The relatively large beta-tubulin gene family of Arabidopsis contains a member with an unusual transcribed 5′ noncoding sequence

We have characterized the beta-tubulin gene family of Arabidopsis thaliana. Five distinct genes were cloned and analyzed by restriction enzyme mapping and cross-hybridization studies. Three of the genes appear to be dispersed, whereas two others are linked within 1.5 kb of one another. The two linked genes are closely related and appear to have resulted from a fairly recent duplication. The three dispersed genes do not cross-hybridize to one another or to the two linked genes under highly stringent hybridization conditions, suggesting that they arose from more ancient duplications. From Southern analysis we estimate that there are a total of between six and ten beta-tubulin genes in Arabidopsis. Additional analyses indicate that the gene family is equal in size or larger than those in other plants, but significantly smaller than those in related Brassica species. Sequence determination of one of the Arabidopsis genes revealed a highly unusual transcribed leader sequence. The leader contains two fairly long tracks of adenines. One is located toward the 5 end of the mRNA and the other is just before the initiation codon. A track of uridines is located between the adenine tracks. This leader can form two different secondary structures that may have regulatory significance.     

A glycan of Ψ-factor from Dictyostelium discoideum contains a bisecting-GlcNAc, an intersecting-GlcNAc, and a core α-1,6-fucose

A secretory glycoprotein named Ψ-factor that we have purified and cloned from Dictyostelium discoideum is prespore cell-inducing factor. To address its functional significance, it is necessary to examine the attached sites and structures of its glycans as well as its protein structure. Here we identified and isolated a tryptic glycosylated peptide with the 71st to 89th amino acids of Ψ-factor that contained the consensus amino acid sequence for an N-linked glycan (N-T-T). MALDI-TOF mass spectrometry indicated that the major protonated molecular ions, [M+H](+), of the glycopeptide were present at m/z 3,806, the minor m/z 3,603 and 3,400 ions corresponding to the loss of one and two N-acetylhexosamines respectively. Digestion of it with N-glycosidase F gave a molecular mass of 1,766.9 for the whole glycan moiety, which accounts for its composition of five hexoses, four N-acetylhexosamines, and a deoxyhexose. Further digestion experiments on the basis of the substrate specificity of α-mannosidase and β-N-acetylhexosaminidase allowed us to elucidate the unique structure of the glycan, which contains a bisecting and an intersecting GlcNAc and a core α1,6-fucosyl moiety.     

Structural and enzymatic characterization of a glycoside hydrolase family 31 α-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification

The desire for improved methods of biomass conversion into fuels and feedstocks has re-awakened interest in the enzymology of plant cell wall degradation. The complex polysaccharide xyloglucan is abundant in plant matter, where it may account for up to 20% of the total primary cell wall carbohydrates. Despite this, few studies have focused on xyloglucan saccharification, which requires a consortium of enzymes including endo-xyloglucanases, α-xylosidases, β-galactosidases and α-L-fucosidases, among others. In the present paper, we show the characterization of Xyl31A, a key α-xylosidase in xyloglucan utilization by the model Gram-negative soil saprophyte Cellvibrio japonicus. CjXyl31A exhibits high regiospecificity for the hydrolysis of XGOs (xylogluco-oligosaccharides), with a particular preference for longer substrates. Crystallographic structures of both the apo enzyme and the trapped covalent 5-fluoro-β-xylosyl-enzyme intermediate, together with docking studies with the XXXG heptasaccharide, revealed, for the first time in GH31 (glycoside hydrolase family 31), the importance of a PA14 domain insert in the recognition of longer oligosaccharides by extension of the active-site pocket. The observation that CjXyl31A was localized to the outer membrane provided support for a biological model of xyloglucan utilization by C. japonicus, in which XGOs generated by the action of a secreted endo-xyloglucanase are ultimately degraded in close proximity to the cell surface. Moreover, the present study diversifies the toolbox of glycosidases for the specific modification and saccharification of cell wall polymers for biotechnological applications.     

