Angiotensin II elicits different responses which affect cardiovascular, neuronal and electrolyte transport regulation. To understand the mechanisms responsible for its various actions, the receptor for angiotensin II has long been sought, but numerous attempts to purify the receptor have been unsuccessful owing to its instability and low concentration. We report here the expression cloning of a complementary DNA encoding a bovine angiotensin II receptor to overcome these difficulties. The receptor cDNA encodes a protein of 359 amino-acid residues with a transmembrane topology similar to that of other G protein-coupled receptors. COS-7 cells transfected with the cDNA expressed specific and high-affinity binding sites for angiotensin II, angiotensin II antagonist and a non-peptide specific antagonist for type-1 receptor. Dithiothreitol inhibited ligand binding. The concentration of intracellular Ca2+ and of inositol-1,4,5-trisphosphate increased in the transfected COS-7 cells in response to angiotensin II or angiotensin III, indicating that this receptor is the type-1 receptor for angiotensin II. Northern blot analysis revealed that the messenger RNA for this receptor is expressed in bovine adrenal medulla, cortex and kidney.

We have isolated three types of cDNAs encoding novel ␤1,3-N-acetylglucosaminyltransferases (designated ␤3Gn-T2, -T3, and -T4) from human gastric mucosa and the neuroblastoma cell line SK-N-MC. These enzymes are predicted to be type 2 transmembrane proteins of 397, 372, and 378 amino acids, respectively. They share motifs conserved among members of the ␤1,3-galactosyltransferase family and a ␤1,3-N-acetylglucosaminyltransferase (designated ␤3Gn-T1), but show no structural similarity to another type of ␤1,3-N-acetylglucosaminyltransferase (iGnT). Each of the enzymes expressed by insect cells as a secreted protein fused to the FLAG peptide showed ␤1,3-N-acetylglucosaminyltransferase activity for type 2 oligosaccharides but not ␤1,3-galactosyltransferase activity. These enzymes exhibited different substrate specificity. Transfection of Namalwa KJM-1 cells with ␤3Gn-T2, -T3, or -T4 cDNA led to an increase in poly-N-acetyllactosamines recognized by an anti-i-antigen antibody or specific lectins. The expression profiles of these ␤3Gn-Ts were different among 35 human tissues. ␤3Gn-T2 was ubiquitously expressed, whereas expression of ␤3Gn-T3 and -T4 was relatively restricted. ␤3Gn-T3 was expressed in colon, jejunum, stomach, esophagus, placenta, and trachea. ␤3Gn-T4 was mainly expressed in brain. These results have revealed that several ␤1,3-Nacetylglucosaminyltransferases form a family with structural similarity to the ␤1,3-galactosyltransferase family. Considering the differences in substrate specificity and distribution, each ␤1,3-N-acetylglucosaminyltransferase may play different roles.A family of human ␤1,3-galactosyltransferases (␤3Gal-Ts) 1 consisting of five members (␤3Gal-T1, -T2, -T3, -T4, and -T5) was recently identified (1-4). The first ␤1,3-galactosyltransferase (␤3Gal-T1), which catalyzes the formation of type 1 oligosaccharides, was isolated by us using an expression cloning approach (1). Expression patterns of ␤3Gal-T1 and type 1 oligosaccharides strongly suggested the existence of ␤3Gal-T1 homologs. For instance, type 1-derived oligosaccharides such as sialyl-Le a were known to be expressed in colon and pancreatic cancer cell lines, whereas expression of ␤3Gal-T1 was detected in brain, but not in cancer cells. Our early approach using Southern hybridization failed to detect the existence of ␤3Gal-T1 homologous genes. However, recent accumulation of nucleotide sequence information on human cDNAs and genes such as expressed sequence tags (ESTs) enabled us to search homologous genes that do not have high similarity as detected by hybridization, but show significant similarity. A homology search based on the nucleotide or amino acid sequence of ␤3Gal-T1 led to the isolation of ␤3Gal-T2, -T3, and -T4, indicating that ␤3Gal-Ts form a family (1-3).␤3Gal-T2 catalyzed a similar reaction, but showed different substrate specificity compared with ␤3Gal-T1. The activity of ␤3Gal-T3 has not been detected, whereas the corresponding mouse enzyme exhibits weak ␤3Gal-T activity for both GlcNAc and GalNAc (5). On the other...

