小鼠α1，2岩藻糖基转移酶基因的克隆及表达

本文报告小鼠GDP岩藻糖：β半乳糖苷α1,2-岩藻糖基转移酶（α1,2-fucosyltansferase,α1,2-FT）基因的克隆,并进行功能鉴定。利用RT—PCR方法克隆小鼠α1,2-岩藻糖基转移酶基因编码区MFUT-II,测序后将其插入表达载体pcDNA3．1的多克隆位点,构建表达载体pcDNA3．1-MFUT-II;采用磷酸钙法将其转染于COS-7细胞进行表达,通过对底物特异性比较研究酶的性质;应用Northern印迹杂交法研究基因在小鼠组织中的表达情况;应用Southern印迹杂交法分析基因存在状态。结果证实MFUT-II为小鼠α1,2-岩藻糖基转移酶基因家族的新成员。含有一个完整的开放读码框。可编码347个氨基酸。其估计分子质量为39kDa,和小鼠日及Sec1基因具有序列同源性。分别与人类Se基因（79．O％）、大鼠Ratrrs（89％）基因、兔Rabbit FT-III基因（77％）具有较高的序列同源性。用MFUT-II基因转染的COS-7细胞具有α1,2-FT活性。MFUT-II可在多种组织中产生3．5kb大小的mRNA转录产物。基因Southern印迹杂交分析结果显示：基因MFUT-II仅为一个拷贝。这些结果证明MFUT-II为小鼠的Se基因。     

Expression of Deltex1 during mouse embryogenesis: comparison with Notch1, 2 and 3 expression.

The Notch signalling pathway defines a phylogenetically conserved cell-cell communication process that enables cell-fate specification in multicellular organisms. Deltex is a component of the Notch signalling network that physically interacts with the ankyrin repeats of Notch. Here, we report on the expression pattern of the Deltex1 gene during mouse embryonic development and, furthermore, we compare its expression with that of the Notch1, 2 and 3 genes. Complementary and combinatorial expression patterns between Deltex1 and the three Notch genes were observed throughout embryogenesis since Deltex1 expression was related either to cytodifferentiation (i.e. neuronal tissues) or to cell proliferation events (i.e. eye, vascular structures, hematopoiesis).     

Stage-specific expression of mucosal addressin cell adhesion molecule-1 during embryogenesis in rats

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is essential for lymphocyte trafficking to gut-associated lymphoid tissues and is implicated in inflammatory disorders in the gut and pancreatic islets. In this study, we examined the functional role of MAdCAM-1 during rat ontogeny using newly generated specific mAb. As previously observed in mice and humans, MAdCAM-1 was preferentially expressed in high endothelial venules (HEV) in gut-associated lymphoid tissues and venules of lamina propria in adult rats. Lymphocyte rolling and adhesion on HEV in Peyer's patches (PP) were completely abrogated with neutralizing anti-MAdCAM-1 mAb, in agreement with the notion that MAdCAM-1 is the principal HEV ligand for lymphocyte rolling and adhesion in adult PP. In the developing gastrointestinal tract, MAdCAM-1 was widely expressed in the venules of the lamina propria of fetal rats. In addition, MAdCAM-1 was also expressed in follicular dendritic cells in the neonatal PP. Interestingly, MAdCAM-1 expression was found also in nonmucosal tissues during ontogeny. MAdCAM-1 was transiently expressed in blood vascular endothelial cells in the fetal skin and neonatal thymus. Notably, MAdCAM-1-positive blood vessels were localized mainly in the cortico-medullary junction in the neonatal thymus and about 10-20% of thymocytes, most of which were either CD4, CD8 double positive or single positive specifically reacted with soluble MAdCAM-1 via integrin alpha4beta7. After birth, MAdCAM-1 expression in thymus blood vessels disappeared and concomitantly, the soluble MAdCAM-1-reactive thymocytes were rapidly down-regulated. Our results suggest that MAdCAM-1 functions as a vascular addressin in not only mucosal, but also nonmucosal lymphoid tissues during ontogeny.     

