We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.

␤1,3-N-acetylglucosaminyltransferase 2 (␤3GnT2) is a polylactosamine synthase that synthesizes a backbone structure of carbohydrate structures onto glycoproteins. Here we generated ␤3GnT2-deficient (␤3GnT2 ؊/؊ ) mice and showed that polylactosamine on N-glycans was markedly reduced in their immunological tissues. In WT mice, polylactosamine was present on CD28 and CD19, both known immune costimulatory molecules. However, polylactosamine levels on these molecules were reduced in ␤3GnT2 ؊/؊ mice. ␤3GnT2 ؊/؊ T cells lacking polylactosamine were more sensitive to the induction of intracellular calcium flux on stimulation with anti-CD3 /CD28 and proliferated more strongly than T cells from WT mice. ␤3GnT2 ؊/؊ B cells also showed hyperproliferation on BCR stimulation. Macrophages from ␤3GnT2 ؊/؊ mice had higher cell surface CD14 levels and enhanced responses to endotoxin. These results indicate that polylactosamine on Nglycans is a putative immune regulatory factor presumably suppressing excessive responses during immune reactions.␤-1,3-N-acetylglucosaminyltransferase ͉ glycosyltransferase ͉ hyperactivation ͉ immune response

EL5, a RING-H2 finger protein, is rapidly induced by N-acetylchitooligosaccharides in rice cell. We expressed the EL5 RING-H2 finger domain in Escherichia coli and determined its structure in solution by NMR spectroscopy. The EL5 RING-H2 finger domain consists of twostranded ␤-sheets (␤1, Ala 147 -Phe 149 ; ␤2, Gly 156 -His 158 ), one ␣-helix (Cys 161 -Leu 166 ), and two large N-and C-terminal loops. It is stabilized by two tetrahedrally coordinated zinc ions. This structure is similar to that of other RING finger domains of proteins of known function. From structural analogies, we inferred that the EL5 RING-H2 finger is a binding domain for ubiquitin-conjugating enzyme (E2). The binding site is probably formed by solvent-exposed hydrophobic residues of the N-and C-terminal loops and the ␣-helix. We demonstrated that the fusion protein with EL5-(96 -181) and maltose-binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a rice E2 protein, OsUBC5b. This supported the idea that the EL5 RING finger domain is essential for ubiquitin-ligase activity of EL5. By NMR titration experiments, we identified residues that are critical for the interaction between the EL5 RING-H2 finger and OsUBC5b. We conclude that the RING-H2 finger domain of EL5 is the E2 binding site of EL5.Upon sensing the invasion of microorganisms, plants evoke a variety of defense reactions, including the synthesis of antimicrobial compounds (phytoalexins) and proteins. Many of these biochemical reactions are based on the activation of defenserelated genes. In some cases, the level of protein accumulation and the rapidity of gene induction in the host plant are correlated to the degree of its disease resistance. Therefore, it might be possible to control disease resistance by modifying the regulatory factors for the expression of defense-related genes.Such regulatory factors could be elements of signal transduction pathways leading from the recognition of invading pathogens to the activation of defense-related genes. Most of the defense responses are reproducible in suspension-cultured cells treated with specific substances called elicitor (1). Chitin fragments (N-acetylchitooligosaccharides) can act as elicitors (2), which induce the transient expression of several "early responsive" genes, such as EL5 (3). EL5 is a RING finger protein, which is structurally related to proteins of the Arabidopsis ATL family. These proteins are characterized by a transmembrane domain (domain I), basic domain (domain II), conserved domain (domain III), and RING-H2 finger domain (domain IV) followed by the C-terminal region with highly diverse amino acid sequences (4). Although some ATL family genes resemble EL5 in being induced in early stages of the defense responses (5), their biochemical function is obscure. Recently, it was shown that the fusion protein of EL5 with maltose-binding protein (MBP) 1 was polyubiquitinated by incubation with ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2). ...

