Determination of major sialylated N-glycans and identification of branched sialylated N-glycans that dynamically change their content during development in the mouse cerebral cortex.

Oligosaccharides of glycoproteins expressed on the cell surface play important roles in cell-cell interactions, particularly sialylated N-glycans having a negative charge, which interact with sialic acid-binding immunoglobulin-like lectins (siglecs). The entire structure of sialylated N-glycans expressed in the mouse brain, particularly the linkage type of sialic acid residues attached to the backbone N-glycans, has not yet been elucidated. An improved method to analyze pyridylaminated sugar chains using high performance liquid chromatography (HPLC) was developed to determine the entire structure of sialylated N-linked sugar chains expressed in the adult and developing mouse cerebral cortices. Three classes of sialylated sugar chains were prevalent: 1) N-glycans containing α(2-3)-sialyl linkages on a type 2 antennary (Galß(1-4)GlcNAc), 2) sialylated N-glycans with α(2-6)-sialyl linkages on a type 2 antennary, and 3) a branched sialylated N-glycan with a [Galß(1-3){NeuAcα(2-6)}GlcNAc-] structure, which was absent at embryonic day 12 but then increased during development. This branched type sialylated N-glycan structure comprised approximately 2 % of the total N-glycans in the adult brain. Some N-glycans (containing type 2 antennary) were found to change their type of sialic acid linkage from α(2-6)-Gal to α(2-3)-Gal. Thus, the linkages and expression levels of sialylated N-glycans change dramatically during brain development.

Effects of amino acid substitutions in the sialylmotifs on molecular expression and enzymatic activities of α2,8-sialyltransferases ST8Sia-I and ST8Sia-VI.

Mouse sialyltransferases are grouped into four families according to the type of carbohydrate linkage they synthesize: ß-galactoside α2,3-sialyltransferases (ST3Gal-I-VI), ß-galactoside α2,6-sialyltransferases (ST6Gal-I and ST6Gal-II), N-acetylgalactosamine α2,6-sialyltransferases (ST6GalNAc-I-VI) and α2,8-sialyltransferases (ST8Sia-I-VI). These sialyltransferases feature a type II transmembrane topology and contain highly conserved motifs termed sialylmotifs L, S, III and VS. Sialylmotifs L and S are involved in substrate binding, whereas sialylmotifs III and VS are involved in catalytic activity. In addition to the conventional sialylmotifs, family and subfamily specific sequence motifs have been proposed. In this study, we analyzed the properties and functions of sialylmotifs in characterizing the enzymatic activity of mouse ST8Sia-I and ST8Sia-VI, both of which are α2,8-sialyltransferases involved in the synthesis of either ganglioside GD3 or disialic acid structures on O-glycans, respectively. The ST8Sia-VI-based chimeric enzymes, whose sialylmotif L sequences were replaced with those of ST8Sia-I and ST8Sia-IV (polysialic acid synthetase), were still active toward O-glycans. However, ST8Sia-VI-based chimeric enzymes lost expression or activity when their sialylmotif L sequences were replaced with those of ST3Gal-I and ST6GalNAc-II, suggesting the existence of an ST8Sia family specific motif in the sialylmotif L. The ST8Sia-I- and ST8Sia-VI-based chimeric enzymes lost enzymatic activity when their sialylmotif S sequences were interchanged. Amino acid substitutions in the sialylmotif S of ST8Sia-I and ST8Sia-VI also affected the enzymatic activity in many cases, indicating the crucial and functional importance of the sialylmotif S in substrate binding, which determines the substrate specificity of sialyltransferase.

Disulphide linkage in mouse ST6Gal-I: determination of linkage positions and mutant analysis.