Plant members of the alpha1-->3/4-fucosyltransferase gene family encode an alpha1-->4-fucosyltransferase, potentially involved in Lewis(a) biosynthesis, and two core alpha1-->3-fucosyltransferases.

Three putative alpha1-->3/4-fucosyltransferase (alpha1-->3/4-FucT) genes have been detected in the Arabidopsis thaliana genome. The products of two of these genes have been identified in vivo as core alpha1-->3-FucTs involved in N-glycosylation. An orthologue of the third gene was isolated from a Beta vulgaris cDNA library. The encoded enzyme efficiently fucosylates Galbeta1-->3GlcNAcbeta1-->3Galbeta1-->4Glc. Analysis of the product by 400 MHz (1)H-nuclear magnetic resonance spectroscopy showed that the product is alpha1-->4-fucosylated at the N-acetylglucosamine residue. In vitro, the recombinant B. vulgaris alpha1-->4-FucT acts efficiently only on neutral type 1 chain-based glycan structures. In plants the enzyme is expected to be involved in Lewis(a) formation on N-linked glycans.     

α1,2-岩藻糖基转移酶基因的克隆及在大肠杆菌中的表达

利用PCR方法从幽门螺旋杆菌(Helicobacter pylori,HP)基因组DNA中获得α1,2-岩藻糖基转移酶(α1,2-fuco-syltransferase,α1,2-fuct)基因,得到大小为906 bp的目的基因,将其定向插入到原核表达载体pET-22b(+)中,得到重组表达载体pET-fuct。将重组表达载体转化到大肠杆菌BL21(DE3)中,25℃,0.1 mmol/L异丙基硫代半乳糖苷(IPTG)诱导表达4 h,并用SDS-PAGE分析目的蛋白的表达情况。结果表明,可表达出相对分子质量为33 kD的蛋白,与预期分子量一致,说明α1,2-岩藻糖基转移酶在大肠杆菌BL21中实现表达,应用HPLC法进行酶活检验,酶活达到了13.21 pmol/(mgPr.h)。     

Differential expression of TGF beta 1, beta 2 and beta 3 genes during mouse embryogenesis

We have examined by Northern analysis and in situ hybridisation the expression of TGF beta 1, beta 2 and beta 3 during mouse embryogenesis. TGF beta 1 is expressed predominantly in the mesodermal components of the embryo e.g. the hematopoietic cells of both fetal liver and the hemopoietic islands of the yolk sac, the mesenchymal tissues of several internal organs and in ossifying bone tissues. The strongest TGF beta 2 signals were found in early facial mesenchyme and in some endodermal and ectodermal epithelial cell layers e.g., lung and cochlea epithelia. TGF beta 3 was strongest in prevertebral tissue, in some mesothelia and in lung epithelia. All three isoforms were expressed in bone tissues but showed distinct patterns of expression both spatially and temporally. In the root sheath of the whisker follicle, TGF beta 1, beta 2 and beta 3 were expressed simultaneously. We discuss the implication of these results in regard to known regulatory elements of the TGF beta genes and their receptors.     

Expression of estrogen receptor alpha and beta during mouse embryogenesis.

In adult mammals numerous target tissues and organs for estrogens exist. Little is known about possible target organs during embryogenesis other than the reproductive tract and the gonads. This is the first report on the expression of estrogen receptor beta (ERbeta) in comparison with ERalpha mRNA during mouse embryogenesis. We found expression of estrogen receptor mRNA in the reproductive tract, but also in the atrial wall, brain, kidney, urethra, bladder neck, mammary gland primordium, midgut, cartilage primordia and perichondria.     

Cloning and expression of the mouse dual-specificity mitogen-activated protein (MAP) kinase phosphatase Mkp3 during mouse embryogenesis

Mitogen-activated protein (MAP) kinase phosphatases (MKPs) constitute a growing family of dual specificity phosphatases, which dephosphorylate both serine/threonine and tyrosine residues of MAP kinases. MAP kinase signaling cascades are involved in the control of cell proliferation, differentiation and apoptosis. In mammals, ten members of the dual-specificity MKP family have so far been identified. In this report, we describe the cloning and expression analysis of the mouse Mkp3 gene. During early development, expression of Mkp3 is most prominent in the primitive streak, presomitic mesoderm and somites, frontonasal prominence, midbrain/hindbrain boundary, branchial arches and limb buds. At later stages, expression is also detected in the tooth primordia, vibrissae, hair follicles, pinna, submandibular gland, mammary gland primordia, lung and kidney. Strong expression was detected in the adult brain.     

