Cloning and functional expression of a novel GDP-6-deoxy-D-talose synthetase from Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that can cause various forms of severe periodontitis and other nonoral infections in human patients. The serotype a-specific polysaccharide antigen of A. actinomycetemcomitans contains solely 6-deoxy-D-talose and its O-2 acetylated modification. This polysaccharide is synthesized from the donor GDP-6-deoxy-D-talose with the relevant talosylation enzyme(s). In the synthesis of GDP-6- deoxy-D-talose, GDP-D-mannose is first converted by GDP-mannose-4,6-dehydratase (GMD) to GDP-4-keto-6-deoxy-D-mannose and then reduced to GDP-6-deoxy-D-talose by GDP-6-deoxy-D-talose synthetase (GTS). In this study, we cloned and overexpressed in Escherichia coli the A. actinomycetemcomitans GTS enzyme responsible for the synthesis of GDP-6-deoxy-D-talose. The recombinant A. actinomycetemcomitans GTS enzyme expressed in E. coli converted the GDP-4-keto-6-deoxy-intermediate to a novel GDP-deoxyhexose. The synthesized GDP-deoxyhexose was shown to be GDP-6-deoxy-D-talose by HPLC, MALDI-TOF MS, and NMR spectroscopy. The functional expression of gts provides another enzymatically defined pathway for the synthesis of GDP-deoxyhexoses, which can be used as donors for the corresponding glycosyltransferases.     

Purification and cDNA cloning of UDP-GlcNAc:GlcNAcbeta1-3Galbeta1-4Glc(NAc)-R [GlcNAc to Gal]beta1,6N-acetylglucosaminyltransferase from rat small intestine: a major carrier of dIGnT activity in rat small intestine

A rat intestinal beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified to apparent homogeneity by successive column chromatographic procedures using an assay wherein pyridylaminated lacto- N-triose II (GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcbeta1-3'LacNAc into GlcNAcbeta1-3'(GlcNAcbeta1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcbeta1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galbeta1-3GalNAcalpha1-O-paranitrophenyl (pNP) and GlcNAcbeta1-3GalNAcalpha1-O-pNP, into Galbeta1-3(GlcNAcbeta1-6) GalNAcalpha1-O-pNP (C2GnT activity) and GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-pNP (C4GnT activity), respectively. Based on the partial amino acid sequences of the purified protein, the cDNA encoding this enzyme was cloned. The COS-1 cells transiently transfected with this cDNA had high dI/C2/C4GnT activities in a ratio of 0.34:1.00:0.90, compared with non- or mock-transfected cells. The primary structure shows a significant homology with human and viral mucin-type core 2 beta1-6GnTs (C2GnT-Ms), indicating that this enzyme is the rat ortholog of human and viral C2GnT-Ms. This is the first identification and purification of this enzyme as a major carrier of dIGnT activity in the small intestine. This rat ortholog should mostly be responsible for making distal I-branch structures on poly-N-acetyllactosamine sequences in this tissue, as well as making mucin core 2 and core 4 structures, given that it also has high C2/C4GnT activities.     

Interaction between the antigen and antibody is controlled by the constant domains: normal mode dynamics of the HEL-HyHEL-10 complex

The antigen binding fragment (Fab) of a monoclonal antibody (HyHEL-10) consists of variable domains (Fv) and constant domains (CL-CH1). Normal modes have been calculated from the three-dimensional structures of hen egg lysozyme (HEL) with Fab, those of HEL with Fv, and so on. Only a small structural change was found between HEL-Fab and HEL-Fv complexes. However, HEL-Fv had a one order of magnitude lower dissociation constant than HEL-Fab. The Calpha fluctuations of HEL-Fab differed from those of HEL-Fv with normal mode calculation, and the dynamics can be thought to be related to the protein-protein interactions. CL-CH1 may have influence not only around local interfaces between CL-CH1 and Fv, but also around the interacting regions between HEL and Fv, which are longitudinally distant. Eighteen water molecules were found in HEL-Fv around the interface between HEL and Fv compared with one water molecule in HEL-Fab. These solvent molecules may occupy the holes and channels, which may occur due to imperfect complementarity of the complex. Therefore, the suppression of atomic vibration around the interface between Fv and HEL can be thought to be related to favorable and compact interface formation by complete desolvation. It is suggested that the ability to control the antigen-antibody affinity is obtained from modifying the CL-CH1. The second upper loop in the constant domain of the light chain (UL2-CL), which is a conserved gene in several light chains, showed the most remarkable fluctuation changes. UL2-CL could play an important role and could be attractive for modification in protein engineering.     

