Multiomics in grape berry skin revealed specific induction of the stilbene synthetic pathway by ultraviolet-C irradiation.

Grape (Vitis vinifera) accumulates various polyphenolic compounds, which protect against environmental stresses, including ultraviolet-C (UV-C) light and pathogens. In this study, we looked at the transcriptome and metabolome in grape berry skin after UV-C irradiation, which demonstrated the effectiveness of omics approaches to clarify important traits of grape. We performed transcriptome analysis using a genome-wide microarray, which revealed 238 genes up-regulated more than 5-fold by UV-C light. Enrichment analysis of Gene Ontology terms showed that genes encoding stilbene synthase, a key enzyme for resveratrol synthesis, were enriched in the up-regulated genes. We performed metabolome analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry, and 2,012 metabolite peaks, including unidentified peaks, were detected. Principal component analysis using the peaks showed that only one metabolite peak, identified as resveratrol, was highly induced by UV-C light. We updated the metabolic pathway map of grape in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and in the KaPPA-View 4 KEGG system, then projected the transcriptome and metabolome data on a metabolic pathway map. The map showed specific induction of the resveratrol synthetic pathway by UV-C light. Our results showed that multiomics is a powerful tool to elucidate the accumulation mechanisms of secondary metabolites, and updated systems, such as KEGG and KaPPA-View 4 KEGG for grape, can support such studies.

Omics studies of citrus, grape and rosaceae fruit trees.

Recent advance of bioinformatics and analytical apparatuses such as next generation DNA sequencer (NGS) and mass spectrometer (MS) has brought a big wave of comprehensive study to biology. Comprehensive study targeting all genes, transcripts (RNAs), proteins, metabolites, hormones, ions or phenotypes is called genomics, transcriptomics, proteomics, metabolomics, hormonomics, ionomics or phenomics, respectively. These omics are powerful approaches to identify key genes for important traits, to clarify events of physiological mechanisms and to reveal unknown metabolic pathways in crops. Recently, the use of omics approach has increased dramatically in fruit tree research. Although the most reported omics studies on fruit trees are transcriptomics, proteomics and metabolomics, and a few is reported on hormonomics and ionomics. In this article, we reviewed recent omics studies of major fruit trees, i.e. citrus, grapevine and rosaceae fruit trees. The effectiveness and prospects of omics in fruit tree research will as well be highlighted.

Cleavage of the interchain disulfide bonds in rituximab increases its affinity for FcγRIIIA.

The Fc region of human IgG1 mediates effector function via binding to Fcγ receptors and complement activation. The H and L chains of IgG1 antibodies are joined by four interchain disulfide bonds. In this study, these bonds within the therapeutic IgG1 rituximab (RTX) were cleaved either by mild reduction followed by alkylation or by mild S-sulfonation; consequently, two modified RTXs - A-RTX (alkylated) and S-RTX (S-sulfonated) - were formed, and both were almost as potent as unmodified RTX when binding CD20 antigen. Unexpectedly, each modified RTX had a higher binding affinity for FcγRIIIA (CD16A) than did unmodified RTX. However, S-RTX and A-RTX were each less potent than RTX in an assay of antibody-dependent cellular cytotoxicity (ADCC). In this ADCC assay, each modified RTX showed decreased secretion of granzyme B, but no change in perforin secretion, from effector cells. These results provide significant information on the structures within IgG1 that are involved in binding FcγRIIIA, and they may be useful in the development of therapeutic antagonists for FcγRIIIA.

Both the Fab and Fc domains of IgG are essential for ROS emission from TNF-α-primed neutrophils by IVIG.

Intravenous immunoglobulin (IVIG) is currently a very important therapeutic used for not only infectious diseases, but also for autoimmune diseases such as idiopathic thrombocytopenic purpura (ITP). Untoward reactions of IVIG have been thought to result from complement activation by aggregated IgG in IVIG. In addition, the aggregates have been known to activate neutrophils, which may result in the untoward reactions. However, the effect and mechanism of IVIG on neutrophils remain unclear. In this study, we investigated the activation of neutrophils by IVIG in terms of their reactive oxygen species (ROS) emission to elucidate the mechanisms. IVIG-induced ROS emission from purified neutrophils was remarkably augmented by TNF-α priming of the cells. The ROS emission from TNF-α-primed neutrophils occurred by activation with whole gammaglobulin (GG) molecules, but not F(ab')(2), Fc, or a mixture of F(ab')(2) and Fc. ROS emission by GG was inhibited by the F(ab')(2) fragment and an inhibitory antibody against FcγRIII. These results suggest that binding of IVIG to not only surface antigen(s), but also FcγRIII on neutrophils, is involved in IVIG-induced ROS emission from TNF-α-primed neutrophils, and contribute to the untoward reactions of IVIG.

