Molecular characterization of a novel UDP-galactose:fucoside alpha3-galactosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium

Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Gal alpha1,Gal alpha1,3Fuc alpha1,2Gal-beta1,3GlcNAc alpha1O-, at a 4-hydroxylated derivative of Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Gal alpha1,3Fuc linkage by transfer of Gal from UDP-alphaGal to Fuc alpha1,2Gal beta1,3GlcNAc alpha1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal beta-propeller domain. Overexpression of GT78 with a His6-tag resulted in a 120-fold increase in GalT-activity in cytosolic extracts, and purified His6-GT78 exhibited alpha3GalT-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 alpha3GalT, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.     

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.     

A bifunctional diglycosyltransferase forms the Fucalpha1,2Galbeta1,3-disaccharide on Skp1 in the cytoplasm of dictyostelium

Skp1 is a subunit of the Skp1 cullin-1 F-box protein (SCF) family of E3 ubiquitin ligases and of other regulatory complexes in the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by a pentasaccharide with the type I blood group H antigen (Fucalpha1,2Galbeta1,3GlcNAc-) at its core. Addition of the Fuc is catalyzed by FT85, a 768-amino acid protein whose fucosyltransferase activity maps to the C-terminal half of the protein. A strain whose FT85 gene is interrupted by a genetic insertion produces a truncated, GlcNAc-terminated glycan on Skp1, suggesting that FT85 may also have beta-galactosyltransferase activity. In support of this model, highly purified native and recombinant FT85 are each able to galactosylate Skp1 from FT85 mutant cells. Site-directed mutagenesis of predicted key amino acids in the N-terminal region of FT85 abolishes Skp1 beta-galactosyltransferase activity with minimal effects on the fucosyltransferase. In addition, a recombinant form of the N-terminal region exhibits beta-galactosyltransferase but not fucosyltransferase activity. Kinetic analysis of FT85 suggests that its two glycosyltransferase activities normally modify Skp1 processively but can have partial function individually. In conclusion, FT85 is a bifunctional diglycosyltransferase that appears to be designed to efficiently extend the Skp1 glycan in vivo.     

Molecular cloning and functional expression of the human Golgi UDP-N-acetylglucosamine transporter.

We have cloned the human UDP-N-acetylglucosamine (UDP-GlcNAc) transporter cDNA, which was recognized through a homology search in the expressed sequence tags database (dbEST) based on its similarity to the human UDP-galactose transporter. The chromosomal location of the UDP-GlcNAc transporter gene was assigned to chromosome 1p21 by fluorescence in situ hybridization (FISH). The transporter was expressed ubiquitously in every tissue so far examined. Expression of the transporter cDNA in CHO-K1 cells in its native and in a C-terminally HA-tagged form indicated that the human UDP-GlcNAc transporter was localized in the Golgi apparatus. The membrane vesicles prepared from yeast cells expressing the cDNA product exhibited UDP-GlcNAc-specific transporting activity. Comparison among UDP-galactose, CMP-sialic acid, and UDP-GlcNAc transporters from several organisms enabled us to identify residues highly conserved among the transporters and residues specific for each group of transporters.     

Specificity of a soluble UDP-galactose: fucoside alpha1,3-galactosyltransferase that modifies the cytoplasmic glycoprotein Skp1 in Dictyostelium

Skp1 is an adaptor-like protein in E3(SCF)-ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Galalpha1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn(2+) and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Galalpha1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to alpha1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K(m) and 35-fold higher V(max) compared with a simple alpha-fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V(max) advantage, but still much less active than Skp1 itself because of a K(m) difference. These findings indicate that alpha-GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Galalpha1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.     

