Evolutionary and functional analysis of fructose bisphosphate aldolase of plant parasitic nematodes

The essential and ubiquitous enzyme fructose bisphosphate aldolase (FBPA) has been a good target for controlling the various types of infections caused by pathogens and parasites. The parasitic infections of nematodes are the major concern of scientific community, leading to biochemical characterization of this enzyme. In this work we have developed a small dataset of all types of FBPA sequences collected from publically available databases (EMBL, NCBI and Uni-Port). The Phylogenetic study shows that evolutionary relationships among sequences of FBPA are clustered into three main groups. FBPA sequences of Globodera rostochiensis (FBPA_GR) and Heterodera glycines (FBPA_HG) are placed in group II, sharing the similar evolutionary relationship. The catalytic mechanism of these enzymes depends upon which class of aldolase, it belongs. The class of enzyme has been confirmed on the basis of sequences and structural similarity with template structure of class I FBPA. To confirm catalytic mechanism of above said model structures, the known substrate fructose-1, 6-bisphosphate (FBP) and competitive inhibitor Mannitol-1, 6 bisphosphate (MBP) were docked at known catalytic site of enzyme of interest. The comparative docking analysis shows that enzyme-substrate complex is forming similar Schiff base intermediate and conducts C₃–C₄ bond cleavage by forming Hydrogen bonding with reaction catalyzing Glu-191, reactive Lys-150, and Schiff base forming Lys-233. On the other hand enzymeinhibitor noncovalent complex is forming cabinolamine precursor and the proton transfer by the formation of hydrogen bond between MBP O₂ with Glu191 enabling stabilization of cabinolamine transition state, which confirms the similar inhibition mechanism. Thus we conclude that Plant Parasitic Nematodes (PPNs) have evolutionary and functional relationship with the class I aldolase enzyme. Hence, FBPA can be targeted to control plant parasitic nematodes.     

Evolution and functional diversification of fructose bisphosphate aldolase genes in photosynthetic marine diatoms

Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked through common, although not necessarily monophyletic, acquisition of plastid endosymbionts of red as well as most likely green algal origin. There is also strong evidence for a relatively high level of lineage-specific bacterial gene acquisition within chromalveolates. Therefore, analyses of gene content and derivation in chromalveolate taxa have indicated particularly diverse origins of their overall gene repertoire. As a single group of functionally related enzymes spanning two distinct gene families, fructose 1,6-bisphosphate aldolases (FBAs) illustrate the influence on core biochemical pathways of specific evolutionary associations among diatoms and other chromalveolates with various plastid-bearing and bacterial endosymbionts. Protein localization and activity, gene expression, and phylogenetic analyses indicate that the pennate diatom Phaeodactylum tricornutum contains five FBA genes with very little overall functional overlap. Three P. tricornutum FBAs, one class I and two class II, are plastid localized, and each appears to have a distinct evolutionary origin as well as function. Class I plastid FBA appears to have been acquired by chromalveolates from a red algal endosymbiont, whereas one copy of class II plastid FBA is likely to have originated from an ancient green algal endosymbiont. The other copy appears to be the result of a chromalveolate-specific gene duplication. Plastid FBA I and chromalveolate-specific class II plastid FBA are localized in the pyrenoid region of the chloroplast where they are associated with β-carbonic anhydrase, which is known to play a significant role in regulation of the diatom carbon concentrating mechanism. The two pyrenoid-associated FBAs are distinguished by contrasting gene expression profiles under nutrient limiting compared with optimal CO2 fixation conditions, suggestive of a distinct specialized function for each. Cytosolically localized FBAs in P. tricornutum likely play a role in glycolysis and cytoskeleton function and seem to have originated from the stramenopile host cell and from diatom-specific bacterial gene transfer, respectively.     

Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action

The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions. The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions. No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle. In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast. Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle. The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe. We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex. A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.     

Factors influencing the expression in vitro of Candida albicans stress mannoproteins reactive with salivary secretory IgA

We have examined the influence of subinhibitory concentrations of several antifungals, the different glucose and ammonium sulphate concentrations in the culture medium as well as the strain variability on the expression in vitro of stress mannoproteins reactive with salivary sIgA in C. albicans and other Candida spp isolates. Irrespective of the conditions used, no reactivity with salivary sIgA was observed in yeast cells grown at 25 °C. However, when grown at 37 °C, all of the 10 C. albicans strains, but only 9 out 28 non-C.albicans isolates studied showed reactivity with salivary sIgA. Cells grown at 37 °C in medium containing maximum concentrations of glucose and ammonium sulphate expressed the antigens reactive with sIgA during longer periods of time than the cells grown in medium with minimal concentrations of the same compounds. The regulatory role showed by the concentration of glucose and ammonium sulphate on the antigenic expression was subordinated, nevertheless, to the most important factor, the temperature of incubation. Only isolates showing low susceptibility expressed the antigens reactive with sIgA under the influence of subinhibitory concentration of antifungals. However, induced resistance to one of the antifungals tested (5 fluorocytosine) allowed the antigenic expression at elevated subinhibitory concentrations even in previous susceptible strains. In conclusion, in addition to the temperature, factors such as characteristics of the strain, the concentration of glucose and ammonium sulphate in the culture medium and the resistance to antifungals played a role on the expression of C. albicans antigens reactive with sIgA, which could be of clinical relevance in the course of infection.This revised version was published online in October 2005 with corrections to the Cover Date.     