All cloned sialyltransferases from vertebrates are classified into four subfamilies and are characterized as having type II transmembrane topology. The catalytic domain has highly conserved motifs known as sialylmotifs. Besides sialylmotifs, each family has several unique conserved cysteine (Cys) residues mainly in the catalytic domain. The number and loci of conserved amino acids, however, differ with each subfamily, suggesting that the conserved Cys-residues and/or disulphide linkages they make may contribute to linkage specificity. Using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF)-mass spectrometry, the present study performed disulphide linkage analysis on soluble mouse ST6Gal-I, which has six Cys-residues. Results confirmed that there were no free Cys-residues, and all six residues contributed to disulphide linkage formation, C(139)-C(403), C(181)-C(332) and C(350)-C(361). Study of single amino acid-substituted mutants revealed that the disulphide linkage C(181)-C(332) was necessary for molecular expression of the enzyme, and that the disulphide linkage C(350)-C(361) was necessary for enzyme activity. The remaining disulphide linkage C(139)-C(403) was not necessary for enzyme expression or for activity, including substrate specificity. Crystallographic study of pig ST3Gal I has recently been reported. Interestingly, the loci of disulphide linkages in ST6Gal-I differ from those in ST3Gal I, suggesting that the linkage specificity of sialyltransferase may results from significant structural differences, including the loci of disulphide linkages.

Adult onset cardiac dilatation in a transgenic mouse line with Galß1,3GalNAc α2,3-sialyltransferase II (ST3Gal-II) transgenes: a new model for dilated cardiomyopathy.

Sugar chain abnormalities in glycolipids and glycoproteins are associated with various diseases. Here, we report an adult onset cardiac dilatation in a transgenic mouse line with Galß1,3GalNAc α2,3-sialyltransferase II (ST3Gal-II) transgenes. The transgenic hearts at the end-stage, at around 7 months old, were enlarged, with enlarged cavities and thin, low-tensile walls, typical of dilated cardiomyopathy. Although no apparent change was found in heart gangliosides, glycosylation of heart proteins was altered. Interestingly, sugar moieties not directly related to the ST3Gal-II catalytic reaction were also changed. Significant increases in calreticulin and calnexin were observed in hearts of the transgenic mice. These results suggest that expression of ST3Gal-II transgenes induces abnormal protein glycosylation, which disorganizes the endoplasmic/sarcoplasmic reticulum quality control system and elevates the calreticulin/calnexin level, resulting in suppression of cardiac function. The transgenic mice showed 100% incidence of adult onset cardiac dilatation, suggesting great potential as a new model for dilated cardiomyopathy.

Analysis of sialyltransferase-like proteins from Oryza sativa.

Sialic acids are widely distributed among living creatures, from bacteria to mammals, but it has been commonly accepted that they do not exist in plants. However, with the progress of genome analyses, putative gene homologs of animal sialyltransferases have been detected in the genome of some plants. In this study, we cloned three genes from Oryza sativa (Japanese rice) that encode sialyltransferase-like proteins, designated OsSTLP1, 2, and 3, and analyzed the enzymatic activity of the proteins. OsSTLP1, 2, and 3 consist of 393, 396, and 384 amino acids, respectively, and each contains sequences similar to the sialyl motifs that are highly conserved among animal sialyltransferases. The recombinant soluble forms of OsSTLPs produced by COS-7 cells were analyzed for sialyltransferase-like activity. OsSTLP1 exhibited such activity toward the oligosaccharide Galbeta1,4GlcNAc and such glycoproteins as asialofetuin, alpha1-acid glycoprotein, and asialo-alpha1-acid glycoprotein; OsSTLP3 exhibited similar activity toward asialofetuin; and OsSTLP2 exhibited no sialyltransferase-like activity. The sialic acid transferred by OsSTLP1 or 3 was linked to galactose of Galbeta1,4GlcNAc through alpha2,6-linkage. This is the first report of plant proteins having sialyltransferase-like activity.

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.

Estimating the stenosis probabilities in arteriosclerosis obliterans using generalized estimating equations.

For each of 211 arteriosclerosis obliterans patients, the degree of stenosis of arteries at four sites were examined at Hiroshima University Hospital to analyse the relationship between the degree of stenosis and age, sex and site. The generalized estimating equations using a proportional odds model for the stenosis probability with exchangeable correlation was applied to this clustered, ordered polytomous data. Since the correlations between the responses were quite small, we could apply the independence estimating equations. The best mean model selected by Akaike information criterion suggested that age dependency of the stenosis probability was not observed in the central sites but was observed in the peripheral sites, and symmetry of the risk of stenosis was seen in the left and right sites. Details of the analysis will be described.