The structure and biosynthesis of poly-Nacetyllactosamine display a dramatic change during development and oncogenesis. Poly-N-acetyllactosamines are also modified by various carbohydrate residues, forming functional oligosaccharides such as sialyl Le x . Herein we describe the isolation and functional expression of a cDNA encoding ␤-1,3-N-acetylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-Nacetyllactosamine. For this expression cloning, Burkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA libraries derived from human melanoma and colon carcinoma cells. Transfected Namalwa cells overexpressing the i antigen were continuously selected by f luorescenceactivated cell sorting because introduced plasmids containing Epstein-Barr virus replication origin can be continuously amplified as episomes. Sibling selection of plasmids recovered after the third consecutive sorting resulted in a cDNA clone that directs the increased expression of i antigen on the cell surface. The deduced amino acid sequence indicates that this protein has a type II membrane protein topology found in almost all mammalian glycosyltransferases cloned to date. iGnT, however, differs in having the longest transmembrane domain among glycosyltransferases cloned so far. The iGnT transcript is highly expressed in fetal brain and kidney and adult brain but expressed ubiquitously in various adult tissues. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the enzyme responsible for the formation of GlcNAc␤1 3 3Gal␤1 3 4GlcNAc 3 R structure and poly-N-acetyllactosamine extension.

The sialyl Lewis a antigen is a well known tumor marker, CA19-9, which is frequently elevated in the serum in gastrointestinal and pancreatic cancers. UDPgalactose:N-acetylglucosamine ␤1,3-galactosyltransferase(s) (␤3Gal-Ts) are required for the synthesis of the sialyl Lewis a epitope. In the present study, a novel ␤3Gal-T, named ␤3Gal-T5, was isolated from a Colo205 cDNA library using a degenerate primer strategy based on the amino acid sequences of the four human ␤3Gal-T genes cloned to date. Transfection experiments demonstrated that HCT-15 cells transfected with the ␤3Gal-T5 gene expressed all the type 1 Lewis antigens. In gastrointestinal and pancreatic cancer cell lines, the amounts of ␤3Gal-T5 transcripts were quite well correlated with the amounts of the sialyl Lewis a antigens. The ␤1,3Gal-T activity toward agalacto-lacto-N-neotetraose was also well correlated with the amounts of ␤3Gal-T5 transcripts in a series of cultured cancer cells, and in Namalwa and HCT-15 cells transfected with the ␤3Gal-T5 gene. Thus, the ␤3Gal-T5 gene is the most probable candidate responsible for the synthesis of the type 1 Lewis antigens in gastrointestinal and pancreatic epithelia and tumor cells derived therefrom. In addition, ␤3Gal-T5 is a key enzyme that determines the amounts of the type 1 Lewis antigens including the sialyl Lewis a antigen.

