNIFICANCE: Sialyltransferases have a wide impact on the biology of cancer and can be the target of innovative therapies. Our unified view provides a conceptual framework to understand the impact of altered glycosylation in cancer.

B4GALNT2 gene expression controls the biosynthesis of Sda and sialyl Lewis X antigens in healthy and cancer human gastrointestinal tract.

The histo blood group carbohydrate Sd(a) antigen and its cognate biosynthetic enzyme B4GALNT2 show the highest level of expression in normal colon. Their dramatic down regulation previously observed in colon cancer tissues could play a role in the concomitant elevation of the selectin ligand sLe(x), involved in metastasis. However, down regulation of sLe(x) expression by B4GALNT2 has been so far demonstrated in vitro, but not in tissues. The human B4GALNT2 gene specifies at least two transcripts, diverging in the first exon, never studied in normal and cancer tissues. The long form contains a 253 nt exon 1L; the short form contains a 38 nt exon 1S. Using qPCR, we showed that cell lines and normal or cancerous colon, expressed almost exclusively the short form, while the long form was mainly expressed by the embryonic colon fibroblast cell line CCD112CoN. Immunochemistry approaches using colon cancer cells permanently expressing either B4GALNT2 cDNAs as controls, led to the observation of several protein isoforms in human normal and cancerous colon, and cell lines. We showed that tissues expressing B4GALNT2 protein isoforms were able to induce Sd(a) and to inhibit sLe(x) expression; both of which are expressed mainly on PNGase F-insensitive carbohydrate chains. Concomitant expression of B4GALNT2 and siRNA-mediated inhibition of FUT6, the major fucosyltransferase involved in sLe(x) synthesis in colon, resulted in a cumulative inhibition of sLe(x). In normal colon samples a significant relationship between sLe(x) expression and the ratio between FUT6/B4GALNT2 activities exists, demonstrating for the first time a role for B4GALNT2 in sLe(x) inhibition in vivo.

The expanding roles of the Sd(a)/Cad carbohydrate antigen and its cognate glycosyltransferase B4GALNT2.

BACKGROUND: The histo-blood group antigens are carbohydrate structures present in tissues and body fluids, which contribute to the definition of the individual immunophenotype. One of these, the Sd(a) antigen, is expressed on the surface of erythrocytes and in secretions of the vast majority of the Caucasians and other ethnic groups. SCOPE OF REVIEW: We describe the multiple and unsuspected aspects of the biology of the Sd(a) antigen and its biosynthetic enzyme ß1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) in various physiological and pathological settings. MAJOR CONCLUSIONS: The immunodominant sugar of the Sd(a) antigen is a ß1,4-linked N-acetylgalactosamine (GalNAc). Its cognate glycosyltransferase B4GALNT2 displays a restricted pattern of tissue expression, is regulated by unknown mechanisms - including promoter methylation, and encodes at least two different proteins, one of which with an unconventionally long cytoplasmic portion. In different settings, the Sd(a) antigen plays multiple and unsuspected roles. 1) In colon cancer, its dramatic down-regulation plays a potential role in the overexpression of sialyl Lewis antigens, increasing metastasis formation. 2) It is involved in the lytic function of murine cytotoxic T lymphocytes. 3) It prevents the development of muscular dystrophy in various dystrophic murine models, when overexpressed in muscular fibers. 4) It regulates the circulating half-life of the von Willebrand factor (vWf), determining the onset of a bleeding disorder in a murine model. GENERAL SIGNIFICANCE: The expression of the Sd(a) antigen has a wide impact on the physiology and the pathology of different biological systems.

Apoptotic cells selectively uptake minor glycoforms of vitronectin from serum.