A 14-kDa Arabidopsis thaliana RNA polymerase III subunit contains two α-motifs flanked by a highly charged C terminus

We have sequenced a cDNA and a gene, AtRPC14, from Arabidopsis thaliana (At) (ecotype Columbia) that encode a protein related to the yeast RNA polymerases (Pol) I and III subunits, yAC19. Polyclonal antibodies raised against the recombinant At polypeptide (AtC14) bind to the Pol I and/or III subunits of about 13–15 kDa, but do not bind to any Pol II subunit in Pol purified from cauliflower, wheat or At. The amino acid (aa) sequence derived from the AtRPC14 cDNA and genomic clones consists of 122 aa, as compared to the 142 aa in the yeast yAC19 subunit and 143 aa in a putative Caenorhabditis elegans CeAC16 subunit. AtC14, yAC19 and CeAC16 contain a conserved sequence of about 85 aa which is related to two motifs in the α subunit of Escherichia coli (Ec) Pol. AtC14 lacks a highly charged N terminus of about 50 aa found in both yAC19 and CeAC16, but has a highly charged C terminus of about 30 aa not found in yAC19 and CeAC16.     

Thermostable α-1,4- and α-1,6-glucosidase enzymes from Bacillus sp. Isolated from a marine environment

Penaeus vannamei (the shrimp) is an omnivorous species and it can be assumed that a high level of carbohydrates is necessary for its growth. -1,4- and 1,6-glucosidases are important enzymes necessary for the ultimate liberation of glucose residues from various carbohydrates, principally starch. However, the shrimp's hepatopancreas produces only -1,4-glucosidases, which limits the growth rate in different sources of starch. In order to identify strains with -1,4- and 1,6-glucosidase enzymes with potential uses in shrimp feed production, Bacillus strains were isolated from marine environments. One strain produced large amounts of an extracellular thermostable -glucosidase that permitted good growth on starch. The organism was identified by polymorphism (restriction-fragment-length polymorphism, RFLP), sequenced, and named B. subtilis LMM-12.     

Hydrolysis of α-1,6-Glucosidic Linkages by an α-Amylase from Thermoactinomyces vulgaris R-47

We studied DNA breakage by phenyl compounds present in foodstuffs in vitro using γDNA and in cultured mammalian cells using RFL and HeLa cells. Strong in vitro activity was detected in o- and p-dihydroxyphenols, but the m-isomer had no activity. The same results were obtained with aminophenols and phenylenediamines. In flavonoids, the 3-OH group seemed to be active in the DNA breakage, in addition to the odiphenol group. Cellular DNA breakage by the compounds was different from their in vitro activity and varied with the cell lines. RFL cells were preferable to HeLa cells for screening for DNA breaking substances, because of their greater sensitivity.     

RNP-T,a ribonucleoprotein from Arabidopsis thaliana,contains two RNP-80 motifs and a novel acidic repeat arranged in an α-helix conformation

We have isolated a 1148 bp long cDNA clone encoding an RNA-binding protein in Arabidopsis. Several partial cDNA clones were isolated by screening an Arabidopsis gt11 expression library for the binding of DNA. One of these clones was used as a probe to isolate a full-length clone. The 329 amino acid protein, termed RNP-T, contains in its carboxy terminus two adjacent RNP-80 motifs, a previously described 80 amino acid long conserved putative RNA-binding domain. Each RNP-80 motif includes both consensus short sequences, RNP1 and RNP2, which are separated by 33 amino acids. We have identified an acidic domain of 54 amino acids, which is located amino-terminal to the RNP-80 motifs. Seven tandem repeats of a hexamer are present within this domain. This acidic domain has a potential -helix conformation. We propose that the acidic patch might play a role in protein-protein interaction.     

Evidence for α-1,6 and α-1,4-glucosidic bond cleavage in highly branched glycogen by amylopullulanase from Thermoanaerobacter ethanolicus

Summary Activity of amylopullulanase from Thermoanaerobacter ethanolicus 39E on -1,6 and -1,4-glucosidic linkages in highly branched mammalian glycogen was analyzed by paper chromatography and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Paper chromatography analysis showed that the glycogen hydrolysate consisted of glucose, maltose, maltotriose and maltotetraose. NMR spectroscopy confirmed that no hydrolysate products of -1,6 linkage were present resulting from treatment with the amylopullulanase. Therefore, the amylopullulanase efficiently hydrolyzed glycogen both at -1,6- and at -1,4-glucosidic linkages into oligosaccharides.     