Common and form-specific cell wall antigens of Candida albicans as released by chemical and enzymatic treatments

In order to investigate the antigenic properties of the proteins and mannoproteins present in the cell surface of Candida albicans, and to identify individual antigenic moieties and their distribution, a number of polyclonal antisera were obtained by immunizing rabbits with chemical and enzymatic cell wall extracts obtained from intact cells from both growth forms (yeast and mycelium) of the fungus. Prior to injection, wall moieties present in the extracts were subjected to different treatments and/or purification procedures such as adsorption onto polystyrenelatex microbeads or electrophoretic separation. When used as probes in indirect immunofluorescence assays, the different antisera gave rise to different fluorescence patterns varying in intensity and topological localization of the reactivity in C. albicans cells. When the different antisera were used as probes in Western blots of wall proteinaceous materials solubilized from both blastospores and germ tubes, differences in reactivity and specificity were readily discernible, allowing to identify a number of common and form-specific cell wall components. Of special interest was the fact that one of the antisera raised, after adsorption onto heat-killed blastospores, specifically recognized medium to low molecular weight moieties present only in the cell wall extracts from germ tubes. When this antiserum was used as probe in immunofluorescence assays, reactivity was confined to the hyphal extensions. Together, these observations seem to indicate that the different antibody preparations described in this report could represent important tools in the study of different aspects of the cell wall biology in C. albicans, including the identification and study of the distribution of common and form-specific cell wall-bound antigens.Abbreviations IIF Indirect immunofluorescence - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PAb polyclonal antibody     

Presentation of Candida albicans and purified protein derivative soluble antigens by Epstein-Barr virus-transformed human lymphoblastoid B-cell lines

Cells other than the macrophage can function as antigen-presenting cells (APCs). These class II-bearing accessory cells include dendritic cells, epidermal Langerhans cells, B cells, murine B-cell tumors, and human Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL). We investigated the ability of EBV-LCL to present two soluble antigens, Candida albicans and purified protein derivative of tuberculin (PPD). The EBV-LCL derived from B cells of two different individuals can present both antigens to bulk cultures of autologous antigen-primed peripheral blood lymphocytes. The responses of PPD-reactive T-cell clones were weaker to PPD when presented by EBV-LCL than by PBL-APCs, with some clones responding only to PPD presented by PBL-APCs. This suggests that EBV-LCL are not equivalent to PBL monocytes in APC function, and that expression of class II major histocompatibility complex antigen is not sufficient in enabling antigen-presenting capability.     

Identification of cyst and trophozoite antigens from Colombian Giardia duodenalis isolates recognized by IgA

Little is known about the role of IgA in the immune response against Giardia duodenalis infection. The current study identified the antigens of Colombian G. duodenalis isolates which stimulate the production of IgA anti-G. dudoenalis. Cyst and trophozoite stage proteins were separated by SDS-PAGE and their antigenicity was determined by Western blot. Without 2-mercapto ethanol (2-ME), the protein profile of the cyst stage showed 24 proteins within a molecular weight range of 23-270 kDa; with 2-ME, 35 polypeptides ranging from 22 to 241 kDa were distinguished. The trophozoite stage protein profile without 2-ME was formed by 16 proteins within the range of 24-270 kDa; with 2-ME, 45 proteins were present between 18 and 241 kDa. The identification of 20 and 29 antigens from the cyst and trophozoite stage, respectively, suggested that G. duodenalis stimulates a specific humoral immune response in the human host. The antigens of 31, 57, 110, 133, and 170 kDa recognized by anti-G duodenalis IgA in both cysts and trophozoites corresponded with G. duodenalis isolates from other geographic regions, whereas those of 35, 38, 43, 45, 49, 52, 60, 62, 65, 72, 82, 99, 145, 155, and 185 kDa seemed specific to Colombian isolates. This indicated that antigens of 57, 65, 145, and 170 kDa, recognized by anti-G. duodenalis IgA antibodies in cysts (with frequencies between 82% and 96%) and trophozoites (with frequencies between 86% and 97%) can be considered identification markers for G. duodenalis infections.     

