Kiyohiko Angata

Polysialyltransferases: major players in polysialic acid synthesis on the neural cell adhesion molecule

Polysialic acid is a unique carbohydrate composed of a linear homopolymer of alpha2,8-linked sialic acid, and is mainly attached to the fifth immunoglobulin-like domain of the neural cell adhesion molecule (NCAM) via a typical N-linked glycan in vertebrate neural system. Polysialic acid plays critical roles in neural development by modulating adhesive property of NCAM such as neural cell migration, neurite outgrowth, neural pathfinding, and synaptogenesis. The expression of polysialic acid is temporally and spatially regulated during neural development. Polysialylation of NCAM is catalyzed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), which belong to the family of six genes encoding alpha 2,8-sialyltransferases. ST8Sia II and IV are expressed differentially in tissue-specific and cell-specific manners, and they apparently have distinct roles in development and organogenesis. The presence of polysialic acid is always associated with expression of ST8Sia II and/or IV, suggesting that ST8Sia II and IV are the key enzymes that control the expression of polysialic acid. Both ST8Sia II and IV can transfer multiple alpha 2,8-linked sialic acid residues to an acceptor N-glycan containing a NeuNAc alpha 2-->3 (or 6) Gal beta 1-->4GlcNAc beta 1-->R structure without participation of other enzymes. The two enzymes differently but cooperatively act on NCAM and the amount of polysialic acid synthesized by both enzymes together is greater than that synthesized by either enzyme alone. The polysialyltransferases are thus important regulators in polysialic acid synthesis and contribute to neural development in the vertebrate.

Human STX polysialyltransferase forms the embryonic form of the neural cell adhesion molecule. Tissue-specific expression, neurite outgrowth, and chromosomal localization in comparison with another polysialyltransferase, PST.

PST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (N-CAM), although it is not known why these two polysialyltransferases exist. In the present study, we have first isolated cDNA encoding human STX, which includes 5′-untranslated sequence. Northern blot analysis, using this cDNA and PST cDNA previously isolated by us, demonstrated that PST and STX are expressed in different fetal and adult tissues. STX is primarily expressed in embryonic tissues, but only modestly in adult heart, brain, and thymus. PST, on the other hand, is continuously expressed in adult heart, brain, thymus, spleen, small and large intestines, and peripheral blood leukocytes. In various parts of adult brain, the relative amount of PST and STX appears to be substantially different depending on the regions. The analysis by in situ hybridization of mouse adult brain, however, suggests that polysialic acid in the hippocampal formation is synthesized by both STX and PST. HeLa cells doubly transfected with the isolated STX cDNA and N-CAM cDNA supported neurite outgrowth much better than HeLa cells expressing N-CAM alone. However, polysialic acid synthesized by PST appears to be a better substratum than that synthesized by STX. Moreover, the genes for PST and STX were found to reside at chromosome 5, band p21 and chromosome 15, band q26, respectively. These results, taken together, strongly suggest that PST and STX are expressed distinctly in tissue-specific and cell-specific manners and that they apparently have distinct roles in development and organogenesis.

Differential and Cooperative Polysialylation of the Neural Cell Adhesion Molecule by Two Polysialyltransferases, PST and STX

PST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (NCAM), and these two polysialyltransferases often exist together in the same tissues. To determine the individual and combined roles of PST and STX in polysialic acid synthesis, in the present study we asked if PST and STX differ in the acceptor requirement and if PST and STX act together in polysialylation of NCAM. We first examined whether PST and STX differ in the requirement of sialic acid and core structures ofN-glycans attached to NCAM. Polysialic acid was formed well on Lec4 and Lec13 cells, which are defective inN-acetylglucosaminyltransferase V and GDP-fucose synthesis, respectively, demonstrating that a side chain elongating from GlcNAcβ1→6Manα1→6R and α-1,6-linked fucose are not required. PST and STX were found to add polysialic acid on NCAM·Fc molecules sialylated by α-2,3- or α-2,6-linkage in vitro, but not on NCAM·Fc lacking either sialic acid. These results indicate that both PST and STX have relatively broad specificity onN-glycan core structures in NCAM and no remarkable difference exists between PST and STX for the requirement of core structures and sialic acid attached to the N-glycans of NCAM. We then, using various N-glycosylation site mutants of NCAM, discovered that PST strongly prefer the sixthN-glycosylation site, which is the closest to the transmembrane domain, over the fifth site. STX slightly prefer the sixth N-glycosylation site over the fifthN-glycosylation site. The results also demonstrated that polysialic acid synthesized by PST is larger than that synthesized by STX in vitro. Moreover, a mixture of PST and STX more efficiently synthesized polysialic acid on NCAM than PST or STX alone. These results suggest that polysialylation of NCAM is influenced by the difference between PST and STX in their preference forN-glycosylation sites on NCAM. The results also suggest that PST and STX form polysialylated NCAM in a synergistic manner.