Engineering nuclear localization signals in modular protein vehicles for gene therapy

Amino acids from 126 to 135 of the SV40 virus T antigen act as efficient nuclear localization signal during infection but also when fused to recombinant proteins. This peptide has been inserted into two alternative acceptor sites of a modified Escherichia coli beta-galactosidase which also displays a DNA-binding domain, a cell-binding motif for integrin alpha(v)beta(3) targeting and cell internalization, and a cryptic nuclear targeting signal naturally present in the bacterial enzyme. In cultured cells, the presence of the SV40 peptide enhances the expression of a delivered DNA up to 30-fold. However, the DNA expression levels are largely depending on the chosen insertion site for the SV40 segment concomitant to the structural impact of peptide accommodation on the protein vehicle. The structural stability of the hybrid protein, apparently critical for efficient gene transfer, is discussed in the context of modular protein engineering to develop non-viral vectors for gene therapy.     

Molecular cloning and expression of the hepatitis delta virus genotype IIb genome

Analysis of hepatitis delta virus (HDV) genome sequences has revealed multiple genotypes with different geographical distributions and associated disease patterns. To date, replication-competent cDNA clones of HDV genotypes I, II, and III have been reported. HDV genotypes I, II, and IIb have been found in Taiwan. Although full-length sequences of genotype IIb have been published, its replication competence in cultured cells has yet to be reported. In order to examine this, we obtained a full-length cDNA clone, Taiwan-IIb-1, from a Taiwanese HDV genotype IIb isolate. Comparison of the complete nucleic acid sequence of Taiwan-IIb-1 with previously published genotype IIb isolates indicated that Taiwan-IIb-1 shares 98% identity with another Taiwanese isolate and 92% identity with a Japanese isolate. Transfection of Taiwan-IIb-1 into COS7 cells resulted in accumulation of the HDV genome and appearance of delta antigens, showing that cloned HDV genotype IIb can replicate in cultured cells.     

Identification of a carcinoembryonic antigen binding protein on monocytes

Tumor-associated monocyte/macrophage cells are important stromal components involved in tumor development. A protein on human monocyte is identified that binds to carcinoembryonic antigen (CEA), a glycoprotein overexpressed in colon tumors. This implicates a role for this protein in CEA processing and establishes a link between monocytes and colon tumor cells. In vitro uptake of 125I-labeled CEA with isolated monocytes showed time and temperature dependence. The binding of 125I-CEA was specific and saturable as it could be inhibited by an excess of unlabeled CEA. To identify the binding protein on monocyte, we used a radiolabeled photoactivable heterobifunctional crosslinking agent and demonstrated that CEA reacts with a 115kDa protein as determined by SDS-polyacrylamide gel electrophoresis and autoradiography. Treatment of human monocytes in vitro with CEA resulted in a several fold increase in the production of proinflammatory cytokines TNF-alpha, IL-1 beta, and IL-6 compared to untreated controls. Binding of CEA to the monocyte protein may have implications in colon tumorigenesis.     

Expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments

AIMS: To study the expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies (MAbs) C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments. METHODS AND RESULTS: The reaction patterns of antibodies to L. monocytogenes held in stressful environments for a short duration (3 h) or grown for extended periods (16-72 h) were investigated. During both short or prolonged exposure to stress environments of high temperature (45 degrees C) and NaCl (>1.5%, w/v), reactions of whole cells of L. monocytogenes to antibodies were severely affected as determined by ELISA and by the reduced expression of the antibody-reactive 66 kDa antigen in the Western blot assay. Conversely, cold (4-15 degrees C) or acid (pH 2-3) stress environments had very little effect on antigen expression or antibody reaction. Additionally, heat-killed cells showed reduced reactions to these antibodies when compared with unheated cells. Artificially created stress environments in hotdog slurry also affected the antigen expression in L. monocytogenes. Immunoelectron microscopy revealed that the antibody-reactive antigens were uniformly present on the surface of the cells. Morphological characteristics following growth in stressed environments revealed that heat stress at 45 degrees C caused L. monocytogenes cells to be elongated and to form clumps; whereas, osmotic stress (5.5% NaCl, w/v) caused filamentous appearance with multiple septa along the length of the cell. CONCLUSIONS: These results indicated that MAb C11E9 or EM-7G1 could detect L. monocytogenes from cold or acid-stress environments; however, they may show weaker reactions with heat or osmotically stressed cells or cells grown at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteria in food are routinely subjected to various stresses, induced by cold, heat, salt or acid during processing and storage. Whether stresses would modify the expression of cellular antigens of L. monocytogenes is of a great concern for immunodetections in food products.     