A new member of the UDP-N-acetylglucosamine:␤-galactose ␤1,3-N-acetylglucosaminyltransferase (␤3Gn-T) family having the ␤3Gn-T motifs was cloned from rat and human cDNA libraries and named ␤3Gn-T5 based on its position in a phylogenetic tree. We concluded that ␤3Gn-T5 is the most feasible candidate for lactotriaosylceramide (Lc 3 Cer) synthase, an important enzyme which plays a key role in the synthesis of lacto-or neolacto-series carbohydrate chains on glycolipids. ␤3Gn-T5 exhibited strong activity to transfer GlcNAc to glycolipid substrates, such as lactosylceramide (LacCer) and neolactotetraosylceramide (nLc 4 Cer; paragloboside), resulting in the synthesis of Lc 3 Cer and neolactopentaosylceramide (nLc 5 Cer), respectively. A marked decrease in LacCer and increase in nLc 4 Cer was detected in Namalwa cells stably expressing ␤3Gn-T5. This indicated that ␤3Gn-T5 exerted activity to synthesize Lc 3 Cer and decrease LacCer, followed by conversion to nLc 4 Cer via endogenous galactosylation. The following four findings further supported that ␤3Gn-T5 is Lc 3 Cer synthase. 1) The ␤3Gn-T5 transcript levels in various cells were consistent with the activity levels of Lc 3 Cer synthase in those cells. 2) The ␤3Gn-T5 transcript was presented in various tissues and cultured cells. 3) The ␤3Gn-T5 expression was up-regulated by stimulation with retinoic acid and down-regulated with 12-O-tetradecanoylphorbol-13-acetate in HL-60 cells. 4) The changes in ␤3Gn-T5 transcript levels during the rat brain development were determined. Points 2, 3, and 4 were consistent with the Lc 3 Cer synthase activity reported previously.To date, three members of the human ␤1,3-N-acetylglucosaminyltransferase (␤3Gn-T) 1 family (␤3Gn-T2, -T3, and -T4) (1, 2) and five members of the human ␤1,3-galactosyltransferase (␤3Gal-T) family (␤3Gal-T1, -T2, -T3, -T4, and -T5) have been identified (3-6). All of them share amino acid motifs (␤3Gn-T motifs or ␤3Gal-T motifs) in three regions of the catalytic domain. The first, ␤3Gn-T, was cloned by an expression cloning method using an anti-i antibody (7). However, this enzyme is unique in that it does not have the ␤3Gn-T motifs although it transfers GlcNAc to Gal with an ␤1,3-linkage, resulting in the synthesis of polylactosamine chains. It was named iGn-T (7). Thereafter, ␤3Gn-T1 was isolated based on structural similarity with the ␤3Gal-T family (2). We previously reported three additional ␤3Gn-Ts, ␤3Gn-T2, -T3, and -T4, which are also structurally related to the ␤3Gn-T family (1). However, the cDNA sequence of ␤3Gn-T1 was recently corrected by Zhou et al. (see Ref. 2). The corrected sequence of ␤3Gn-T1 was identical to that of ␤3Gn-T2 which was isolated * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank TM /EBI Data Bank with the accession num...

The 3-fucosyl-N-acetyllactosamine (Lewis x, CD15, SSEA-1) carbohydrate epitope is widely distributed in many tissues and is developmentally expressed in some rodent and human tissues, i.e. brain and lung, and mouse early embryo. In such tissues, the Lewis x epitope is considered to be involved in cell-cell interactions. We isolated a novel mouse ␣1,3-fucosyltransferase gene, named mFuc-TIX, from an adult mouse brain cDNA library using the expression cloning method. On flow cytometric analysis, Namalwa cells transfected stably with the mFuc-TIX gene showed a marked increase in Lewis x epitopes but not sialyl Lewis x epitopes. As seen experiments involving oligosaccharides as acceptor substrates, mFuc-TIX transfers a fucose to lacto-N-neotetraose but not to either ␣2,3-sialyl lacto-N-neotetraose or lacto-N-tetraose. The substrate specificity of mFuc-TIX was similar to that of mouse myeloid-type ␣1,3-fucosyltransferase (mFuc-TIV). The deduced amino acid sequence of mFuc-TIX, consisting of 359 residues, indicated a type II membrane protein and shows low degrees of homology to the previously cloned ␣1,3-fucosyltransferases, i.e. mFuc-TIV (48.4%), mouse Fuc-TVII (39.1%), and human Fuc-TIII (43.0%), at the amino acid sequence level. A phylogenetic tree of the ␣1,3-fucosyltransferases constructed by the neighbor-joining method showed that mFuc-TIX is quite distant from the other ␣1,3-fucosyltransferases. Thus, mFuc-TIX does not belong to any subfamilies of known ␣1,3Fuc-Ts. The mFuc-TIX transcript was mainly detected in brain and kidney with the Northern blotting and competitive reverse transcription-polymerase chain reaction methods, whereas the mFuc-TIV transcript was not detected in brain with these methods. On in situ hybridization, the mFuc-TIX transcript was detected in neuronal cells but not in the glial cells including astrocytes. These results strongly indicated that mFuc-TIX participates in the Lewis x synthesis in neurons of the brain and may be developmentally regulated.

