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N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of ␤1,6-GlcNAc branching of N-glycans, which contributes to metastasis. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulting in the suppression of metastasis. It has long been hypothesized that the suppression of GnT-V product formation by the action of GnT-III would also exist in vivo, which will consequently lead to the inhibition of biological functions of GnT-V. To test this, we draw a comparison among MKN45 cells, which were transfected with GnT-III, GnT-V, or both, respectively. We found that ␣3␤1 integrin-mediated cell migration on laminin 5 was greatly enhanced in the case of GnT-V transfectant. This enhanced cell migration was significantly blocked after the introduction of GnT-III. Consistently, an increase in bisected GlcNAc but a decrease in ␤1,6-GlcNAcbranched N-glycans on integrin ␣3 subunit was observed in the double transfectants of GnT-III and GnT-V. Conversely, GnT-III knockdown resulted in increased migration on laminin 5, concomitant with an increase in ␤1,6-GlcNAc-branched N-glycans on the ␣3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the ␣3 subunit may be an explanation for why GnT-III inhibits GnT-V-induced cell migration. Taken together, our results demonstrate for the first time that GnT-III and GnT-V can competitively modify the same target glycoprotein and furthermore positively or negatively regulate its biological functions.Malignant transformation is accompanied by increased ␤1,6-GlcNAc branching of N-glycans attached to Asn-X-Ser/ Thr sequences in mature glycoproteins (1-3). N-Acetylglucosaminyltransferase V (GnT-V)3 catalyzes the addition of ␤1,6-linked GlcNAc (see Fig. 8 ) and defines this subset of N-glycans (4, 5). A relation between GnT-V and cancer metastasis has been reported by Dennis et al. (6) and Yamashita et al. (1).Studies on transplantable tumors in mice indicate that the product of GnT-V directly contributes to the growth of cancer and subsequent metastasis (7,8). On the other hand, somatic tumor cell mutants that are deficient in GnT-V activity produce fewer spontaneous metastases and grow more slowly than wildtype cells (6, 9). The suppression of tumor growth and metastasis has been reported in GnT-V-deficient mice (3). Moreover, Partridge et al. (10) reported that GnT-V-modified N-glycans with poly-N-acetyllactosamine, the preferred ligand for galectin-3, on surface receptors oppose their constitutive endocytosis and result in promoting intracellular signaling and consequently cell migration and tumor metastasis. These results indicate that inhibition of GnT-V might be useful in the treatment of malignancies by targeting their roles in metastasis.N-Acetylglucosaminyltransferase III (GnT-III) participates in the branching of N-glycans (see Fig. 8), catalyzing the formation of a unique sugar chain structure-bisecting GlcNAc (11). GnT-III is g...

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The core fucosylation (␣1,6-fucosylation) of glycoprotein is widely distributed in mammalian tissues. Recently ␣1,6-fucosylation has been further reported to be very crucial by the study of ␣1,6-fucosyltransferase (Fut8)-knock-out mice, which shows the phenotype of emphysema-like changes in the lung and severe growth retardation. In this study, we extensively investigated the effect of core fucosylation on ␣3␤1 integrin and found for the first time that Fut8 makes an important contribution to the functions of this integrin. The role of core fucosylation in ␣3␤1 integrin-mediated events has been studied by using Fut8 ؉/؉ and Fut8 ؊/؊ embryonic fibroblasts, respectively. We found that the core fucosylation of ␣3␤1 integrin, the major receptor for laminin 5, was abundant in Fut8 ؉/؉ cells but was totally abolished in Fut8 ؊/؊ cells, which was associated with the deficient migration mediated by ␣3␤1 integrin in Fut8 ؊/؊ cells. Moreover integrin-mediated cell signaling was reduced in Fut8؊/؊ cells. The reintroduction of Fut8 potentially restored laminin 5-induced migration and intracellular signaling. Collectively, these results suggested that core fucosylation is essential for the functions of ␣3␤1 integrin.␣1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue from GDP-fucose to position 6 of the innermost GlcNAc residue of the hybrid and complex types of N-linked oligosaccharides on the glycoproteins (Fig.

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The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.