Chemically dimerized intravenous immunoglobulin has potent ameliorating activity in a mouse immune thrombocytopenic purpura model.

High-dose intravenous immunoglobulin (IVIG) preparations are currently used for the treatment of autoimmune diseases such as immune thrombocytopenic purpura (ITP). Although the mechanisms of IVIG efficacy remain enigmatic, some clinical and laboratory studies suggest that interaction of the Fc domain of IgG, especially the Fc domain of dimeric IgG, with its receptors (Fc gamma receptors; FcγRs) plays an essential role. In this study, IVIG was dimerized with chemical crosslinkers to augment its therapeutic efficacy. Dimerized IVIG was found to have a much higher affinity for FcγRs than monomeric IVIG. In a mouse ITP model, chemically dimerized IVIG abrogated the decrease in platelet numbers in the blood that was caused by an anti-platelet antibody at a dose that was one tenth of the required dose of IVIG. These results suggest that chemical dimerization of IVIG should greatly improve the efficacy of IVIG therapy of ITP.

Leptospira infection at the University of Peradeniya Teaching Hospital, Sri Lanka: clinical and laboratory investigations.

To help formulate a local intervention for leptospirosis in Sri Lanka, we determined the serogroups of leptospiral species among 97 patients diagnosed with leptospirosis at the University of Peradeniya Teaching Hospital, Sri Lanka. Ninety-two point eight percent of the patients were men; nearly two-thirds were > or = 35 years old; the majority had secondary or higher education level, half were farmers or laborers; and 57.7% presented in the acute-phase of the illness. Twenty-five patients (25.8%) were confirmed to have leptospirosis by a positive laboratory method; 17 and 8 cases were confirmed with a positive test by quantitative MAT and nested PCR, respectively. Of the 17 MAT positive cases, infection occurred in a variety of serogroups, but the predominant groups were Sejroe and Tarassovi. Of the 8 nested PCR positive cases, 7 were seen among those with a MAT titer <200 and 1 occurred in a patient with a MAT titer > or = 200 but <400. Of the 8 PCR positive cases, 7 were infected with the leptospiral species L. interrogans. Approximately 26% of the clinically diagnosed patients were confirmed by the two laboratory methods. Laboratory positivity was based on the time of blood collection after the onset of fever. Further studies are warranted to refine the clinical diagnostic criteria and to develop more efficient and accurate diagnostic tests for leptospirosis in resource limited settings.

Evola: Ortholog database of all human genes in H-InvDB with manual curation of phylogenetic trees.

Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Currently, with the rapid growth of transcriptome data of various species, more reliable orthology information is prerequisite for further studies. However, detection of orthologs could be erroneous if pairwise distance-based methods, such as reciprocal BLAST searches, are utilized. Thus, as a sub-database of H-InvDB, an integrated database of annotated human genes (http://h-invitational.jp/), we constructed a fully curated database of evolutionary features of human genes, called 'Evola'. In the process of the ortholog detection, computational analysis based on conserved genome synteny and transcript sequence similarity was followed by manual curation by researchers examining phylogenetic trees. In total, 18 968 human genes have orthologs among 11 vertebrates (chimpanzee, mouse, cow, chicken, zebrafish, etc.), either computationally detected or manually curated orthologs. Evola provides amino acid sequence alignments and phylogenetic trees of orthologs and homologs. In 'd(N)/d(S) view', natural selection on genes can be analyzed between human and other species. In 'Locus maps', all transcript variants and their exon/intron structures can be compared among orthologous gene loci. We expect the Evola to serve as a comprehensive and reliable database to be utilized in comparative analyses for obtaining new knowledge about human genes. Evola is available at http://www.h-invitational.jp/evola/.

The Rice Annotation Project Database (RAP-DB): 2008 update.