The CD15 carbohydrate epitope is expressed in mature human neutrophils, monocytes, and promyelocytes. We aimed to determine the ␣1,3-fucosyltransferase responsible for the expression of CD15 in each subpopulation of leukocytes. Three ␣1,3-fucosyltransferases, FUT4, FUT7, and FUT9, are expressed in human leukocytes. We demonstrated that FUT9 exhibits 20-fold stronger activity for CD15 synthesis than FUT4, whereas FUT4 exhibits 4.5-fold stronger activity for CDw65 synthesis than FUT9. By competitive reverse transcriptase-polymerase chain reaction, FUT9 was found to be strongly expressed in mature granulocytes and peripheral blood mononuclear cell, but not in monocytes. CD34؉ and CD15 ؉ cells in cord blood and myeloid cell lines (HL-60 and U937) did not express FUT9 at all. FUT4 transcripts were ubiquitously expressed in all blood cells and all cultured cell lines, with HL-60 and U937 cells in particular expressing a number of FUT4 transcripts. Transfection of the FUT9 gene into Jurkat and U937 cells demonstrated that FUT9 has the potential to express CD15 in myeloid and lymphoid cells. These findings suggest that the expression of CD15 in mature granulocytes is directed by FUT9, whereas it is determined in promyelocytes and monocytes by FUT4. Measurement of CD15 synthesizing activity in cell homogenates of each cell population using the polylactosamine acceptor further supported these conclusions.There are three CD 1 markers of human leukocytes comprising fucosylated carbohydrate epitopes. As listed in Fig. 1 below, the distal lactosamine unit (LN; type 2 chain), Gal␤1,4GlcNAc, of the polylactosamine chain is fucosylated through ␣1,3-fucosyltransferase (␣1,3FUT) activity to form the CD15 (Lewis x; Le X ) epitope (1, 2). The CD15s (sialylated CD15; sialyl Le X (sLe X )) and CDw65 (VIM-2) epitopes are also fucosylated structures related to CD15, i.e. CD15s is formed by ␣2,3-sialylation prior to the fucosylation of the distal LN unit of polylactosamine by ␣1,3FUT, and CDw65 is formed by fucosylation of the inner LN unit of ␣2,3-sialylated polylactosamine by ␣1,3FUT (2, 3).The CD15 epitope is expressed in some tissues, such as epithelial cells of intestinal tissues (4 -6), certain neurons and glial cells in the central nervous system (7,8). In human leukocytes, CD15 is expressed preferentially in monocytes, mature neutrophils, and all myeloid cells from the promyelocyte stage onwards, making it a useful cell surface marker (9 -11). CD15 is considered to be involved in neutrophil functions, that is, cell-cell interactions, phagocytosis, stimulation of degranulation, and respiratory burst, although the function of CD15 is not clear (12)(13)(14)(15)(16).Six human ␣1,3FUT genes have been cloned to date, which are FUT3 (Fuc-TIII), FUT4 (Fuc-TIV), FUT5 (Fuc-TV), FUT6 (Fuc-TVI), FUT7 (Fuc-TVII), and FUT9 (Fuc-TIX) (1, 17-23). FUT9, a new member of the human ␣1,3FUT family, which we have recently cloned, is expressed in human leukocytes, glandular compartments of the stomach, and forebrain (23). The FUT9 gene was mapped on ch...

The amino acid sequence of Fuc-TIX is very highly conserved between mouse and human. The number of nonsynonymous nucleotide substitutions of the Fuc-TIX gene between human and mouse was strikingly low, and almost equivalent to that of the K K-actin gene. This indicates that Fuc-TIX is under a strong selective pressure of preservation during evolution. The human Fuc-TIX (hFuc-TIX) showed a unique characteristics, i.e. hFuc-TIX was not activated by Mn 2+ and Co 2+ , whereas hFuc-TIV and hFuc-TVI were activated by the cations. The hFuc-TIX transcripts were abundantly expressed in brain and stomach, and interestingly were detected in spleen and peripheral blood leukocytes.z 1999 Federation of European Biochemical Societies.