Molecular cloning and enzymatic characterization of a UDP-GalNAc:GlcNAc(beta)-R beta1,4-N-acetylgalactosaminyltransferase from Caenorhabditis elegans

A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Gal(beta)4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAc(beta)4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding beta4GalTs responsible for LN synthesis have been reported, the beta4GalNAcT(s) responsible for LDN synthesis has not been identified. Here we report the identification of a gene from Caenorhabditis elegans encoding a UDP-GalNAc:GlcNAc(beta)-R beta1,4-N-acetylgalactosaminyltransferase (Ce(beta)4GalNAcT) that synthesizes the LDN structure. Ce(beta)4GalNAcT is a member of the beta4GalT family, and its cDNA is predicted to encode a 383-amino acid type 2 membrane glycoprotein. A soluble, epitope-tagged recombinant form of Ce(beta)4GalNAcT expressed in CHO-Lec8 cells was active using UDP-GalNAc, but not UDP-Gal, as a donor toward a variety of acceptor substrates containing terminal beta-linked GlcNAc in both N- and O-glycan type structures. The LDN structure of the product was verified by co-chromatography with authentic standards and (1)H NMR spectroscopy. Moreover, Chinese hamster ovary CHO-Lec8 and CHO-Lec2 cells expressing Ce(beta)4GalNAcT acquired LDN determinants on endogenous glycoprotein N-glycans, demonstrating that the enzyme is active in mammalian cells as an authentic beta4GalNAcT. The identification and availability of this novel enzyme should enhance our understanding of the structure and function of LDN-containing glycoconjugates.     

Molecular cloning and functional characterization of the bovine (Bos taurus) organic anion transporting polypeptide Oatp1a2 (Slco1a2)

We describe the cloning, functional characterization and tissue localization of a novel membrane transporter of the OATP/Oatp-gene family obtained from liver and kidney of cattle (Bos taurus). The carrier protein exhibits highest sequence identity to the human OATP1A2 (previously called OATP-A) and is, therefore, named bovine Oatp1a2. Bovine Oatp1a2 received the gene symbol Slco1a2 that is identical to the SLC classification of human OATP1A2 (SLCO1A2, previously called SLC21A3) and is likely an orthologue of the human gene. Two different full-length bOatp1a2 cDNAs of 2316-bp and 3504-bp were obtained and encoded for a 666 amino acid membrane protein, which contains twelve putative transmembrane spanning domains. Bovine Oatp1a2 expression was detected in liver, kidney, brain and adrenal gland. Uptake studies in cRNA-injected oocytes demonstrated that bOatp1a2 transports estrone-3-sulfate and taurocholate, with K(m) values of 9.6 microM and 51 microM, respectively, and estradiol-17beta-glucuronide. However, the structurally-related heart glycosides ouabain (1 microM) and digoxin (1 microM) are neither transported by bovine Oatp1a2 nor by human OATP1A2. We conclude that based on the tested substrates bovine Oatp1a2 shows functional homology to human OATP1A2.     

Control of glycoprotein synthesis. Detection and characterization of a novel branching enzyme from hen oviduct, UDP-N-acetylglucosamine:GlcNAc beta 1-6 (GlcNAc beta 1-2)Man alpha-R (GlcNAc to Man) beta-4-N-acetylglucosaminyltransferase VI

