Cloning and functional characterization of an α-1,3-fucosyltransferase from Bacteroides fragilis

Bacteroides fragilis is a clinically important anaerobic pathogen present in the human gastrointestinal tract and is involved in a high number of anaerobic peritoneal infections. The complete genome sequence of B. fragilis NCTC 9343 revealed the presence of several putative fucosyltransferase gene homologues known as alpha-1,3-fucosyltransferases (α-1,3-FucTs). However, their expression and functional activities have not been studied. Here, we report the molecular cloning, functional expression, and characterization of the alpha-1,3-fucosyltransferase 3 (α-1,3-FucT3) enzyme from B. fragilis NCTC 9343. The polymerase chain reaction (PCR)-based approach was used to clone the 331 amino acid long (MW, ～39 kDa) PCR product encoding fucosyltransferase enzyme. The enzyme had low identity of 30–40% with other known α-1,3-FucTs from Azospirillum sp, Rickettsia bellii, and different strains of Helicobacter pylori. An in vitro enzyme reaction analysis showed the ability of the enzyme to transfer the fucose moiety from guanosine-5′-diphosphate β-l-fucose to the N-acetyllactosamine to produce Lewis X. The reaction product, Lewis X was confirmed by thin layer chromatography, liquid chromatography-mass spectroscopy, and ¹H-nuclear magnetic resonance analyses.     

Schistosoma japonicum: Cloning, expression and characterization of a gene encoding the α5-subunit of the proteasome

The development of an effective vaccine against the schistosome is thought to be the most desirable means to control schistosomiasis, even though there is an effective means of chemotherapy with praziquantel. A full-length cDNA encoding the Schistosoma japonicum proteasome subunit alpha type 5 protein (SjPSMA5) was first isolated from 18-day-schistosomulum cDNAs. The cDNA had an open reading frame (ORF) of 747 bp and encoded 248 amino acids. Real-time quantitative RT-PCR analysis revealed that SjPSMA5 is up-regulated in 18-day and 32-day schistosomes, and the level of expression in male is around fourfold higher than that in female worms at 42 days. The SjPSMA5 was subcloned into pET28a(+) and expressed as inclusion bodies in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjPSMA5 (rSjPSMA5) was immunogenic. After immunization of BALB/c mice with rSjPSMA5, reductions of 23.29% and 35.24% were obtained in the numbers of worms and eggs in the liver, respectively. The levels of specific IgG antibodies and cells were significantly higher (P < 0.01) in the group vaccinated with rSjPSMA5 combined with Seppic 206 adjuvant than in the other groups, as detected by enzyme linked immunosorbent assay (ELISA) and flow cytometry. The study suggested that rSjPSMA5 induced partial immunoprotection against S. japonicum in BALB/c mice, and it could be a potential vaccine candidate against schistosomiasis.     

Effects of anti-juvenile hormone “ETB” on the development and metamorphosis of the silkworm, Bombyx mori

As in the tobacco hornworm Manduca sexta, the synthetic juvenile hormone analogue ETB (ethyl 4-[2-(tert-buthylcarbonyloxy)butoxy]benzoate) showed both juvenile hormone-like and anti-juvenile hormone activities in the silkworm, Bombyx mori. When ETB was topically applied to allatectomized 4th-instar larvae, the compound counteracted the effects of allatectomy, such as induction of precocious metamorphosis and black pigmentation in the larval markings. Therefore, ETB had juvenile hormone activity, but it could neither induce brown pigmentation in the markings nor induce an extra-larval moult as can juvenile hormone.When intact 3rd-instar larvae were treated with the compound, the majority underwent precocious metamorphosis in the 4th-instar, and later formed fertile miniature adults. Some moulted into larval-pupal intermediates or 5th-instar larvae with darkened larval markings and/or with abnormality of specific regions of the silk-gland. The optimal dose for such anti-juvenile effects was about 1–10 μg/larva, and higher doses showed less activity. Such anti-juvenile hormone effects of ETB were counteracted by administration of the juvenile hormone analogue, methoprene, before a certain critical time in the 4th-instar. The corpora allata of treated larvae appeared cytologically normal, and the corpora allata from ETB-induced miniature moths secreted juvenile hormone when implanted into allatectomized 4th-instar larvae.     