ABSTRACTcDNA clones encoding chum salmon (Oncorhynchus keta) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)+ RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putative signal sequence of 22 amino acids. The 5' and 3' untranslated regions of the message were 64 and 426 bases long, respectively. Mature sGH was efficiently expressed in Escherichia coli carrying a plasmid in which the sGH cDNA was under control of the E. coli trp promoter; sGH comprised about 15% of the total cellular protein in such bacteria. The partially purified sGH from E. coli stimulated the growth of rainbow trout and the activity was indistinguishable from that of natural sGH.Human growth hormone now can be produced by genetically engineered organisms and can be used as a therapeutic agent. Salmon growth hormone (sGH) can be synthesized by use of similar techniques, and the massive supply of sGH may be extremely important to fish culture. Growth hormone (GH), together with prolactin and chorionic somatomammotropin (placental lactogen), forms a set of proteins that are structurally related and have partially overlapping biological activities (1). Primary structure analysis of the peptides and of the genes suggests that these hormone genes evolved from a common ancestral origin (1-5). Therefore, these genes provide an excellent model system for studying structurefunction relationships, evolution, and regulation of expression. GH genes have been isolated from several mammalian species and characterized in detail (3,(6)(7)(8). To obtain information about the evolution and the mechanisms of organization of this set of genes, it is essential to compare the structures of these hormone genes isolated from many organisms at various evolutionary stages. No information, however, has been available about lower vertebrates such as fish.

HNK-1 (human natural killer-1) glyco-epitope, a sulfated glucuronic acid attached to N-acetyllactosamine on the nonreducing termini of glycans, is highly expressed in the nervous system. Our previous report showed that mice lacking a glucuronyltransferase (GlcAT-P), a key enzyme for biosynthesis of the HNK-1 epitope, showed reduced long term potentiation at hippocampal CA1 synapses. In this study, we identified an ␣-amino-3-hydroxy-5-methylisoxazole propionate (AMPA)-type glutamate receptor subunit, GluR2, which directly contributes to excitatory synaptic transmission and synaptic plasticity, as a novel HNK-1 carrier molecule. We demonstrated that the HNK-1 epitope is specifically expressed on the N-linked glycan(s) on GluR2 among the glutamate receptors tested, and the glycan structure, including HNK-1 on GluR2, was determined using liquid chromatography-tandem mass spectrometry. As for the function of HNK-1 on GluR2, we found that the GluR2 not carrying HNK-1 was dramatically endocytosed and expressed less on the cell surface compared with GluR2 carrying HNK-1 in both cultured hippocampal neurons and heterologous cells. These results suggest that HNK-1 stabilizes GluR2 on neuronal surface membranes and regulates the number of surface AMPA receptors. Moreover, we showed that the expression of the HNK-1 epitope enhanced the interaction between GluR2 and N-cadherin, which has important roles in AMPA receptor trafficking. Our findings suggest that the HNK-1 epitope on GluR2 regulates cell surface stability of GluR2 by modulating the interaction with N-cadherin.HNK-1 glyco-epitope (HSO 3 -3GlcA␤1-3Gal␤1-4GlcNAc) is characteristically expressed on some cell adhesion molecules (NCAM, L1, and MAG, etc.) and extracellular matrix molecules (tenascin-R and phosphacan, etc.) in the nervous system (1). It has been reported that HNK-1 mediates the interaction of these adhesion molecules, thereby controlling their functions, including cell-to-cell adhesion (2), migration (3), and neurite extension (4). The unique structural feature of the HNK-1 epitope is the sulfated glucuronic acid, because sialic acids are usually attached to the terminal galactose residue of the inner N-acetyllactosamine structure (Gal␤1-4GlcNAc) on various glycoproteins. HNK-1 is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) 3 (5, 6) and a sulfotransferase (HNK-1ST) (7). These enzymes are thought to localize and function in the Golgi apparatus, especially the trans-Golgi to trans-Golgi network, like most sialyltransferases and galactosyltransferases (8).We previously demonstrated that mice deficient in GlcAT-P showed an almost complete loss of HNK-1 expression in the brain and exhibited reduced LTP in hippocampal CA1 synapses (9). Similarly, HNK-1ST-deficient mice also exhibited a reduction of LTP, and several other studies also revealed that HNK-1 is associated with neural plasticity (10 -12). A recent study showed that ␤4-galactosyltransferase-2 synthesizes the glycan backbone structure of HNK-1, Gal␤1-4GlcNAc. ...