Enhanced insulin sensitivity in mice lacking ganglioside GM3.

Gangliosides are sialic acid-containing glycosphingolipids that are present on all mammalian plasma membranes where they participate in recognition and signaling activities. We have established mutant mice that lack GM3 synthase (CMP-NeuAc:lactosylceramide alpha2,3-sialyltransferase; EC 2.4.99.-). These mutant mice were unable to synthesize GM3 ganglioside, a simple and widely distributed glycosphingolipid. The mutant mice were viable and appeared without major abnormalities but showed a heightened sensitivity to insulin. A basis for the increased insulin sensitivity in the mutant mice was found to be enhanced insulin receptor phosphorylation in skeletal muscle. Importantly, the mutant mice were protected from high-fat diet-induced insulin resistance. Our results show that GM3 ganglioside is a negative regulator of insulin signaling, making it a potential therapeutic target in type 2 diabetes.

Localization of phosphorylated cAMP response element-binding protein in immature neurons of adult hippocampus.

Neurogenesis continues to occur in the adult hippocampus, although many of the newborn cells degenerate 1-2 weeks after birth. The number and survival of newborn cells are regulated by a variety of environmental stimuli, but very little is known about the intracellular signal transduction pathways that control adult neurogenesis. In the present study, we examine the expression of the phosphorylated cAMP response element-binding protein (pCREB) in immature neurons in adult hippocampus and the role of the cAMP cascade in the survival of new neurons. The results demonstrate that virtually all immature neurons, identified by triple immunohistochemistry for bromodeoxyuridine (BrdU) and polysialic acid-neural cell adhesion molecule (PSA-NCAM), are also positive for pCREB. In addition, upregulation of cAMP (via pharmacological inhibition of cAMP breakdown or by antidepressant treatment) increases the survival of BrdU-positive cells. A possible role for pCREB in the regulation of PSA-NCAM, a marker of immature neurons involved in neuronal remodeling and neurite outgrowth, is supported by cell culture studies demonstrating that the cAMP-CREB pathway regulates the expression of a rate-limiting enzyme responsible for the synthesis of PSA-NCAM. These findings indicate that the cAMP-CREB pathway regulates the survival, and possibly the differentiation and function, of newborn neurons.

Characterization of the second type of human beta-galactoside alpha 2,6-sialyltransferase (ST6Gal II), which sialylates Galbeta 1,4GlcNAc structures on oligosaccharides preferentially. Genomic analysis of human sialyltransferase genes.

A novel member of the human beta-galactoside alpha2,6-sialyltransferase (ST6Gal) family, designated ST6Gal II, was identified by BLAST analysis of expressed sequence tags and genomic sequences. The sequence of ST6Gal II encoded a protein of 529 amino acids, and it showed 48.9% amino acid sequence identity with human ST6Gal I. 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 activities toward some glycoproteins and glycolipids, respectively. It is concluded that ST6Gal II is an oligosaccharide-specific enzyme compared with ST6Gal I, which exhibits broad substrate specificities, and is mainly involved in the synthesis of sialyloligosaccharides. The expression of the ST6Gal II gene was significantly detected by reverse transcription PCR in small intestine, colon, and fetal brain, whereas the ST6Gal I gene was ubiquitously expressed, and its expression levels were much higher than those of the ST6Gal II gene. The ST6Gal I gene was also expressed in all tumors examined, but no expression was observed for the ST6Gal II gene in these tumors. The ST6Gal II gene is located on chromosome 2 (2q11.2-q12.1), and it spans over 85 kb of human genomic DNA consisting of at least eight exons and shares a similar genomic structure with the ST6Gal I gene. In this paper, we have shown that ST6Gal I, which has been known as the sole member of the ST6Gal family, also has the counterpart enzyme (ST6Gal II) like other sialyltransferases.

Molecular cloning and expression of a sixth type of alpha 2,8-sialyltransferase (ST8Sia VI) that sialylates O-glycans.