Rôle of the midgut,crop, and maxillae of Bombyx mori in the production of cocoon-digesting enzyme

The rôle of the midgut, crop, and maxillae in the production and utilization of the cocoon-digesting enzyme was investigated in the silkworm, Bombyx mori.About a sixtyfold purified preparation of midgut protease was obtained by ammonium sulphate precipitation and column chromatography.Immunological studies by the agar diffusion method of Ouchterlony revealed that the crop and midgut proteases of the pharate adult are antigenically identical whereas that of the maxillary protease is different.From the results of extirpation experiments and previous studies it was shown that the midgut, crop, and maxillae play important rôles in the escape of moths from their cocoons.     

Extracellular expression and biochemical characterization of α-cyclodextrin glycosyltransferase from Paenibacillus macerans

The cgt gene encoding α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01 was expressed in Escherichia coli as a C-terminal His-tagged protein. After 90 h of induction, the activity of α-CGTase in the culture medium reached 22.5 U/mL, which was approximately 42-fold higher than that from the parent strain. The recombinant α-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and hydrophobic interaction chromatography. Then, the purified enzyme was characterized in detail with respect to its cyclization activity. It is a monomer in solution. Its optimum reaction temperature is 45 °C, and half-lives are approximately 8 h at 40 °C, 1.25 h at 45 °C and 0.5 h at 50 °C. The recombinant α-CGTase has an optimum pH of 5.5 with broad pH stability between pH 6 and 9.5. It is activated by Ca²⁺, Ba²⁺, and Zn²⁺ in a concentration-dependent manner, while it is dramatically inhibited by Hg²⁺. The kinetics of the α-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation.     

Identification,characterization, and crystal structure of an aldo–keto reductase (AKR2E4) from the silkworm Bombyx mori

A new member of the aldo–keto reductase (AKR) superfamily with 3-dehydroecdysone reductase activity was found in the silkworm Bombyx mori upon induction by the insecticide diazinon. The amino acid sequence showed that this enzyme belongs to the AKR2 family, and the protein was assigned the systematic name AKR2E4. In this study, recombinant AKR2E4 was expressed, purified to near homogeneity, and kinetically characterized. Additionally, its ternary structure in complex with NADP⁺ and citrate was refined at 1.3 Å resolution to elucidate substrate binding and catalysis. The enzyme is a 33-kDa monomer and reduces dicarbonyl compounds such as isatin and 17α-hydroxy progesterone using NADPH as a cosubstrate. No NADH-dependent activity was detected. Robust activity toward the substrate inhibitor 3-dehydroecdysone was observed, which suggests that this enzyme plays a role in regulation of the important molting hormone ecdysone. This structure constitutes the first insect AKR structure determined. Bound NADPH is located at the center of the TIM- or (β/α)₈-barrel, and residues involved in catalysis are conserved.     

Cloning and expression of a chicken α-amylase gene

We have isolated and sequenced a genomic clone for a pancreatic α-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY) of 512 aa that is 83% identical to the human pancreatic α-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the α-amylase produced by the yeast cells was identical to that of the native chicken enzyme.     

Phosphorylation of glycogen synthase kinase-3β in relation to diapause processing in the silkworm, Bombyx mori

Glycogen synthase kinase-3 (GSK-3) is a multifunctional protein kinase that plays important roles in regulating both glycogen synthesis and protein synthesis. In the present study, we investigated GSK-3β phosphorylation of silkworm eggs by immunoblotting with a conserved phospho-specific antibody to GSK-3β. Results showed that the temporal changes in GSK-3β phosphorylation were closely related to changes in glycogen levels previously reported by other researchers. In diapause eggs, an abrupt decrease in phosphorylation of GSK-3β was found with the onset of diapause, and phosphorylation level of GSK-3β reached a minimum level within 1 week after oviposition. However, when diapause eggs were incubated at 25 °C for 15 days and then transferred to 5 °C, a great increase in GSK-3β phosphorylation was observed 5 days after transfer to 5 °C and high levels were maintained throughout the chilling period. In both non-diapause eggs and eggs whose diapause initiation was prevented by HCl, levels of GSK-3β phosphorylation appeared to remain relatively high for several days and then greatly decreased 2 or 3 days before hatching. Moreover, GSK-3β phosphorylation dramatically increased when dechorionated eggs were incubated in medium. The addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, U0126, did not inhibit GSK-3β phosphorylation in dechorionated eggs, although U0126 dose-dependently inhibited ERK phosphorylation. This result showed that ERK phosphorylation is not involved in upstream signaling for GSK-3β phosphorylation and that there may be two distinct signaling pathways involved in diapause processing in Bombyx mori eggs.     