Hen oviduct membranes were shown to contain high activity of a novel enzyme, UDP-GlcNac:GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-R (GlcNAc to Man) beta 4-GlcNAc-transferase VI. The enzyme was shown to transfer GlcNAc in beta 1-4 linkage to the D-mannose residue of GlcNAc beta 1-6 (GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or methyl. Radioactive enzyme products were purified by several chromatographic steps, including high performance liquid chromatography, and structures were determined by proton nmr, fast atom bombardment-mass spectrometry, and methylation analysis to be GlcNAc beta 1-6 ([14C]GlcNAc beta 1-4) (GlcNAc beta 1-2) Man alpha-R. The enzyme is stimulated by Triton X-100 and has optimum activity at a relatively high MnCl2 concentration of about 100 mM; Co2+, Mg2+, and Ca2+ could partially substitute for Mn2+. A tissue survey demonstrated high GlcNAc-transferase VI activity in hen oviduct and lower activity in chicken liver and colon, duck colon, and turkey intestine. No activity was found in mammalian tissues. Hen oviduct membranes cannot act on GlcNAc beta 1-6Man alpha-R but have a beta 4-GlcNAc-transferase activity that converts GlcNAc beta 1-2Man alpha-R to GlcNAc beta 1-4(GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or 1-6Man beta methyl. The latter activity is probably due to GlcNAc-transferase IV which preferentially adds GlcNAc in beta 1-4 linkage to the Man alpha 1-3 arm of the GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn core structure of asparagine-linked glycans. The minimum structural requirement for a substrate of beta 4-GlcNAc-transferase VI is therefore the trisaccharide GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-; this trisaccharide is found on the Man alpha 6 arm of many branched complex asparagine-linked oligosaccharides. The data suggest that GlcNAc-transferase VI acts after the synthesis of the GlcNAc beta 1-2Man alpha 1-3-, GlcNAc beta 1-2Man alpha 1-6-, and GlcNAc beta 1-6 Man alpha 1-6-branches by GlcNAc-transferases I, II, and V, respectively, and is responsible for the synthesis of branched oligosaccharides containing the GlcNAc beta 1-6(GlcNAc beta 1-4)(GlcNAc beta 1-2)Man alpha 1-6Man beta moiety.     

Molecular cloning and expression profile of Xenopus calcineurin A subunit(1)

We have cloned a cDNA encoding a catalytic subunit of calcineurin (CnA) expressed in Xenopus oocytes. The deduced amino acid sequence indicates 96.3% and 96.8% identities with the mouse and human CnAalpha isoforms, respectively. Xenopus CnA (XCnA) RNA and protein are expressed as maternal and throughout development. Recombinant XCnA protein interacted with calmodulin in the presence of Ca(2+). Deletion of calmodulin binding domain and auto-inhibitory domain revealed calcium independent phosphatase activity, thereby showing that XCnA is likely to be modulated by both calmodulin and calcium.     

松墨天牛漆酶基因MaLac1的克隆、原核表达及表达谱分析

【目的】本研究旨在克隆并鉴定松墨天牛Monochamus alternatus内源漆酶基因MaLac1,分析其在松墨天牛不同发育阶段的表达水平,为进一步明确MaLac1功能提供依据。【方法】基于松墨天牛肠道转录组测序数据,通过RACE克隆松墨天牛MaLac1基因的全长cDNA序列,并对其进行生物信息学分析;将该基因与pET-32a载体链接构建表达载体pET-MaLac1,导入大肠杆菌Escherichia coli Rosetta (DE3)使其表达;使用qPCR检测MaLac1基因在松墨天牛不同发育阶段(低龄幼虫、老熟幼虫、蛹、雌成虫和雄成虫)肠道中的表达差异。【结果】克隆获得松墨天牛MaLac1的cDNA全长序列(GenBank登录号:KY073340)。MaLac1开放阅读框全长2 067 bp,编码一个含688个氨基酸的蛋白质,预测分子量为78.34 kD,等电点为5.30。SignalP 4.1 Server预测MaLac1在N端包含一个15个氨基酸的信号肽。序列比对分析表明,MaLac1具有典型的昆虫漆酶基因特征,与赤拟谷盗Tribolium castaneum漆酶基因的氨...     