A novel member of the mouse alpha2,8-sialyltransferase (ST8Sia) family, designated ST8Sia VI, was identified by BLAST analysis of expressed sequence tags. The sequence of ST8Sia VI encodes a protein of 398 amino acids and shows 42.0 and 38.3% amino acid sequence identities to mouse alpha2,8-sialyltransferases ST8Sia I (GD3 synthase) and ST8Sia V (GD1c, GT1a, GQ1b, and GT3 synthases), respectively. The recombinant soluble form of ST8Sia VI expressed in COS-7 cells exhibited alpha2,8-sialyltransferase activity toward both glycolipids and glycoproteins that have the NeuAcalpha2,3(6)Gal sequence at the nonreducing end of their carbohydrate groups. This enzyme formed NeuAcalpha2,8NeuAc structures, but not oligosialic or polysialic acid structures. Analysis of the fetuin sialylated by ST8Sia VI indicated that ST8Sia VI prefers O-glycans to N-glycans as acceptor substrates. Substrate specificities and kinetic properties also showed that ST8Sia VI prefers O-glycans to glycolipids as acceptor substrates. ST8Sia VI also exhibited activity toward oligosaccharides such as sialyllactose and sialyllactosamine, and the structure of the minimal acceptor substrate for ST8Sia VI was determined as the NeuAcalpha2,3(6)Gal sequence. The expression of the ST8Sia VI gene was ubiquitous, and the highest expression was observed in kidney, with three major transcripts of 8.2, 3.8, and 2.7 kb. This is the first report of a mammalian alpha2,8-sialyltransferase that sialylates O-glycans preferentially.

Identification and functional characterization of a human GalNAc [alpha]2,6-sialyltransferase with altered expression in breast cancer.

BACKGROUND: We sought to identify genes with altered expression during human breast cancer progression by applying mRNA comparisons of normal and tumor mammary cell lines with increasingly malignant phenotypes. The gene encoding a new sialyltransferase (STM) was found to be down-regulated in tumor cells. Abnormal expression and enzymatic activities of sialyltransferases in tumor cells result in the formation of tumor-associated carbohydrate antigens that can be used for the better understanding of the disease process and are applied for tumor diagnosis and immunotherapy. Altered glycosylation patterns of the MUC1 mucin, in particular, is a target antigen for immunotherapy of breast and other cancers. MATERIALS AND METHODS: Total RNAs from multiple normal mammary epithelial cell strains and tumor cell lines were compared by differential display and the differential expression of selected cDNAs was confirmed by Northern analyses. Recombinant STM was expressed in COS-7 cells. The substrate and linkage specificity of STM was examined using various oligosaccharides and O-glycosylated proteins as acceptor substrates. The chromosomal localization of the SIATL1 gene was assigned by somatic cell hybrid analysis. RESULTS: A human sialyltransferase gene was identified by differential display as being down-regulated in breast tumor cell lines as compared to normal mammary epithelial cell strains, and the corresponding full-length cDNA (stm) was cloned. The encoded protein of 374 amino acid residues contained the L- and S-sialylmotifs, two catalytic regions conserved in all functional sialyltransferases. Recombinant STM is an active GalNAc alpha2,6-sialyltransferase with Gal beta 1,3 GalNAc-O-Ser/Thr and (+/- Neu5Ac alpha 2,3) Gal beta 1,3GalNAc-O-Ser/Thr acceptor specificity. The SIATL1 gene, encoding STM, was mapped to the long arm of human chromosome 17 at q23-qter, a region that is nonrandomly deleted in human breast cancers. However, Southern analyses indicated that SIATL1 is usually not grossly rearranged in breast tumors. Northern analyses showed that the gene was widely expressed in normal human tissues, as well as in normal breast and prostate epithelial cell lines, but significantly down-regulated or absent in corresponding tumor cell lines. CONCLUSIONS: Our findings suggest that aberrant expression of STM sialyltransferase in tumors could be a feature of the malignant phenotype. In breast cancers, the MUC1 mucin is overexpressed and contains shorter O-glycans as compared to the normal mucin. Because STM catalyzes the synthesis of O-glycans, cloning and characterization of its substrate specificity will contribute to the understanding of the molecular mechanisms underlying the aberrant glycosylation patterns of O-glycans and the formation of mucin-related antigens in human breast cancers.