Tumor suppressor function of laminin-binding α-dystroglycan requires a distinct β3-N-acetylglucosaminyltransferase

α-Dystroglycan (α-DG) represents a highly glycosylated cell surface molecule that is expressed in the epithelial cell-basement membrane (BM) interface and plays an essential role in epithelium development and tissue organization. The α-DG–mediated epithelial cell-BM interaction is often impaired in invasive carcinomas, yet roles and underlying mechanisms of such an impaired interaction in tumor progression remain unclear. We report here a suppressor function of laminin-binding glycans on α-DG in tumor progression. In aggressive prostate and breast carcinoma cell lines, laminin-binding glycans are dramatically decreased, although the amount of α-DG and β-dystroglycan is maintained. The decrease of laminin-binding glycans and consequent increased cell migration were associated with the decreased expression of β3-N-acetylglucosaminyltransferase-1 (β3GnT1). Forced expression of β3GnT1 in aggressive cancer cells restored the laminin-binding glycans and decreased tumor formation. β3GnT1 was found to be required for laminin-binding glycan synthesis through formation of a complex with LARGE, thus regulating the function of LARGE. Interaction of the laminin-binding glycans with laminin and other adhesive molecules in BM attenuates tumor cell migratory potential by antagonizing ERK/AKT phosphorylation induced by the components in the ECM. These results identify a previously undescribed role of carbohydrate-dependent cell-BM interaction in tumor suppression and its control by β3GnT1 and LARGE.

Polysialic Acid-Directed Migration and Differentiation of Neural Precursors Are Essential for Mouse Brain Development

ABSTRACT Polysialic acid, which is synthesized by two polysialyltransferases, ST8SiaII and ST8SiaIV, plays an essential role in brain development by modifying the neural cell adhesion molecule (NCAM). It is currently unclear how polysialic acid functions in different processes of neural development. Here we generated mice doubly mutant in both ST8SiaII and ST8SiaIV to determine the effects of loss of polysialic acid on brain development. In contrast to NCAM-deficient, ST8SiaII-deficient, or ST8SiaIV-deficient single mutant mice, ST8SiaII and ST8SiaIV double mutants displayed severe defects in anatomical organization of the forebrain associated with apoptotic cell death. Loss of polysialic acid affected both tangential and radial migration of neural precursors during cortical development, resulting in aberrant positioning of neuronal and glial cells. Glial cell differentiation was aberrantly increased in vivo and in vitro in the absence of polysialic acid. Consistent with these findings, polysialic acid-deficient mice exhibited increased expression of the glial cell marker glial fibrillary acidic protein and a decrease in expression of Pax6, a transcription factor regulating neural cell migration. These results indicate that polysialic acid regulates cell migration and differentiation of neural precursors crucial for brain development.

Polysialic acid, a unique glycan that is developmentally regulated by two polysialyltransferases, PST and STX, in the central nervous system: From biosynthesis to function

Polysialic acid is a developmentally regulated carbohydrate composed of a linear homopolymer of a‐2,a‐linked sialic acid residues. This unique glycan is mainly attached to the neural cell adhesion molecule (N‐CAM) and implicated in many morphogenic events of the neural cells by modulating the adhesive property of N‐CAM. Recently, the cDNA that encodes polysialyltransferase, which is responsible for the polysialylation of N‐CAM, was successfully cloned from three mammalian species. This review focuses on the molecular cloning of human polysialyltransferase, designated PST. it then describes the number of enzymes actually required for the polysialylation of N‐CAM using an in vitro polysialyltransferase assay. Comparisons between PST and another polysialyltransferase, sialyltransferase X (STX), are made and it Is demonstrated that both enzymes can independently form polysiatic acid In vitro, but that during neural development they coordinately but distinctly synthesize polysialic acid on N‐CAM. The role of polysialic acid in the central nervous system is also discussed. Finally, evidence that the two polysialyltransferases, PST and STX, apparently have distinct roles in the development of neural cells is provided by using a neurite outgrowth assay.