The Rice Annotation Project Database (RAP-DB) was created to provide the genome sequence assembly of the International Rice Genome Sequencing Project (IRGSP), manually curated annotation of the sequence, and other genomics information that could be useful for comprehensive understanding of the rice biology. Since the last publication of the RAP-DB, the IRGSP genome has been revised and reassembled. In addition, a large number of rice-expressed sequence tags have been released, and functional genomics resources have been produced worldwide. Thus, we have thoroughly updated our genome annotation by manual curation of all the functional descriptions of rice genes. The latest version of the RAP-DB contains a variety of annotation data as follows: clone positions, structures and functions of 31 439 genes validated by cDNAs, RNA genes detected by massively parallel signature sequencing (MPSS) technology and sequence similarity, flanking sequences of mutant lines, transposable elements, etc. Other annotation data such as Gnomon can be displayed along with those of RAP for comparison. We have also developed a new keyword search system to allow the user to access useful information. The RAP-DB is available at: http://rapdb.dna.affrc.go.jp/ and http://rapdb.lab.nig.ac.jp/.

The H-Invitational Database (H-InvDB), a comprehensive annotation resource for human genes and transcripts.

Here we report the new features and improvements in our latest release of the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/), a comprehensive annotation resource for human genes and transcripts. H-InvDB, originally developed as an integrated database of the human transcriptome based on extensive annotation of large sets of full-length cDNA (FLcDNA) clones, now provides annotation for 120 558 human mRNAs extracted from the International Nucleotide Sequence Databases (INSD), in addition to 54 978 human FLcDNAs, in the latest release H-InvDB_4.6. We mapped those human transcripts onto the human genome sequences (NCBI build 36.1) and determined 34 699 human gene clusters, which could define 34 057 (98.1%) protein-coding and 642 (1.9%) non-protein-coding loci; 858 (2.5%) transcribed loci overlapped with predicted pseudogenes. For all these transcripts and genes, we provide comprehensive annotation including gene structures, gene functions, alternative splicing variants, functional non-protein-coding RNAs, functional domains, predicted sub cellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs, co-localization with orphan diseases, gene expression profiles, orthologous genes, protein-protein interactions (PPI) and annotation for gene families. The current H-InvDB annotation resources consist of two main views: Transcript view and Locus view and eight sub-databases: the DiseaseInfo Viewer, H-ANGEL, the Clustering Viewer, G-integra, the TOPO Viewer, Evola, the PPI view and the Gene family/group.

Practical preparation of UDP-apiose and its applications for studying apiosyltransferase.

UDP-apiose, a donor substrate of apiosyltransferases, is labile because of its intramolecular self-cyclization ability, resulting in the formation of apiofuranosyl-1,2-cyclic phosphate. Therefore, stabilization of UDP-apiose is indispensable for its availability and identifying and characterizing the apiosyltransferases involved in the biosynthesis of apiosylated sugar chains and glycosides. Here, we established a method for stabilizing UDP-apiose using bulky cations as counter ions. Bulky cations such as triethylamine effectively suppressed the degradation of UDP-apiose in solution. The half-life of UDP-apiose was increased to 48.1 ±â€¯2.4 h at pH 6.0 and 25 °C using triethylamine as a counter cation. UDP-apiose coordinated with a counter cation enabled long-term storage under freezing conditions. UDP-apiose was utilized as a donor substrate for apigenin 7-O-ß-D-glucoside apiosyltransferase to produce the apiosylated glycoside apiin. This apiosyltransferase assay will be useful for identifying genes encoding apiosyltransferases.

Genome-wide analysis of ATP binding cassette (ABC) transporters in tomato.

ATP binding cassette (ABC) transporters are proteins that actively mediate the transport of a wide range of molecules, such as organic acids, metal ions, phytohormones and secondary metabolites. Therefore, ABC transporters must play indispensable roles in growth and development of tomato, including fruit development. Most ABC transporters have transmembrane domains (TMDs) and belong to the ABC protein family, which includes not only ABC transporters but also soluble ABC proteins lacking TMDs. In this study, we performed a genome-wide identification and expression analysis of genes encoding ABC proteins in tomato (Solanum lycopersicum), which is a valuable horticultural crop and a model plant for studying fleshy fruits. In the tomato genome, a total of 154 genes putatively encoding ABC transporters, including 9 ABCAs, 29 ABCBs, 26 ABCCs, 2 ABCDs, 2 ABCEs, 6 ABCFs, 70 ABCGs and 10 ABCIs, were identified. Gene expression data from the eFP Browser and reverse transcription-semi-quantitative PCR analysis revealed their tissue-specific and development-specific expression profiles. This work suggests physiological roles of ABC transporters in tomato and provides fundamental information for future studies of ABC transporters not only in tomato but also in other Solanaceae species.

Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis.

The Fc domain of human IgG1 binds to Fcγ receptors (FcγRs) to induce effector functions such as phagocytosis. There are four interchain disulfide bonds between the H and L chains. In this study, the disulfide bonds within the IgG1 trastuzumab (TRA), which is specific for HER2, were cleaved by mild S-sulfonation or by mild reduction followed by S-alkylation with three different reagents. The cleavage did not change the binding activities of TRA to HER2-bearing SK-BR-3 cells. The binding activities of TRA to FcγRIIA and FcγRIIB were greatly enhanced by modification with mild reduction and S-alkylation with ICH2CONH2 or N-(4-aminophenyl) maleimide, while the binding activities of TRA to FcγRI and FcγRIIIA were decreased by any of the four modifications. However, the interchain disulfide bond cleavage by the different modifications did not change the antibody-dependent cell-mediated phagocytosis (ADCP) of SK-BR-3 cells by activated THP-1 cells. The order of FcγR expression levels on the THP-1 cells was FcγRII > FcγRI > FcγRIII and ADCP was inhibited by blocking antibodies against FcγRI and FcγRII. These results imply that the effect of the interchain disulfide bond cleavage on FcγRs binding and ADCP is dependent on modifications of the cysteine residues and the FcγR isotypes.

Simultaneous paresthesia of the lingual nerve and inferior alveolar nerve caused by a radicular cyst.

The inferior alveolar nerve is sometimes affected by periapical pathoses and mandibular cysts. However, mandibular intraosseous lesions have not been reported to disturb the lingual nerve. A case of simultaneous paresthesia of the right lingual nerve and the right inferior alveolar nerve is presented. The possible mechanisms of this extremely uncommon condition are discussed.

M(3) receptor antagonism by the novel antimuscarinic agent solifenacin in the urinary bladder and salivary gland.

The antimuscarinic profile of the experimental drug solifenacin/YM905 [(+)-(1 S,3' R)-quinuclidin-3'-yl 1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-carboxylate] for the treatment of overactive bladder was compared with the commonly prescribed agent oxybutynin. In radioligand binding assays, p K(i) values of solifenacin for M(1), M(2), and M(3) receptors were 7.6, 6.9, and 8.0, respectively. These values for oxybutynin were 8.6 (M(1)), 7.7 (M(2)), and 8.9 (M(3)). Solifenacin and oxybutynin antagonized the contractile effect of carbachol (CCh) on isolated guinea pig urinary bladder smooth muscle (detrusor), displaying the negative logarithm of antagonist apparent affinity constant (p K(b) value) of 7.1 for solifenacin and 7.4 for oxybutynin. To study the tissue selectivity between bladders and salivary glands, guinea pig detrusor and mouse submandibular gland cells were stimulated with CCh and monitored for intracellular Ca2+, as determined by Fura 2 fluorescence. Ca2+ mobilization of detrusor cells was inhibited equipotently by solifenacin (p K(i)=8.4) and oxybutynin (p K(i)=8.6), whereas that of the gland cells was antagonized less potently by solifenacin (p K(b)=7.4) than by oxybutynin (p K(b)=8.8), although the M(3) subtype mediated both cell responses. In anesthetized rats, solifenacin (63-2100 nmol kg(-1) or 0.03-1 mg kg(-1)) dose-dependently inhibited CCh-stimulated increases in urinary bladder pressure, while its inhibitory effects on salivation and bradycardia were apparent only at a dose of 2100 nmol kg(-1). In contrast, oxybutynin within a dose range of 77-770 nmol kg(-1) (0.03-0.3 mg kg(-1)) inhibited responses of the bladder and salivary gland slightly more potently than that of the heart. In addition, inhibitory effects of darifenacin indicated a major role of M(3) receptors in the bladder and salivary gland. Therefore, M(3) receptor antagonism by solifenacin could be bladder-selective. This selectivity remains to be elucidated and may provide new approaches to the pharmacotherapy of overactive bladder.

Darexaban: anticoagulant effects in mice and human plasma in vitro, antithrombotic effects in thrombosis and bleeding models in mice and effects of anti-inhibitor coagulant complex and recombinant factor VIIa.