Apoptosis profoundly alters the carbohydrate layer coating the membrane of eukaryotic cells. Previously we showed that apoptotic cells became reactive with the α2,6-sialyl-specific lectin from Sambucus nigra agglutinin (SNA), regardless of their histological origin and the nature of the apoptotic stimulus. Here we reveal the basis of the phenomenon by showing that in apoptotic cancer cell lines SNA reactivity was mainly associated with a 67 kDa glycoprotein which we identified by MALDI-TOF/TOF and immunoblot analysis as bovine vitronectin (bVN). bVN was neither present in non-apoptotic cells, nor in cells induced to apoptosis in serum-free medium, indicating that its uptake from the cell culture serum occurred only during apoptosis. The bVN molecules associated with apoptotic cancer cell lines represented minor isoforms, lacking the carboxyterminal sequence and paradoxically containing a few α2,6-linked sialic acid residues. Despite their poor α2,6-sialylation, these bVN molecules were sufficient to turn apoptotic cells to SNA reactivity, which is a late apoptotic event occurring in cells positive to both annexin-V and propidium iodide. Unlike in cancer cell lines, the major bVN form taken up by apoptotic neutrophils and mononuclear cells was a 80 kDa form. In apoptotic SW948 cells we also detected the α2,6-sialylated forms of the stress-70 mitochondrial precursor (mortalin) and of tubulin-ß2C. These data indicate that the acquisition of vitronectin isoforms from the environment is a general, although cell specific phenomenon, potentially playing an important role in post-apoptotic events and that the α2,6-sialylation of intracellular proteins is a new kind of posttranslational modification associated with apoptosis.

Mechanisms of cancer-associated glycosylation changes.

Cell membrane glycoconjugates undergo characteristic changes as a consequence of neoplastic transformation. The cancer-associated carbohydrate structures play key roles in cancer progression by altering the cell-cell and cell-environment interactions. In this review, we will discuss some of the most relevant cancer-associated carbohydrate structures, including the ß1,6-branching of N-linked chains, the sialyl Lewis antigens, the α2,6-sialylated lactosamine, the Thomsen-Friedenreich-related antigens and gangliosides. We will describe the mechanisms leading to the expression of these structures and their interactions with sugar binding molecules, such as selectins and galectins. Finally, we will discuss how the glycosylation machinery of the cell is controlled by signal transduction pathways, epigenetic mechanisms and responds to hypoxia.

The biosynthesis of the selectin-ligand sialyl Lewis x in colorectal cancer tissues is regulated by fucosyltransferase VI and can be inhibited by an RNA interference-based approach.

Sialyl Lewis x (sLex) is a selectin ligand whose overexpression in epithelial cancers mediates metastasis formation. The molecular basis of sLex biosynthesis in colon cancer tissues is still unclear. The prerequisite for therapeutic approaches aimed at sLex down-regulation in cancer, is the identification of rate-limiting steps in its biosynthesis. We have studied the role of α1,3-fucosyltransferases (Fuc-Ts) potentially involved in sLex biosynthesis in specimens of normal and cancer colon as well as in experimental systems. We found that: (i) in colon cancer, but not in normal mucosa where the antigen was poorly expressed, sLex correlated with a Fuc-T which, like Fuc-TVI, was active on 3'sialyllactosamine at a low concentration (Fuc-T(SLN)); (ii) competitive RT-PCR analysis revealed that the level of Fuc-T mRNA expression in both normal and cancer colon was Fuc-TVI>Fuc-TIII>Fuc-TIV; Fuc-TV and Fuc-TVII expression was negligible; (iii) sLex was expressed only by the gastrointestinal cell lines displaying both Fuc-TVI mRNA and Fuc-T(SLN) activity, but not by those expressing only Fuc-TIII mRNA; (iv) transfection with Fuc-TVI cDNA, but not with Fuc-TIII cDNA, induced sLex expression in gastrointestinal cell lines; (v) Fuc-TVI knock-down with specific siRNA induced down-regulation of Fuc-TVI mRNA and Fuc-T(SLN) activity and a dramatic inhibition of sLex expression. These data indicate that in colon cancer tissues Fuc-TVI is a key regulator of sLex biosynthesis which can be the target of RNA-interference-based gene knock-down approaches.

Exposure of alpha2,6-sialylated lactosaminic chains marks apoptotic and necrotic death in different cell types.