Molecular cloning and characterization of a novel alpha 1,2-fucosyltransferase (CE2FT-1) from Caenorhabditis elegans

Here we report the discovery of a unique fucosyltransferase (FT) in Caenorhabditis elegans. In studying the activities of FTs in extracts of adult C. elegans, we detected activity toward the unusual disaccharide acceptors Galbeta1-4Xyl-R and Galbeta1-6GlcNAc-R to generate products with the general structure Fucalpha1-2Galbeta1-R. We identified a gene encoding a unique alpha1,2FT (designated CE2FT-1), which contains an open reading frame encoding a predicted protein of 355 amino acids with the type 2 topology and domain structure typical of other glycosyltransferases. The predicted cDNA for CE2FT-1 has very low identity (5-10%) at the amino acid level to alpha1,2FT sequences in humans, rabbits, and mice. Recombinant CE2FT-1 expressed in human 293T cells has high alpha1,2FT activity toward the simple acceptor Galbeta-O-phenyl acceptor to generate Fucalpha1-2Galbeta-R, which in this respect resembles mammalian alpha1,2FTs. However, CE2FT-1 is otherwise completely different from known alpha1,2FTs in its acceptor specificity, since it is unable to fucosylate either Galbeta1-4Glcbeta-R or free lactose and prefers the unusual acceptors Galbeta1-4Xylbeta-R and Galbeta1-6GlcNAc-R. Promoter analysis of the CE2FT-1 gene using green fluorescent protein reporter constructs demonstrates that CE2FT-1 is expressed in single cells of early stage embryos and exclusively in the 20 intestinal cells of L(1)-L(4) and adult worms. These and other results suggest that multiple fucosyltransferase genes in C. elegans may encode enzymes with unique activities, expression, and developmental roles.     

Tissue specific characterisation of Lim-kinase 1 expression during mouse embryogenesis

The Lim-kinase (LIMK) proteins are important for the regulation of the actin cytoskeleton, in particular the control of actin nucleation and depolymerisation via regulation of cofilin, and hence may control a large number of processes during development, including cell tensegrity, migration, cell cycling, and axon guidance. LIMK1/LIMK2 knockouts disrupt spinal cord morphogenesis and synapse formation but other tissues and developmental processes that require LIMK are yet to be fully determined. To identify tissues and cell-types that may require LIMK, we characterised the pattern of LIMK1 protein during mouse embryogenesis. We showed that LIMK1 displays an expression pattern that is temporally dynamic and tissue-specific. In several tissues LIMK1 is detected in cell-types that also express Wilms' tumour protein 1 and that undergo transitions between epithelial and mesenchymal states, including the pleura, epicardium, kidney nephrons, and gonads. LIMK1 was also found in a subset of cells in the dorsal retina, and in mesenchymal cells surrounding the peripheral nerves. This detailed study of the spatial and temporal expression of LIMK1 shows that LIMK1 expression is more dynamic than previously reported, in particular at sites of tissue-tissue interactions guiding multiple developmental processes.     