Does N‐acetyl Cystein Affect the Sensitivity and Specificity of Helicobacter pylori Stool Antigen Test?

Background. N‐acetyl cystein, a mucolytic agent, might make Helicobacter pylori antigens shed more easily to stool, and might therefore contribute to the diagnostic accuracy of the Helicobacter pylori stool antigen test. The aim of this study is to investigate if N‐acetyl cystein contributes to the diagnostic accuracy of the Helicobacter pylori stool antigen test by increasing the sensitivity and specificity of the test. Materials and Methods. 107 patients were separated into treatment and placebo groups. The AC group (n = 53) was given 5 ml of acetyl cystein (4%) t.i.d. and the Placebo group (n = 54) was given placebo, for 3 days. Helicobacter pylori status was determined by both histology and CLOtest. Stool samples were assayed using a specific ELISA kit for Helicobacter pylori stool antigen. Results. The sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of Helicobacter pylori stool antigen test were 76%, 79%, 90%, 55%, and 77%, respectively, in AC group; and 85%, 89%, 93%, 76% and 86%, respectively, in placebo group. Conclusions. N‐acetyl cystein did not increase, and actually decreased, the sensitivity and specificity of the Helicobacter pylori stool antigen test according to our results. We believe that this finding can be taken into consideration when setting up the exclusion criteria for future studies, which will use Helicobacter pylori stool antigen tests.     

Mapping of the bovine blood group systems J,N', R', and Z show evidence for oligo-genetic inheritance

Genes determining the bovine erythrocyte antigens were mapped by linkage analysis. In total 9591 genotypes of 20 grandsire families with 1074 sires from a grand-daughter design were elucidated for the genes determining the erythrocyte antigens EAA, EAB, EAC, EAF, EAJ, EAL, EAM, EAN', EAR', EAS, EAT', and EAZ according to standard paternity testing procedures in the blood typing laboratories. Linkage analyses were performed with 248 microsatellite markers, eight SSCP markers and four polymorphic proteins and enzymes covering the 29 autosomes and the pseudoautosomal region of the sex chromosomes. The number of informative meioses for the blood group systems ranged from 76 to 947. Blood group systems EAM and EAT' were non-informative. Most of the erythrocyte antigen loci showed significant linkage to a single chromosome and were mapped unequivocally. The genes determining erythrocyte antigen EAA, EAB, EAC, EAL, and EAS were mapped to chromosomes 15, 12, 18, 3, and 21, respectively. Lod-score values ranged from 11.43 to 107.83. Moreover, the EAF system could be mapped to chromosome 17. However, the EAN' system previously known as part of the EAF system could be mapped to chromosome 5. In addition, the blood group systems EAJ, the new EAN', EAR', and EAZ, showed significant linkage to microsatellite markers on various chromosomes and also to other blood groups. The appearance of a single blood group system might be therefore either dependent on the existence of other blood group systems or because of an interaction between different loci on various chromosomes as is known in humans and in pigs.     

How do two unrelated antibodies,HyHEL‐10 and F9.13.7, recognize the same epitope of hen egg‐white lysozyme?

The anti-hen egg-white lysozyme (HEWL) antibodies HyHEL-10 and F9.13.7 recognize a common epitope. The structures of the complexes differ, however, in the numbers of electrostatic and hydrogen-bond interactions and in the distributions of contacts between the light and heavy chains. The equilibria and kinetics characterizing the F9.13.7 complex formation were evaluated for both wild-type and mutant derivatives of HEWL to help to understand how the different contacts are effectively used in the complexes with the two antibodies. Three epitope hot spots, Y20, K96, and R73 (destabilization > 4 kcal/mole), were found by alanine scanning mutagenesis. The first two constitute two of the three hot spots in the HyHEL-10 complex. The hot spots of the HyHEL-10 paratope are centered on the HEWL epitope; whereas R73 (HEWL), the only important light-chain-contacting residue, is clearly separated from the other hot spots of the F9.13.7 complex. The larger number of epitope warm plus hot spots found in the F9.13.7 complex compared with that of HyHEL-10 shows that the specificity of the former is greater even though the K(D) value is 20-fold larger. Conservative mutations showed that the specificity enhancement is related to the greater number of functional polar and hydrogen bond interactions in the F9.13.7 complex. Alanine scanning mutagenesis would not have illuminated these distinctions. It is shown that the concept of antigen specificity, as defined by cross-reactivity with natural variant antigens, is flawed by phylogenetic bias, and that specificity can only be defined by the use of unbiased epitopes, which are conveniently accessed by site-directed mutagenesis.