Many observations have reported glycosylation changes associated with apoptosis in different biological systems, although none of these has shown any general significance. In this work, we show that in cell lines from different histological origin, (colon, breast, pancreas, and bladder cancer) as well as in normal human and mice neutrophils, apoptosis is accompanied by the exposure of sugar chains recognized by the lectin from Sambucus nigra (SNA), specific for Sia alpha 2,6Gal/GalNAc structures. Also, cells undergoing primary necrosis induced by heat treatment (56 degrees C, 30 min) expose specifically binding sites for SNA. While this modification is recognized also by the lectin from the mushroom Polyporus squamosus, which is highly specific for alpha2,6-sialylated lactosamine, no significant changes were detected in the binding of lectins specific for other carbohydrate structures, such as those from Phaseolus vulgaris, Arachis hypogea, and Maackia amurensis. The binding of SNA to apoptotic/necrotic cells is inhibited by neuraminidase treatment and by alpha2,6-sialylated compounds. In apoptotic, but not in necrotic SW948 cells, SNA reactivity is specifically associated with 65, 69, and 87 kDa glycoproteins. The exposure of SNA-reactive chains by apoptotic/necrotic cells occurs also in cells not expressing sialyltransferases ST6Gal.1 or ST6Gal.2 and is largely independent of the presence of alpha2,6-sialylated lactosaminic chains on the surface of preapoptotic cells. In neutrophils from ST6Gal.1 knock-out mice, the apoptosis-related increase in SNA reactivity is reduced but not abolished. These data demonstrate that apoptosis and primary necrosis induce a specific glycosylation change independent of the cell type and nature of the stimulus.

Biosynthesis and expression of the Sda and sialyl Lewis x antigens in normal and cancer colon.

The carbohydrate determinants Sd(a) and sialyl Lewis x (sLex) both result from substitution of an alpha2,3-sialylated type 2 chain: the first with an N-acetylgalactosamine (GalNAc) beta1,4-linked to Gal and the second by an alpha1,3-linked fucose on N-acetylglucosamine. The Sd(a) antigen is synthesized by Sd(a) beta1,4-N-acetylgalactosaminyltransferase II (beta4GalNAcT-II), which is downregulated in colon cancer, whereas sLex is a cancer-associated antigen. In view of the possible competition between beta4GalNAcT-II and the fucosyltransferases (FucTs) synthesizing the sLex antigen, we investigated whether beta4GalNAcT-II acts as a negative regulator of sLex expression in colon cancer. beta4GalNAcT-II cDNA, when expressed in LS174T colon cancer cells, induces the expression of the Sd(a) antigen, a dramatic inhibition of sLex expression on cell membranes, and the replacement of sLex with the Sd(a) antigen on 290 kDa glycoproteins. Unexpectedly, in colorectal cancer specimens, beta4GalNAcT-II and sLex show a direct relation. The reasons appear to be (i) Sd(a) and sLex antigens are expressed by different glycoproteins of 340 and 290 kDa, respectively; (ii) the activity of alpha1,3-FucTs on 3'-sialyllactosamine parallels that of beta4GalNAcT-II; and (iii) both beta4GalNAcT-II and FucT activities parallel sLex expression. Quantitative reverse transcription-polymerase chain reaction analysis reveals that the transcripts of beta4GalNAcT-II and those of FucT-III and FucT-VII are positively correlated. These data indicate that in colon cancer tissues, the sLex antigen is regulated mainly by the total FucT activity on 3'-sialyllactosamine acceptors and that beta4GalNAcT-II can inhibit sLex expression in an experimental model, although not in colon cancer tissues.

Beta-galactoside alpha2,6-sialyltransferase and the sialyl alpha2,6-galactosyl-linkage in tissues and cell lines.

beta-Galactoside alpha2,6-sialyltransferase (ST6Gal.I) is the principal sialyltransferase responsible for the biosynthesis of the sialyl alpha2,6-galactosyl linkage. This enzyme and its cognate glycosidic structure are overexpressed in several malignancies and are related to cancer progression. The expression of the enzyme is regulated primarily through the expression of three principal mRNA species differing in the 5'-untranslated exons. The form known as YZ is considered associated with the basal expression of the gene, while forms H and X are specific to the liver and B-lymphocytes, respectively. The authors have studied the expression of ST6Gal.I activity by two different methods using a panel of human cancer cell lines: the expression of alpha2,6-sialylated sugar chains by the lectin from Sambucus nigra (SNA), and the expression of the different mRNA species by RT-PCR using oligonucleotide primers complementary to the isoform-specific regions. Very high levels of ST6Gal.I activity result in high levels of SNA reactivity and are associated with the expression of the H transcript in colon and liver cell lines, and of the X transcript in B cells.

Phenotypic changes induced by expression of beta-galactoside alpha2,6 sialyltransferase I in the human colon cancer cell line SW948.

Beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I), the enzyme which adds sialic acid in alpha2,6-linkage on lactosaminic termini of glycoproteins, is frequently overexpressed in cancer, but its relationship with malignancy remains unclear. In this study, we have investigated the phenotypic changes induced by the expression of alpha2,6-sialylated lactosaminic chains in the human colon cancer cell line SW948 which was originally devoid of ST6Gal.I. Clones derived from transfection with the ST6Gal.I cDNA were compared with untransfected cells and mock transfectants. The ST6Gal.I-expressing clones show (1) increased adherence to fibronectin and collagen IV but not to hyaluronic acid. Treatment with Clostridium perfrigens neuraminidase reduces the binding to fibronectin and collagen IV of ST6Gal.I-expressing cells but not that of ST6Gal.I-negative cells; (2) accumulation and more focal distribution of beta1 integrins on the cell surface; (3) different distribution of actin fibers; (4) flatter morphology and reduced tendency to multilayer growth; (5) improved ability to heal a scratch wound; (6) reduced ability to grow at the subcutaneous site of injection in nude mice. Our data suggest that the presence of alpha2,6-linked sialic acid on membrane glycoconjugates increases the binding to extracellular matrix components, resulting in a membrane stabilization of beta1 integrins, further strengthening the binding. This mechanism can provide a basis for the flatter morphology and the reduced tendency to multilayer growth, resulting in a more ordered tissue organization. These data indicate that in the cell line SW948, the effect of ST6Gal.I expression is consistent with the attenuation of the neoplastic phenotype.

Expression of beta-galactoside alpha2,6 sialyltransferase and of alpha2,6-sialylated glycoconjugates in normal human liver, hepatocarcinoma, and cirrhosis.

beta-Galactoside alpha2,6-sialyltransferase (ST6Gal.I) mediates the addition of alpha2,6-linked sialic acid to glycoproteins. ST6Gal.I is strongly expressed by the liver and is up-regulated in several cancers, but little is known of its regulation in human liver diseases. We have investigated the expression of ST6Gal.I and its product, the alpha2,6-sialylated lactosamine, in normal human liver, hepatocarcinoma (HCC), and cirrhosis. We found that both ST6Gal.I activity and mRNA can undergo up- or down-regulation in different HCC patients. At the mRNA level, the groups of specimens showing the highest expression were HCC of grade 2, HCC developed without preexisting cirrhosis, and HCC of male patients. The lectin from Sambucus nigra (SNA) reveals a significative overexpression of alpha2,6-sialylated glycoconjugates in HCC tissue homogenates and their intracellular accumulation in HCC histological sections, even though in a few cases the extent of alpha2,6-sialylation dramatically decreases. Transcription of the gene occurs through at least two different promoters, resulting in two differentially expressed mRNA species. RNA in situ hybridization reveals that the ST6Gal.I mRNA can be expressed at a quantitatively heterogeneous level among the neoplastic cells. Neither ST6Gal.I expression nor alpha2,6-sialylation are altered in cirrhosis. These data indicate that neoplastic transformation but not cirrhosis can alter the process of alpha2,6-sialylation of liver glycoproteins.

Molecular cloning of the human beta1,4 N-acetylgalactosaminyltransferase responsible for the biosynthesis of the Sd(a) histo-blood group antigen: the sequence predicts a very long cytoplasmic domain.

The Sd(a) antigen is a carbohydrate determinant expressed on erythrocytes, the colonic mucosa and other tissues. This epitope, whose structure is Siaalpha2,3[GalNAcbeta1,4]Gal beta1,4GlcNAc, is synthesized by a beta1,4 N-acetylgalactosaminyltransferase (beta4GalNAc-T) that transfers a beta1,4-linked GalNAc to the galactose residue of an alpha2,3-sialylated chain. We have cloned from human colon carcinoma Caco2 cells a cDNA whose transfection in COS cells induces a GalNAc-T active on sialylated but not on asialylated fetuin and putatively represents the human Sd(a) beta4GalNAc-T. The cDNA predicts a 566 aa protein showing 66.6% and 39% identity with mouse CT beta4GalNAc-T and human GM2/GD2 synthase, respectively, with a typical type II glycosyltransferase organization, no potential N-glycosylation sites and a 67 aa cytoplasmic tail, which is probably the longest among the glycosyltransferases cloned to date. The gene maps in chromosome 17q23, and is composed of at least 11 exons. Exons 2-11 are homologous to exons 2-11 of the previously cloned CT beta4GalNAc-T from murine cytotoxic T lymphocytes while exons 1 of the two enzymes are totally different. The mRNA is expressed at a high level in differentiated Caco2 cells and in colonic mucosa and at a much lower level in lymphocytes and other colon cancer cell lines.