Generation of α-1,3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes

Recent progress in genetic manipulation of pigs designated for xenotransplantation ha6s shown considerable promise on xenograft survival in primates. However, genetic modification of multiple genes in donor pigs by knock-out and knock-in technologies, aiming to enhance immunological tolerance against transplanted organs in the recipients, has not been evaluated for health issues of donor pigs. We produced transgenic Massachusetts General Hospital piglets by knocking-out the α-1,3-galactosyltransferase (GT) gene and by simultaneously knocking-in an expression cassette containing five different human genes including, DAF, CD39, TFPI, C1 inhibitor (C1-INH), and TNFAIP3 (A20) [GT^{?(DAF/CD39/TFPI/C1-INH/TNFAIP3)/+}] that are connected by 2A peptide cleavage sequences to release individual proteins from a single translational product. All five individual protein products were successfully produced as determined by western blotting of umbilical cords from the newborn transgenic pigs. Although gross observation and histological examination revealed no significant pathological abnormality in transgenic piglets, hematological examination found that the transgenic piglets had abnormally low numbers of platelets and WBCs, including neutrophils, eosinophils, basophils, and lymphocytes. However, transgenic piglets had similar numbers of RBC and values of parameters related to RBC compared to the control littermate piglets. These data suggest that transgenic expression of those human genes in pigs impaired hematopoiesis except for erythropoiesis. In conclusion, our data suggest that transgenic expression of up to five different genes can be efficiently achieved and provide the basis for determining optimal dosages of transgene expression and combinations of the transgenes to warrant production of transgenic donor pigs without health issues.     

Purification and properties of a thermostable pullulanase from Clostridium thermosulfurogenes EM1 which hydrolyses both α-1,6 and α-1,4-glycosidic linkages

Summary A novel thermostable pullulanase, secreted by the thermophilic anaerobic bacterium Clostridium thermosulfurogenes EM1, was purified and characterized. Applying anion exchange chromatography and gel filtration the enzyme was purified 47-fold and had a specific activity of 200 units/mg. The molecular mass of this thermostable enzyme was determined to be 102 000 daltons and consisted of a single subunit. The enzyme was able to attack specifically the -1,6-glycosidic linkages in pullulan and caused its complete hydrolysis to maltotriose. Surprisingly and unlike the enzyme from Klebsiella pneumoniae, the purified enzyme from this anaerobic thermophile exhibited, in addition to its debranching and pullulanase activity, an -1,4 hydrolysing activity as well. By the action of this single polypeptide chain various branched and linear polysaccharides were completely converted to two major products, namely maltose and maltotriose. The K _m values of this enzyme for pullulan and amylose were determined to be 1.33 mg/ml and 0.38 mg/ml, respectively. This debranching enzyme displays a temperature optimum at 60°–65° C and a pH optimum at 5.5–6.0. The application of this new class of pullulanase (pullulanase type II) in industry will significantly enhance the starch saccharification process. Offprint requests to: G. Antranikian     

Drosophila α-1,4-glycosyltransferase (α-4GT1) inhibits reaper-mediated apoptosis in the eye

In a genetic screen, α-4GT1 has been identified as a potential enhancer of Hairless-mediated cell death in the eye of Drosophila. α-4GT1 encodes an α-1,4-glycosyltransferase, known to catalyze the fifth step in a series of ceramide glycosylation events. As reported for other enzymes involved in the glycosylation of ceramide, α-4GT1 is strongly expressed during oogenesis and is deposited maternally in the egg. Moreover, the protein is enriched at cell membranes. Unexpectedly, overexpression of α-4GT1 does not enhance Hairless-mediated cell death; instead, Hairless enhancement is caused by an allele of Scutoid ¹ present on the α-4GT1 chromosome. Interestingly, the downregulation of α-4GT1 during eye development amplifies cell death induction by the pro-apoptotic gene reaper. Accordingly, overexpression of α-4GT1 represses reaper-induced cell death. Thus, α-4GT1 appears to be an inhibitor of apoptosis, as has previously been observed for other ceramide glycosylating enzymes, suggesting that it likewise contributes to ceramide anchoring in the membrane. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by a grant from the German Science Foundation (DFG) to A.C.N. (NA427/1—2). The authors declare that they have no conflict of interest.     