斜纹夜蛾水通道蛋白1（AQP1）基因的克隆、分子特性和表达分析

水通道蛋白(aquaporin, AQP)是生物体中一种重要的跨膜通道蛋白, 它通过一些中性小分子化合物的运输, 从而参与了昆虫对食物中水分再吸收、抗寒和抗干燥等重要生理机制。为研究斜纹夜蛾中水通道蛋白的基因特征和时空表达特征, 本研究利用同源克隆和RACE技术获得了斜纹夜蛾水通道蛋白1（AQP1）基因的两个转录异构体, 并将其分别命名为SL-AQP1A （GenBank登录号： KC999953）和SL-AQP1B （GenBank登录号： KC999954）,其中SL-AQP1B比SL-AQP1A在推导的编码区5′端连续性缺失81个碱基, 而其他序列完全一致; 同源分析显示推导的SL-AQP1与家蚕为代表的水通道蛋白1具有较高的同源性。拓扑学和三级结构模拟显示其有经典的6个全跨膜结构域、2个半跨膜结构域、2个保守的NPA (asparagine-proline-alanine, 天冬氨酸-脯氨酸-丙氨酸)结构基序及选择性水孔构件ar/R （aromatic arginine, 芳香族精氨酸）。qRT-PCR结果显示, SL-AQP1整体时空表达差异性十分明显, 并且SL-AQP1B的表达量显著高于SL-AQP1A; SL-AQP1在卵期和预蛹期有较高的表达量, 在中肠、马氏管、血淋巴、消化腺中有相对较高的表达量, 暗示其在斜纹夜蛾中存在重要的渗透压调节作用。本文结果为进一步研究水通道蛋白在斜纹夜蛾中的作用提供了一定的分子基础。     

Molecular cloning and expression study of Xenopus latent TGF-beta binding protein-1 (LTBP-1)

Latent transforming growth factor β binding protein-1 (LTBP-1) is important in regulating the localization and activation of transforming growth factor β. In this paper is reported the isolation of the full-length Xenopus LTBP-1 cDNA from screening a neurula embryo cDNA library. Sequence analysis of XLTBP-1 cDNA revealed an open reading frame of 4518 bp encoding a 1398 amino acid protein with a molecular mass of 154.1 kDa and an isoelectric point of 4.65. The Xenopus XLTBP-1 shares 61 and 65% amino acid identity with the mouse and human LTBP-1, respectively. It contains 17 epidermal growth factor-like motifs and four eight-cysteine repeats (8-Cys). RNase protection assay revealed that XLTBP-1 is a maternal and zygotic gene, while whole-mount in situ hybridization analysis performed on embryos at different stages showed that during early Xenopus development, XLTBP-1 mRNA is expressed in the Spemann organizer, prechordal and chordal mesoderm, and later on in the organizer derived tissues. These findings suggest an important role for XLTBP-1 in embryo axis formation.     

Molecular cloning and baculovirus expression of the rabbit corneal aldehyde dehydrogenase (ALDH1A1) cDNA

Most mammalian species express high concentrations of ALDH3A1 in corneal epithelium with the exception of the rabbit, which expresses high amounts of ALDH1A1 rather than ALDH3A1. Several hypotheses that involve catalytic and/or structural functions have been postulated regarding the role of these corneal ALDHs. The aim of the present study was to characterize the biochemical properties of the rabbit ALDH1A1. We have cloned and sequenced the rabbit ALDH1A1 cDNA, which is 2,073 bp in length (excluding the poly(A+) tail), and has 5' and 3' nontranslated regions of 46 and 536 bp, respectively. This ALDH1A1 cDNA encodes a protein of 496 amino acids (Mr = 54,340) that is: 86-91% identical to mammalian ALDH1A1 proteins, 83-85% identical to phenobarbital-inducible mouse and rat ALDH1A7 proteins, 84% identical to elephant shrew ALDH1A8 proteins (eta-crystallins), 69-73% identical to vertebrate ALDH1A2 and ALDH1A3 proteins, 65% identical to scallop ALDH1A9 protein (omega-crystallin), and 55-57% to cephalopod ALDH1C1 and ALDH1C2 (omega-crystallins). Recombinant rabbit ALDH1A1 protein was expressed using the baculovirus system and purified to homogeneity with affinity chromatography. We found that rabbit ALDH1A1 is catalytically active and efficiently oxidizes hexanal (Km = 3.5 microM), 4-hydroxynonenal (Km = 2.1 microM) and malondialdehyde (Km = 14.0 microM), which are among the major products of lipid peroxidation. Similar kinetic constants were observed with the human recombinant ALDH1A1 protein, which was expressed and purified using similar experimental conditions. These data suggest that ALDH1A1 may contribute to corneal cellular defense against oxidative damage by metabolizing toxic aldehydes produced during UV-induced lipid peroxidation.