Biochemical characterization of Silene alba α4-fucosyltransferase and Lewis a products

1,4-Fucosylation has been recently detected in Arabidopsis thaliana [Léonard et al. (2002), Glycobiology 12: 299–306], and corresponding enzymes have also been characterized in Beta vulgaris [Bakker et al. (2001), FEBS Lett, 507: 307–312], and Lycopersicum aesculentum [Wilson (2001), Glycoconjugate J., 18: 439–447]. Here we demonstrated fucosyltransferase activity (FucT) in Silene alba cells and tissues. The Fuc linkage to GlcNAc residues of the lactosamine moiety of the Type I acceptor was confirmed by mass spectrometry experiments. Le^a-glycoconjugates are found in the Golgi apparatus and plasma membrane of plant cells. In planta, the highest levels of activity were detected in seedlings, young roots and male flowers. The enzyme was stable up to 45C and the optimum pH of reaction was 8.0. The enzyme required Mg²⁺ or Mn²⁺ for activity and was inhibited by Zn²⁺ and ethylenediaminetetraacetic acid. Chemical modification of the enzyme with group-selective reagents revealed that selective modifications of arginine and lysine residues had no effect on enzyme activity. However the enzyme contains histidine and tryptophan residues that are essential for its activity. In contrast to human FUT3, the S. alba 4-FucT was insensitive to N-ethylmaleimide (NEM) treatment. Measurement of enzyme activity in S. alba cell fractions indicated that the enzyme is bound to microsomal membranes, furthermore a soluble isoform of the protein may be present. Published in 2005.     

Cloning and characterization of chicken α5 integrin: Endogenous and experimental expression in early chicken embryos

Key roles for fibronectin and its integrin receptors have been postulated in the multiple cell-matrix interactions essential for chick embryo morphogenesis. However, mechanistic studies of these processes have been hampered by the current absence of sequence data and chicken cDNA clones for the major fibronectin receptor subunit, integrin α5 (ITGA5). We report here the sequence, endogenous expression pattern, and transfection of full-length chicken integrin α5. During early chicken embryonic development, α5 is highly expressed in cranial neural folds and migrating neural crest cells, suggesting potential roles in neural crest formation and migration. In fact, over-expression of this integrin in early neural tube selectively induces BMP5, a growth factor recently implicated in neural crest formation. Availability of these α5 integrin tools should facilitate studies of its functions in early embryonic development.     

Cloning,expression and some properties of α-carbonic anhydrase from Helicobacter pylori

The α-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO₂ hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25°C are k_cat=2.4×10⁵ s⁻¹, K_M=17 mM and k_cat/K_M=1.4×10⁷ M⁻¹ s⁻¹. The pH dependence of k_cat/K_M fits with a simple titration curve with pK_a=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO₂ hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, k_enz, of 24 M⁻¹ s⁻¹ at pH 8.8 and 25°C. However, with 2-nitrophenyl acetate as substrate a k_enz value of 665 M⁻¹ s⁻¹ was obtained under similar conditions.     

Cloning,characterisation and expression of the α-tubulin genes of the leech,Hirudo medicinalis

We have isolated two α-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two α-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo α-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech α-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod α-tubulins. The protein sequences of the leech α-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.     

Cloning, expression and characterization of human tissue-specific DNA polymerase λ2*

DNA polymerase (POL) λ plays an important role during DNA repair and DNA nonhomologous recom-bination processes. A novel POL λ variant was cloned from a human liver cDNA library and named POL λ2 (GenBank Accession No. AY302442). POL λ2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL λ2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL λ2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL λ and POL λ2 in normal liver tissues and hepato-cellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL λ. This abnormality might be involved in hepato-carcinogenesis. The recombinant POL λ2 with His-tag was expressed as a soluble active protein in E. coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chro-matography in an FPLC system. The analysis using isotope α-³²P-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.     