Expression, purification and characterization of low-glycosylation influenza neuraminidase in α-1,6-mannosyltransferase defective Pichia pastoris

Influenza A viruses expose two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Although N-glycosylation is essential for many glycoproteins, the glycoproteins expressed in yeast are sometimes hyper-glycosylated, which maybe a primary hindrance to the exploitation of therapeutic glycoprotein production because glycoproteins decorated with yeast-specific glycans are immunogenic and show poor pharmacokinetic properties in humans. To elucidate the NA with different glycosylation in interaction with immunogenicity, here we reported the heterologous expression of influenza NA glycoprotein derived from influenza virus A/newCaledonia/20/99(H1N1) in wide-type Pichia pastoris, α-1,6-mannosyltransferase (och1)-defective P. pastoris and Escherichia coli. We also assessed the immunogenicity of hyper-glycosylated NA expressed in the wide-type, low-glycosylated NA expressed in och1-defective P. pastoris strain and non-glycosylated NA produced in E. coli. Recombinant NA was expressed in wide-type P. pastoris as a 59–97 above kDa glycoprotein, 52–57 kDa in the och1 defective strain, and as a 45 kDa non-glycoprotein in E. coli. The antibody titers of Balb/c mice were tested after the mice were immunized three times with 0.2, 1, or 3 μg purified recombinant NA. Our results demonstrated that after the second immunization, the antibody titer elicited with 1 μg low-glycosylated NA was 1:5,500, while it was 1:10 and 1:13 when elicited by 1 μg hyper-glycosylated and non-glycosylated NA. In the 0.2 μg dose groups, a high antibody titer (1:4,900) was only found after third immunization by low-glycosylated NA, respectively. These results suggest that low-glycosylation in och1-defective P. pastoris enhances the immunogenicity of recombinant NA and elicits similar antibody titers with less antigen when compared with hyper- and non-glycosylated NA. Thus, och1-defective P. pastoris may be a better yeast expression system for production of glycoproteins to research immunogenic characterization.     

Structural Elucidation of the Cyclization Mechanism of α-1,6-Glucan by Bacillus circulans T-3040 Cycloisomaltooligosaccharide Glucanotransferase

Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase belongs to the glycoside hydrolase family 66 and catalyzes an intramolecular transglucosylation reaction that produces cycloisomaltooligosaccharides from dextran. The crystal structure of the core fragment from Ser-39 to Met-738 of B. circulans T-3040 cycloisomaltooligosaccharide glucanotransferase, devoid of its N-terminal signal peptide and C-terminal nonconserved regions, was determined. The structural model contained one catalytic (β/α)₈-barrel domain and three β-domains. Domain N with an immunoglobulin-like β-sandwich fold was attached to the N terminus; domain C with a Greek key β-sandwich fold was located at the C terminus, and a carbohydrate-binding module family 35 (CBM35) β-jellyroll domain B was inserted between the 7th β-strand and the 7th α-helix of the catalytic domain A. The structures of the inactive catalytic nucleophile mutant enzyme complexed with isomaltohexaose, isomaltoheptaose, isomaltooctaose, and cycloisomaltooctaose revealed that the ligands bound in the catalytic cleft and the sugar-binding site of CBM35. Of these, isomaltooctaose bound in the catalytic site extended to the second sugar-binding site of CBM35, which acted as subsite −8, representing the enzyme·substrate complex when the enzyme produces cycloisomaltooctaose. The isomaltoheptaose and cycloisomaltooctaose bound in the catalytic cleft with a circular structure around Met-310, representing the enzyme·product complex. These structures collectively indicated that CBM35 functions in determining the size of the product, causing the predominant production of cycloisomaltooctaose by the enzyme. The canonical sugar-binding site of CBM35 bound the mid-part of isomaltooligosaccharides, indicating that the original function involved substrate binding required for efficient catalysis.     