Molecular Cloning and Expression of a Novel Human β-Gal-3-O-sulfotransferase That Acts Preferentially onN-Acetyllactosamine in N- andO-Glycans

A novel cDNA-encoding galactose 3-O-sulfotransferase was cloned by screening the expressed sequence tag data base using the previously cloned cDNA encoding a galactosyl ceramide 3-O-sulfotransferase, which we term Gal3ST-1. The newly isolated cDNA encodes a novel 3-O-sulfotransferase, termed Gal3ST-3, that acts exclusively on N-acetyllactosamine present inN-glycans and core2-branched O-glycans. These conclusions were confirmed by analyzing CD43 chimeric proteins in Chinese hamster ovary cells expressing core2 β1,6-N-acetylglucosaminyltransferase. The acceptor specificity of Gal3ST-3 contrasts with that of the recently cloned galactose 3-O-sulfotransferase (Honke, K., Tsuda, M., Koyota, S., Wada, Y., Iida-Tanaka, N., Ishizuka, I., Nakayama, J., and Taniguchi, N. (2001) J. Biol. Chem. 276, 267–274), which we term Gal3ST-2 in the present study because the latter enzyme can also act on core1 O-glycan and type 1 oligosaccharides, Galβ1→3GlcNAc. Moreover, Gal3ST-3 but not Gal3ST-2 can act on Galβ1→4(sulfo→6)GlcNAc, indicating that disulfated sulfo→3Galβ1→4(sulfo→6) GlcNAc→R may be formed by Gal3ST-3 in combination with GlcNAc 6-O-sulfotransferase. Although both Gal3ST-2 and Gal3ST-3 do not act on galactosyl ceramide, Gal3ST-3 is only moderately more homologous to Gal3ST-2 (40.1%) than to Gal3ST-1 (38.0%) at the amino acid level. Northern blot analysis demonstrated that transcripts for Gal3ST-3 are predominantly expressed in the brain, kidney, and thyroid where the presence of 3′-sulfation ofN-acetyllactosamine has been reported. These results indicate that the newly cloned Gal3ST-3 plays a critical role in 3′-sulfation of N-acetyllactosamine in both O- and N-glycans.

Fer kinase regulates cell migration through α-dystroglycan glycosylation

This is the first report on the role of Fer kinase in down-regulating the expression of laminin-binding glycans that suppress cell migration. The data show a novel biochemical interaction between glycan-based adhesion and cell migration, mediated by a tyrosine kinase.

Cellular and molecular analysis of neural development of glycosyltransferase gene knockout mice.

Recent studies demonstrate that carbohydrates synthesized by specific glycosyltransferases play important roles in the development of the central nervous system. Among these carbohydrates, polysialic acid is a unique glycan that modulates functions of the neural cell adhesion molecule (NCAM) by attenuating NCAM-mediated interaction between neural cells. During brain development, polysialic acid is synthesized in a specific spatiotemporal pattern by two polysialyltransferases, ST8SiaII and ST8SiaIV. To study in vivo the roles of polysialic acid synthesized by each respective enzyme, we generated ST8SiaII and ST8SiaIV knockout mice. Single knockout ST8SiaII or ST8SiaIV mice show polysialic acid expression patterns differing from wild type, and those patterns indicate different roles of each gene during neural development. In this chapter, we discuss methods used to analyze polysialyltransferase knockout mice using immunohistochemistry of brain and primary cultures of neurons.

HNK-1 glycan functions as a tumor suppressor for astrocytic tumor

Astrocytic tumor is the most prevalent primary brain tumor. However, the role of cell surface carbohydrates in astrocytic tumor invasion is not known. In a previous study, we showed that polysialic acid facilitates astrocytic tumor invasion and thereby tumor progression. Here, we examined the role of HNK-1 glycan in astrocytic tumor invasion. A Kaplan-Meier analysis of 45 patients revealed that higher HNK-1 expression levels were positively associated with increased survival of patients. To determine the role of HNK-1 glycan, we transfected C6 glioma cells, which lack HNK-1 glycan expression, with β1,3-glucuronyltransferase-P cDNA, generating HNK-1-positive cells. When these cells were injected into the mouse brain, the resultant tumors were 60% smaller than tumors emerging from injection of the mock-transfected HNK-1-negative C6 cells. HNK-1-positive C6 cells also grew more slowly than mock-transfected C6 cells in anchorage-dependent and anchorage-independent assays. C6-HNK-1 cells migrated well after treatment of anti-β1 integrin antibody, whereas the same treatment inhibited cell migration of mock-transfected C6 cells. Similarly, α-dystroglycan containing HNK-1 glycan is different from those containing the laminin-binding glycans, supporting the above conclusion that C6-HNK-1 cells migrate independently from β1-integrin-mediated signaling. Moreover, HNK-1-positive cells exhibited attenuated activation of ERK 1/2 compared with mock-transfected C6 cells, whereas focal adhesion kinase activation was equivalent in both cell types. Overall, these results indicate that HNK-1 glycan functions as a tumor suppressor.