Here, we investigated the anticoagulant effects of darexaban in mice and human plasma in vitro, effects of darexaban in thrombosis and bleeding models in mice, and reversal effects of anti-inhibitor coagulant complex (ACC) and recombinant factor VIIa (rFVIIa) on anticoagulant effects of darexaban. In mice, darexaban inhibited FXa activity in plasma with an ED50 value of 24.8 mg/kg. Both darexaban and warfarin prolonged prothrombin time (PT) at 3 mg/kg and 0.3 mg/kg/day, respectively. PT and activated partial thromboplastin time (aPTT) prolonged by darexaban were dose-dependently reversed by intravenously-administered rFVIIa, significantly so at 1 mg/kg. In a pulmonary thromboembolism (PE) mouse model, both darexaban and warfarin dose-dependently reduced the mortality rate, significantly so at 10 mg/kg and 3 mg/kg/day, respectively. In a FeCl3-induced venous thrombosis (VT) mouse model, darexaban (0.3-10 mg/kg) dose-dependently decreased the thrombus protein content, significantly so at doses of 3 mg/kg or higher. In a tail-transection mouse model, darexaban had no significant effect on the amount of blood loss at doses up to 10 mg/kg, while warfarin showed a dose-dependent increase in blood loss, significantly so from 1 mg/kg/day. Darexaban and its metabolite darexaban glucuronide significantly prolonged PT and aPTT in human plasma in vitro, and while rFVIIa concentration-dependently reversed the prolonged PT in this plasma, ACC dose-dependently reversed both PT and aPTT changes prolonged by darexaban. Taken together, these results suggest that darexaban has a potential to be an oral anticoagulant with a better safety profile than warfarin, and that rFVIIa and ACC may be useful as antidotes to darexaban in cases of overdose.

PCDq: human protein complex database with quality index which summarizes different levels of evidences of protein complexes predicted from h-invitational protein-protein interactions integrative dataset.

BACKGROUND: Proteins interact with other proteins or biomolecules in complexes to perform cellular functions. Existing protein-protein interaction (PPI) databases and protein complex databases for human proteins are not organized to provide protein complex information or facilitate the discovery of novel subunits. Data integration of PPIs focused specifically on protein complexes, subunits, and their functions. Predicted candidate complexes or subunits are also important for experimental biologists. DESCRIPTION: Based on integrated PPI data and literature, we have developed a human protein complex database with a complex quality index (PCDq), which includes both known and predicted complexes and subunits. We integrated six PPI data (BIND, DIP, MINT, HPRD, IntAct, and GNP_Y2H), and predicted human protein complexes by finding densely connected regions in the PPI networks. They were curated with the literature so that missing proteins were complemented and some complexes were merged, resulting in 1,264 complexes comprising 9,268 proteins with 32,198 PPIs. The evidence level of each subunit was assigned as a categorical variable. This indicated whether it was a known subunit, and a specific function was inferable from sequence or network analysis. To summarize the categories of all the subunits in a complex, we devised a complex quality index (CQI) and assigned it to each complex. We examined the proportion of consistency of Gene Ontology (GO) terms among protein subunits of a complex. Next, we compared the expression profiles of the corresponding genes and found that many proteins in larger complexes tend to be expressed cooperatively at the transcript level. The proportion of duplicated genes in a complex was evaluated. Finally, we identified 78 hypothetical proteins that were annotated as subunits of 82 complexes, which included known complexes. Of these hypothetical proteins, after our prediction had been made, four were reported to be actual subunits of the assigned protein complexes. CONCLUSIONS: We constructed a new protein complex database PCDq including both predicted and curated human protein complexes. CQI is a useful source of experimentally confirmed information about protein complexes and subunits. The predicted protein complexes can provide functional clues about hypothetical proteins. PCDq is freely available at http://h-invitational.jp/hinv/pcdq/.

Biochemical and pharmacological profile of darexaban, an oral direct factor Xa inhibitor.