<Emphasis Type="Italic">AXY3</Emphasis> encodes a α-xylosidase that impacts the structure and accessibility of the hemicellulose xyloglucan in Arabidopsis plant cell walls

Xyloglucan is the most abundant hemicellulose in the walls of dicots such as Arabidopsis. It is part of the load-bearing structure of a plant cell and its metabolism is thought to play a major role in cell elongation. However, the molecular mechanism by which xyloglucan carries out this and other functions in planta is not well understood. We performed a forward genetic screen utilizing xyloglucan oligosaccharide mass profiling on chemically mutagenized Arabidopsis seedlings to identify mutants with altered xyloglucan structures termed axy-mutants. One of the identified mutants, axy3.1, contains xyloglucan with a higher proportion of non-fucosylated xyloglucan subunits. Mapping revealed that axy3.1 contains a point mutation in XYLOSIDASE1 (XYL1) known to encode for an apoplastic glycoside hydrolase releasing xylosyl residues from xyloglucan oligosaccharides at the non-reducing end. The data support the hypothesis that AXY3/XYL1 is an essential component of the apoplastic xyloglucan degradation machinery and as a result of the lack of function in the various axy3-alleles leads not only to an altered xyloglucan structure but also a xyloglucan that is less tightly associated with other wall components. However, the plant can cope with the excess xyloglucan relatively well as the mutant does not display any visible growth or morphological phenotypes with the notable exception of shorter siliques and reduced fitness. Taken together, these results demonstrate that plant apoplastic hydrolases have a larger impact on wall polymer structure and function than previously thought.     

A Novel Glucanotransferase from a Bacillus circulans Strain That Produces a Cyclomaltopentaose Cyclized by an α-1,6-Linkage

A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an α-1,6-linkage [ICG5; cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS–PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50 °C, and stable from pH 4.5 to 9.0 at up to 35 °C. The addition of 1 mM Ca²⁺ enhanced the thermal stability of the enzyme up to 40 °C. It acted on maltooligosaccharides that have degrees of polymerization of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular α-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular α-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme.     

Hydrolysis of β-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-β-glucanase from the basidiomycete Phanerochaete chrysosporium

When Phanerochaete chrysosporium was grown with laminarin (a β-1,3/1,6-glucan) as the sole carbon source, a β-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear β-1,3-glucan, branched β-1,3/1,6-glucan, and β-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-β-glucanase (EC 3.2.1.6) with broad substrate specificity for β-1,3-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (β-d-Glcp-(1–>6)-β-d-Glcp-(1–>3)-β-d-Glcp-(1–>3)-d-Glc) and 4-O-glucosyl-laminaribiose (β-d-Glcp-(1–>4)-β-d-Glcp-(1–>3)-d-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes β-d-Glcp-(1–>3)-d-Glcp at subsites −2 and −1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a β-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched β-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular β-1,3-glucanases.     

Two new human hemoglobin variants caused by unusual mutational events: Hb Zaïre contains a five residue repetition within the α-chain and Hb Duino has two residues substituted in the β-chain

Summary With rare exceptions, the more than 600 human hemoglobin variants described are caused by a single point mutation. Other abnormal features, such as unequal crossing-over, frameshift mutagenesis or double mutations in the same polypeptide chain, have seldom been encountered. We report two new variants caused by such rare mutational events. Hb Zaïre [116(GH4)-His-Leu-Pro-Ala-Glu-117 (GH5)] is the second example in which a short amino acid sequence is inserted within the -chain. This abnormal hemoglobin results from a tandem repetition of 5 amino-acid residues, from sequence 112 through 116, at the end of the GH corner. Hb Duino is an unstable hemoglobin. It presents within the same -chain, the association of two rare point mutations; these substitutions are those found in Hb Newcastle [92(F8)HisPro] and in Hb Camperdown [104(G6)ArgSer]. Family studies demonstrated that the Hb Newcastle abnormality was a de novo mutation of a gene already carrying the Hb Camperdown substitution.     

α°- and β°- Thalassemia in a Thai family: unusually mild homozygous β°-thalassemia without α-globin gene deletion

Summary Alpha-globin genes were analyzed by the direct method of DNA mapping using - and -globin specific probes in a Thai family in which the proposita was an unusually mild °-thalessemia homozygote. °-Thalessemia was found to be segregating in the family, inherited from the proposita's father by one of her younger sisters. However, °-thatlessemia was not detected by this DNA mapping in the proposita. The mild homozygous °-thalessemia in this family may result from interactions of a non-deletion -thalassemia, a gene responsible for high proteolytic activity permitting more balanced globin-chain levels, or from an unusually active hemoglobin F production in the proposita.