EL5 is a rice ubiquitin-protein isopeptide ligase (E3) containing a RING-H2 finger domain that interacts with Oryza sativa (Os) UBC5b, a rice ubiquitin carrier protein. We introduced point mutations into the EL5 RING-H2 finger so that residues that functionally interact with OsUBC5b could be identified when assayed for ubiquitination activity in vitro. The residue positions were selected based on the results of an EL5 RING-H2 finger/OsUBC5b NMR titration experiment. These RING-H2 finger residues form or are adjacent to a shallow groove that is recognized by OsUBC5b. The E3 activity of EL5 is shown to be dependent on a Trp located at the center of the groove. We classified rice RING fingers according to the type of metal-chelating motif, i.e. RING-H2 or RING-HC, and according to the presence or absence of a conserved EL5-like Trp. We discuss the probable relationship between E3 activity and the conserved Trp.The RING finger motif, found in many functionally distinct proteins, was first identified as the protein product of the human gene RING1 (Really Interesting New Gene 1) (1). The RING finger motif is defined by the consensus sequence Cys-X 2 -Cys-X 9 -39 -Cys-X 1-3 -His-X 2-3 -(Cys/ His)-X 2 -Cys-X 4 -48 -Cys-X 2 -Cys, where X is any amino acid and the number of X residues varies in different fingers. Two types of RING finger motifs are distinguished by a cysteine (RING-HC) or histidine (RING-H2) as the fifth metal-chelating residue. A RING finger typically binds two zinc atoms, with its Cys and/or His side chains in a unique "cross-brace" arrangement. The nearly invariant spacing between the second and third pairs of Cys/His residues probably conserves the distance between the two metal-chelating sites (2). RING fingers are commonly found in proteins that are involved in cell growth and differentiation (3). Some RING fingers may be required for protein association, e.g. homo-or heterodimerization, whereas others are needed for ubiquitination (4 -6). The ubiquitination product is an isopeptide bond between the C-terminal carboxyl of ubiquitin (Gly 76 ) and a substrate lysine ⑀-amino group. Ubiquitination requires three sequential enzymatic reactions: (i) a ubiquitin-activating enzyme (E1) 2 forms a thiol ester between one of its cysteines and the ubiquitin Gly 76 carboxyl; (ii) then a conjugating enzyme (ubiquitin carrier protein (E2)) transiently carries the ubiquitin (as a thiol ester); and (iii) a ubiquitin-protein isopeptide ligase (E3) helps to transfer the activated ubiquitin from E2 to the substrate Lys. Generally, eukaryotic cells contain a single type of E1, multiple types of E2, and many different E3 enzymes. Efficient and targeted ubiquitination depends on E3. All known E3 enzymes have one of two E2-binding domains: the RING finger domain or the HECT domain (7). The three-dimensional structures of E3-type RING fingers have been determined by NMR spectroscopy (8 -13) or x-ray diffraction (14 -16). These structures all have a groove formed by the first zincbinding loop (N-loop; Cys 134 -Cys 137 of the...

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In a previous study, we demonstrated that β1,3-N-acetylglucosaminyltransferase 5 (B3gnt5) is a lactotriaosylceramide (Lc 3 Cer) synthase that synthesizes a precursor structure for lacto/neolacto-series glycosphingolipids (GSLs) in in vitro experiments. Here, we generated B3gnt5-deficient (B3gnt5 −/− ) mice to investigate the in vivo biological functions of lacto/neolacto-series GSLs. In biochemical analyses, lacto/neolacto-series GSLs were confirmed to be absent and no Lc 3 Cer synthase activity was detected in the tissues of these mice. These results demonstrate that β3GnT5 is the sole enzyme synthesizing Lc 3 Cer in vivo. Ganglioside GM1, known as a glycosphingolipid-enriched microdomain (GEM) marker, was found to be upregulated in B3gnt5 −/− B cells by flow cytometry and fluorescence microscopy. However, no difference in the amount of GM1 was observed by TLC-immunoblotting analysis. The GEM-stained puncta on the surface of B3gnt5 −/− resting B cells were brighter and larger than those of WT cells. These results suggest that structural alteration of GEM occurs in B3gnt5 −/− B cells. We next examined whether BCR signaling-related proteins, such as BCR, CD19, and the signaling molecule Lyn, had moved into or out of the GEM fraction. In B3gnt5 −/− B cells, these molecules were enriched in the GEM fraction or adjacent fraction. Moreover, B3gnt5 −/− B cells were more sensitive to the induction of intracellular phosphorylation signals on BCR stimulation and proliferated more vigorously than WT B cells. Together, these results suggest that lacto/neolacto-series GSLs play an important role in clustering of GEMs and tether-specific proteins, such as BCR, CD19, and related signaling molecules to the GEMs.A lmost all organisms possess lipids and proteins to which a broad range of carbohydrate chains are linked. Some carbohydrate structures are known to participate in vital processes, such as the molecules responsible for cell-cell, receptor-ligand, and carbohydrate-carbohydrate interactions. It is known that glycosphingolipids (GSLs) have an important role in biological functions. In mammals, GSLs can be classified into several major classes, such as globo-, isoglobo-, ganglio-, and lacto/neolacto-