A novel alpha1,2-fucosyltransferase (CE2FT-2) in Caenorhabditis elegans generates H-type 3 glycan structures

The 1,2-fucosyltransferase family (1,2FT) is the largest familyof glycosyltransferases in the genome of the free-living nematodeCaenorhabditis elegans, and early evidence suggests that eachmember may have a unique activity. Here we describe a C. elegansgene (designated CE2FT-2) encoding an 1,2FT that has the potentialto generate the sequence Fuc1-2Galβ1-3GalNAc-R, which isthe H-type 3 blood group structure. The CE2FT-2 cDNA encodesa putative transmembrane protein that shows 42% amino acid identityto a previously cloned C. elegans 1,2FT (termed CE2FT-1), buthas a very low identity (16–20%) to 1,2FT sequences inhumans, rabbits, and mice. A recombinant form of CE2FT-2 expressedin human 293T cells has a high 1,2FT activity toward Galβ1-3GalNAc-O-pNP,but unexpectedly, the enzyme is inactive toward the acceptorGalβ-O-phenyl. Thus, CE2FT-2 differs from all other 1,2FTspreviously described from animals that all utilize Galβ-O-phenyl.CE2FT-2 is expressed at all stages of worm development, butremarkably, promoter analysis of the CE2FT-2 gene using greenfluorescent protein reporter constructs indicates that the CE2FT-2is expressed exclusively in pharyngeal cells of the worm fromembryo to an adult stage. Because pharyngeal cells are knownto secrete their glycoconjugates to the nematode surface, theseresults may indicate that products of CE2FT-2 contribute tointeractions of the nematode with its environment or are usedas ligands for bacterial attachment. These findings, along withthose on other 1,2FTs in C. elegans, suggest that each 1,2FTin this organism may have a unique acceptor specificity, expressionpattern, and biological function.     

A non-Golgi alpha 1,2-fucosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium.

Skp1 is a subunit of the SCF-E3 ubiquitin ligase that targets cell cycle and other regulatory factors for degradation. In Dictyostelium, Skp1 is modified by a pentasaccharide containing the type 1 blood group H trisaccharide at its core. To address how the third sugar, fucose alpha1,2-linked to galactose, is attached, a proteomics strategy was applied to determine the primary structure of FT85, previously shown to copurify with the GDP-Fuc:Skp1 alpha 1,2-fucosyltransferase. Tryptic-generated peptides of FT85 were sequenced de novo using Q-TOF tandem mass spectrometry. Degenerate primers were used to amplify FT85 genomic DNA, which was further extended by a novel linker polymerase chain reaction method to yield an intronless open reading frame of 768 amino acids. Disruption of the FT85 gene by homologous recombination resulted in viable cells, which had altered light scattering properties as revealed by flow cytometry. FT85 was necessary and sufficient for Skp1 fucosylation, based on biochemical analysis of FT85 mutant cells and Escherichia coli that express FT85 recombinantly. FT85 lacks sequence motifs that characterize all other known alpha 1,2-fucosyltransferases and lacks the signal-anchor sequence that targets them to the secretory pathway. The C-terminal region of FT85 harbors motifs found in inverting Family 2 glycosyltransferase domains, and its expression in FT85 mutant cells restores fucosyltransferase activity toward a simple disaccharide substrate. Whereas most prokaryote and eukaryote Family 2 glycosyltransferases are membrane-bound and oriented toward the cytoplasm where they glycosylate lipid-linked or polysaccharide precursors prior to membrane translocation, the soluble, eukaryotic Skp1-fucosyltransferase modifies a protein that resides in the cytoplasm and nucleus.     

Molecular cloning and heterologous expression of beta1,2-xylosyltransferase and core alpha1,3-fucosyltransferase from maize

Maize is considered a promising alternative production system for pharmaceutically relevant proteins. However, like in all other plant species asparagine-linked oligosaccharides of maize glycoproteins are modified with beta1,2-xylose and core alpha1,3-fucose sugar residues, which are considered to be immunogenic in mammals. This altered N-glycosylation when compared to mammalian cells may reduce the potential of maize as a production system for heterologous glycoproteins. Here we report the cloning and characterization of the cDNA sequences coding for the maize enzymes beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT). The cloned XylT and FucT cDNAs were shown to encode enzymatically active proteins, which were independently able to convert a mammalian acceptor glycoprotein into an antigen binding anti-plant N-glycan antibodies. The complete sequence of the XylT gene was determined. Evidence for the presence of at least three XylT and FucT gene loci in the maize genome was obtained. The identification of the two enzymes and their genes will allow the targeted downregulation or even elimination of beta1,2-xylose and core alpha1,3-fucose addition to recombinant glycoproteins produced in maize.     