Cloning and characterization of α-L-arabinofuranosidase and bifunctional α-L-arabinopyranosidase/β-D-galactopyranosidase from Bifidobacterium longum H-1

Aims: This study focused on the cloning, expression and characterization of recombinant α‐l ‐arabinosidases from Bifidobacterium longum H‐1. Methods and Results: α‐l ‐Arabinofuranosidase (AfuB‐H1) and bifunctional α‐l ‐arabinopyranosidase/β‐d ‐galactosidase (Apy‐H1) from B. longum H‐1 were identified by Southern blotting, and their recombinant enzymes were overexpressed in Escherichia coli BL21 (DE3). Recombinant AfuB‐H1 (rAfuB‐H1) was purified by single‐step Ni²⁺‐affinity column chromatography, whereas recombinant Apy‐H1 (rApy‐H1) was purified by serial Q‐HP and Ni²⁺‐affinity column chromatography. Enzymatic properties and substrate specificities of the two enzymes were assessed, and their kinetic constants were calculated. According to the results, rAfuB‐H1 hydrolysed p‐nitrophenyl‐α‐l ‐arabinofuranoside (pNP‐αL‐Af) and ginsenoside Rc, but did not hydrolyse p‐nitrophenyl‐α‐l ‐arabinopyranoside (pNP‐αL‐Ap). On the other hand, rApy‐H1 hydrolysed pNP‐αL‐Ap, p‐nitrophenyl‐β‐d ‐galactopyranoside (pNP‐βD‐Ga) and ginsenoside Rb2. Conclusions: Ginsenoside‐metabolizing bifidobacterial rAfuB‐H1 and rApy‐H1 were successfully cloned, expressed, and characterized. rAfuB‐H1 specifically recognized the α‐l ‐arabinofuranoside, whereas rApy‐H1 had dual functions, that is, it could hydrolyse both β‐d ‐galactopyranoside and α‐l ‐arabinopyranoside. Significance and Impact of the Study: These findings suggest that the biochemical properties and substrate specificities of these recombinant enzymes differ from those of previously identified α‐l ‐arabinosidases from Bifidobacterium breve K‐110 and Clostridium cellulovorans.     

Entamoeba histolytica: Identification and partial characterization of α-mannosidase activity

Despite their well recognized importance in pathogenesis of Entamoeba histolytica there are few studies dealing with the assembly and secretion of glycoproteins that participate in the adhesion to target cells and in the dissemination of the parasite in infected tissues. Some of these studies refer to the identification and, in some cases, the characterization of glycosyl transferases and glycosidases involved in the biosynthesis of these macromolecules as well as to compartments involved in the amoeba dolichol-linked glycosylation pathway. While an N-glycan trimming α-mannosidase has been demonstrated in E. histolytica, little is known on its cellular distribution and properties. Here we describe the presence and partial biochemical characterization of soluble and MMF-associated forms of α-mannosidase and the separation of at least three internal membrane structures enriched with this glycosidase. Results are discussed in terms of the possible identity of α-mannosidase activity and the potential precursor-product relationship between the two enzyme forms.     

Cloning of α-amylase gene fromSchwanniomyces occidentalis and expression inSaccharomyces cerevisiae

The cloning of α-amylase gene ofS. occidentalis and the construction of starch digestible strain of yeast,S. cerevisiae AS. 2. 1364 with ethanol-tolerance and without auxotrophic markers used in fermentation industry were studied. The yeast/E.coli shuttle plasmid YCEp1 partial library ofS. occidentalis DNA was constructed and α-amylase gene was screened in S.cerevisiae by amylolytic activity. Several transformants with amylolysis were obtained and one of the fusion plasmids had an about 5.0 kb inserted DNA fragment, containing the upstream and downstream sequences of α-amylase gene fromS. occidentalis. It was further confirmed by PCR and sequence determination that this 5.0 kb DNA fragment contains the whole coding sequence of α-amylase. The amylolytic test showed that when this transformant was incubated on plate of YPDS medium containing 1 % glum and 1 % starch at 30°C for 48 h starch degradation zones could be visualized by staining with iodine vapour. α-amylase activity of the culture filtratate is 740–780 mU/mL and PAGE shows that the yeast harboring fusion plasmids efficiently secreted α-amylase into the medium, and the amount of the recombinant α-amylase is more than 12% of the total proteins in the culture filtrate. These results showed that α-amylase gene can be highly expressed and efficiently secreted inS. cerevisiae AS. 2.1364, and the promotor and the terminator of α-amylase gene fromS. occidentalis work well inS. cercvisiac AS. 2.1364.