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In North China and Mongolia, Gueldenstaedtia multiflora BGE. (Leguminosae) is used as an internal or external antiphlogistic, analgesics, and antiicterus agent. Only one reference 1) describing the isolation of soyasapogenols B and E, and flavonoids from G. multiflora was found. Therefore, since details are required for our systematic search for constituents of leguminous plants to discover new compounds, 2) we have started to isolate the constituents in this plant and obtained 15 compounds (GM-1-GM-15) including six new triterpene glycosides from the whole plants and roots. Results and DiscussionThe MeOH extract of the whole plants of G. mulutiflora (1.27 kg) was separated using Diaion HP-20 to give fr. 1 (60% MeOH eluate) and 2 (80% MeOH eluate). The 60% eluate was subjected to HPLC (ODS) to afford GM-11. The 80% MeOH was chromatographed on a Sephadex LH 20 column with 80% MeOH to provide the total isoflavonoid fraction and total saponin fraction, the latter of which was then separated by using MCI gel (60% MeOH-80% MeOH), ODS, and silica gel chromatography to give GM-1-GM-10. The MeOH extract (111.35 g) of the roots of this plant (1.5 kg) was passed through Diaion HP-20 (eluted first with water and next MeOH). A part (10 g) of the MeOH eluate (43.35 g) was subjected to MCI gel chromatography (eluted successively with 40% MeOH-50% MeOH-60% MeOH-70% MeOH-80% MeOH). The 60% MeOH eluate was further separated with ODS, silica gel and Sephadex LH-20 chromatography to afford GM-3, GM-12, GM-13, and GM-14. The 70% MeOH eluate gave GM-1 and GM-3 upon ODS separation. From the 80% MeOH eluate, GM-1 and GM-15 were obtained by using silica gel separation. GM-1, GM-2, GM-3, GM-4, GM-6, GM-7, GM-12, GM-13, and GM-15 were identified as soyasaponin I, azukisaponin V, comploside II, kudzusaponin SB 1 , 22-dehydroazukisaponin V, dehydrosoyasaponin I, medicalpin 3-O-b-D-glucopyranoside, subproside V, and soyasaponin Eg, respectively, by comparison with the various data including the 13 C-NMR spectral data. 16.4, 16.9, 18.1, 19.4, 21.2, 26.1, 26.3, 30.1, 30.8, 32.2, 34.2, 36.8, 36.9, 38.3, 39.2, 41.1, 42.8, 43.5, 47.4, 47.7, 49.8, 50.9, 57.0, 63.9, 81.0, 110.0, 150.9, and 175.2; and five anomeric-carbon signals at d 95.1, 101.5, 102.5, 103.7, and 104.5. The HMBC (Fig. 1) From the whole plants and the roots of Gueldenstaedtia multiflora, which has been used in traditional Chinese medicine, five new oleanane glycosides and one lupane glycoside were isolated together with eight known oleanane glycosides and a medicarpin derivative. These structures were determined based on MS and 2D-NMR spectra.