Darexaban (YM150) is an oral factor Xa inhibitor developed for the prophylaxis of venous and arterial thromboembolic disease. This study was conducted to investigate the biochemical and pharmacological profiles of darexaban and its active metabolite darexaban glucuronide (YM-222714), which predominantly determines the antithrombotic effect after oral administration of darexaban. In vitro activity was evaluated by enzyme and coagulation assays, and a prothrombin activation assay using reconstituted prothrombinase or whole blood clot. In vivo effects were examined in venous thrombosis, arterio-venous (A-V) shunt thrombosis, and bleeding models in rats. Both darexaban and darexaban glucuronide competitively and selectively inhibited human factor Xa with Ki values of 0.031 and 0.020 µM, respectively. They showed anticoagulant activity in human plasma, with doubling concentrations of darexaban and darexaban glucuronide for prothrombin time of 1.2 and 0.95 µM, respectively. Anticoagulant activity was independent of antithrombin. Darexaban and darexaban glucuronide inhibited the prothrombin activation induced by prothrombinase complex or whole blood clot with similar potency to free factor Xa. In contrast, prothrombinase- and clot-induced prothrombin activation were resistant to inhibition by enoxaparin. In venous and A-V shunt thrombosis models in rats, darexaban strongly suppressed thrombus formation without affecting bleeding time, with ID50 values of 0.97 and 16.7 mg/kg, respectively. Warfarin also suppressed thrombus formation in these models, but caused a marked prolongation of bleeding time at antithrombotic dose. In conclusion, darexaban is a selective and direct factor Xa inhibitor and a promising oral anticoagulant for the prophylaxis and treatment of thromboembolic diseases.

Carcinoma ex pleomorphic adenoma of the submandibular gland: report of a case with an unusual malignant component of clear cell squamous cell carcinoma.

A large carcinoma ex pleomorphic adenoma (CXPA) with regional lymph node metastasis occurred in the left submandibular gland of a 64-year-old man. Initially, the patient underwent tumor extirpation and ipsilateral radical neck dissection. Histologic examination revealed the malignant component of CXPA was clear cell squamous cell carcinoma (SCC), which is quite uncommon in head and neck region. In the metastatic regional lymph nodes, aggregates of clear cells were observed. To the best of our knowledge, this is the first case of CXPA of which the malignant component is clear cell SCC. Four months after the postoperative radiotherapy, delayed regional metastasis became evident in the contralateral side of the neck. Radical neck dissection was carried out, and, microscopically, moderately differentiated SCC was identified. The patient recovered well and remained free of disease for 29 months after the second surgery.

Identification of epitope on DNA-binding protein expressed in insect cell infected by baculovirus.

DNA-binding protein (DBP) is an early gene product produced during viral replication. Polyclonal anti-DBP was produced using rabbit by intradermal injections of Escherichia coli-expressed purified recombinant DBP. Prepared anti-DBP completely blocked the replication of baculovirus in insect cells. The anti-DBP binding to DBP was confirmed by both Western blotting with Tn-5B1-4 insect cell lysates as well as immunostained baculovirus-infected Tn-5B1-4 insect cells. To determine the anti-DBP epitope 12 peptides were synthesized and their specific-binding activities were measured using ELISA. Based on specific-binding activity against anti-DBP the epitope was predicted to be between amino acid residues 248-265 (QRMSVEDFDRLFEMDKID). Especially from 18 amino acid residues it was further to be narrowed between amino acid residues 260-265 (EMDKID) which showed a critical role in specific-binding activity.

The effects of N-glycosylation sites and the N-terminal region on the biological function of beta1,3-N-acetylglucosaminyltransferase 2 and its secretion.

Human beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is thought to be an enzyme that extends the polylactosamine acceptor chains, but its function and structure analysis are unknown. To obtain insight into the structure of beta3GnT2, the effects of N-glycosylation on its biological function were evaluated using the addition of inhibitors, site-directed mutagenesis of potential N-glycosylation sites, and deletion of its N-terminal region using a fusion protein with GFP(uv) in a baculovirus expression system. Four of five potential N-glycosylation sites were found to be occupied, and their biological function and secretion were inhibited with the treatment of N-glycosylation inhibitor, tunicamycin. The N-glycosylation at Asn219 was necessary for the beta3GnT activity; moreover, N-glycosylation at Asn127 and Asn219 was critical for efficient protein secretion. When Ser221 was replaced with Thr, fusion protein was expressed as a single band, indicating that the double band of the expressed fusion protein was due to the heterogeneity of the glycosylation at Asn219. The truncated protein consisting of amino acids 82-397 (GFP(uv)-beta3GnT2Delta83), which lacked both one N-glycosylation site at Asn79 and the stem region of glycosyltransferase, was expressed as only a small form and showed no beta3GnT activity. These results suggest that the N-glycosylation site at Asn219, which is conserved throughout the beta1,3-glycosyltransferase family, is indispensable not only with regard to its biological function, but also to its secretion. The N-terminal region, which belongs to a stem region of glycosyltransferase, might also be important to the active protein structure.