The expression of plexins during mouse embryogenesis

Plexins are large transmembrane proteins that are receptors for semaphorins, either alone or in a complex with neuropilin-1 or -2. Nine different mouse plexins have been found: Plexin-A1-4, -B1-3, -C1 and -D1. The expression and function of plexins in non-neuronal tissues has been poorly characterized, although Plexin-A1 has been shown to have a role during lung and cardiac morphogenesis. We have done an extensive non-radioactive in situ hybridisation survey of Plxna1-a4, Plxnb1 -b3 and Plxnc1 in E14 mouse embryo. At E14, Plxnb3 expression could not be detected by in situ hybridisation. All other plexins studied are widely expressed both in neuronal and non-neuronal tissues. We have also followed the expression patterns of plexins during the development of the kidney, tooth and testis. Plxnb1 and Plxnb2 are expressed in the immature glomeruli and mesenchyme of the developing kidney. In the tooth bud, Plxna1 and Plxnb1 are expressed in the oral epithelium, enamel knot and in both the inner and outer enamel epithelium, whereas the expression of Plxnb2 is more restricted to the inner enamel epithelium. In the testis, Plxna1, Plxnb1 and Plxnc1 are expressed in the developing sex chords. This study shows that during development, plexins are expressed in specific and distinct patterns also in non-neuronal tissues.     

Purification of the beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase from human serum.

A beta-N-Acetylglucosaminide alpha 1----3-fucosyltransferase was purified from human serum by ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-hexanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration. Gel filtration chromatography of the native enzyme revealed a Mr of 45,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein also appeared as a single molecular species of Mr 45,000. In contrast to the multisubunit beta-galactoside alpha 1----2-fucosyltransferases with an apparent Mr of 150,000, present in human serum, the native beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase is a monomer with a Mr of 45,000. The enzyme is glycosylated, as revealed by wheat germ agglutinin binding properties. The alpha 1----3 linkage formed by the enzyme between alpha-L-fucose and the penultimate beta-N-acetylglucosamine by the purified enzyme was confirmed by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide product. The specificity of the purified enzyme is restricted to type 2 structures, as revealed by its reactivity with different substrates and from the Km values calculated from the initial rate data using various oligosaccharide acceptors. The enzyme has the ability to utilize the N-acetyl-beta-lactosamine determinant (Gal beta 1----4GlcNAc) and the sialylated (NeuAc alpha 2----3Gal beta 1----4GlcNAc) and fucosylated (Fuc alpha 1----2Gal beta 1----4GlcNAc) derivatives of N-acetyl-beta-lactosamine and thus is distinct from both the human Lewis gene-encoded enzyme and the alpha 1----3-fucosyltransferase of the myeloid cell type.     

A bisubstrate analog inhibitor for alpha(1----2)-fucosyltransferase

Porcine submaxillary beta-galactoside alpha(1----2)-fucosyltransferase is known to transfer a fucosyl residue from guanosine 5'-diphosphofucose (GDP-fucose) to the 2-OH group of beta-D-galactopyranosides with inversion of configuration at the fucopyranosyl anomeric carbon. A bisubstrate analog (1) of the postulated transition-state for this reaction, which has O-2 of phenyl beta-D-galactopyranoside attached to the terminal phosphorous of GDP through a flexible ethylene bridge, has been chemically synthesized and evaluated as an inhibitor of this enzyme. Compound 1 was found to be a competitive inhibitor with respect to both GDP-fucose and phenyl beta-D-galactopyranoside for both the membrane-bound and soluble forms of the fucosyltransferase. It was also a competitive inhibitor with respect to the alternate acceptor beta DGal(1----3)beta DGlcNAcO(CH2)8-COOMe. The Ki values were in the range 2.3-16 microM. Compound 1 is the first example of a bisubstrate analog inhibitor for a glycosyltransferase which binds to both the acceptor and donor recognition sites of the enzyme. The potential of a bisubstrate analog strategy for the production of specific glycosyltransferase inhibitors is discussed.