Concentration and partitioning of intermediates in the fructose bisphosphate aldolase reaction. Comparison of the muscle and liver enzymes

Chemical analysis of enzyme reaction intermediates has been used to compare the liver and muscle isozymes of rabbit aldolase at equilibrium and in their steady states to determine if they have properties that favor the direction of flow of glycolytic intermediates in their tissues of origin. For both enzymes at saturating concentrations of fructose 1,6-P2, the sum of intermediates in the steady state agreed with the total active enzyme calculated to be present. The two half-reactions, characterized by fructose 1,6-bisphosphate(Fru-P2):aldehyde exchange and DHAP:proton exchange were found to be of different importance in determining the rate of reaction with Fru-P2 with the liver enzyme being much more limited in the processing of DHAP. The chemical interconversions within each half-reaction are generally rapid compared with the release of products. The greater sensitivity of liver aldolase to inhibition by aldehydes in Fru-P2 cleavage seems to be a normal consequence of the higher level of the eneamine of DHAP in the forward steady state with the liver enzyme and probably should not be ascribed to a greater intrinsic affinity. An earlier report (Grazi, E., and Trombetta, G. (1979) Eur. J. Biochem. 100, 197-202) purporting to show a special interaction of glyceraldehyde-3-P with liver enzyme prior to proton abstraction from DHAP could not be reproduced. Examples are presented from the data that validate the use of the analytical methods used for analysis of intermediates in the case of the Schiff's base aldolases.     

Immunochemical characterization of two antigens recognized by new monoclonal antibodies against human colon carcinoma

Two different monoclonal antibodies (MAb), called L-D1 and L-C5, were produced after immunization with either intact cells or the methanol phase of glycolipid extracts, respectively, from the same human colon carcinoma line, LoVo. As determined by an antibody-binding radioimmunoassay (RIA) on intact cells, MAb L-D1 and MAb L-C5 were highly reactive with all five colon carcinoma lines tested and with only one out of the 21 cell lines of various tissue origin tested. No reactivity of either MAb was observed with peripheral blood lymphocytes, granulocytes, or erythrocytes from healthy donors of various blood groups. Both MAb were tested in competitive binding experiments with an anti-CEA MAb from our laboratory (CEA 35) and with two previously described anti-colon carcinoma MAb from the Wistar Institute called 1083-17-1A (17-1A) and NS-19.9. In competitive binding experiments, MAb L-D1 was inhibited by MAb 17-1A and reciprocally, whereas MAb L-C5 was not inhibited by any of the other MAb tested. MAb L-D1 precipitated a major protein band with an apparent molecular weight (MW) of 41 kilodaltons (kD); interestingly, MAb 17-1A, which was reported to react with an uncharacterized antigen, precipitated the same protein band of 41 kD. This was confirmed with immunodepletion experiments. Furthermore, after treatment of the colon carcinoma cell line with tunicamycin, both MAb L-D1 and 17-1A precipitated a protein band of 35 kD. This shift of 6 kD suggests that the glycoprotein recognized by these 2 MAb contains two to three N-linked carbohydrate side chains. MAb L-C5 precipitated a group of three to four protein bands ranging from 43 to 53 kD that were not modified by tunicamycin treatment. A preliminary study conducted by using immunoperoxidase labeling on frozen sections of primary colon carcinoma showed that the two new MAb react strongly with these tumors, but also weakly with the normal adjacent mucosa, as did the other anti-colon carcinoma MAb tested.     

Induction of salivary antibodies to inhibit Candida albicans adherence to human epithelial cells by tonsillar immunization in rabbits

To examine the possibility of a vaccine for Candida albicans infection in the oral cavity, we induced salivary antibodies by immunization of killed-C. albicans ATCC 18804 on the palatine tonsils of rabbits. The enzyme-linked immunosorbent assay reaction of salivary antibodies was high against C. albicans serotype A. The saliva antibodies greatly inhibited C. albicans adherence to cloned epithelial cells from human gingiva. Tonsillar immunizations of C. albicans ATCC 18804 induce salivary antibodies that prevent C. albicans adherence to epithelial cells, and thus should prove useful in the prevention of oral candidiasis caused by C. albicans serotype A.     

Re-expression by Candida albicans germ tubes of antigens lost during subculture of blastospores

The effect of germ tube induction on the antigenic variability in C. albicans was studied in strains from blood cultures (Group I) and superficial candidiasis (Group II). When compared by immunoblotting with a rabbit antiserum, antigenic extracts from Group I strains grown as blastospores showed a higher reactivity than that of Group II strains. Major bands in Group I strains (45–47, 33, 30 kDa) were continuously expressed through the subcultures in vitro but, with the exception of the 45 kDa band, the reactivity of all of them decreased or disappeared after the tenth subculture in Group II strains. The induction of the germ tubes produced the re-expression of the antigens lost during subculture in the yeast form, the effect being very clear in Group II strains. The re-expression by C. albicans germ tubes of antigens lost during subculture of blastospores in vitro and the higher reactivity shown by Group I strains grown in mycelial phase should be taken into consideration when a test to detect anti-C. albicans antibodies is to be developed.Abbreviations GYE glucose-yeast extract agar     

Purification and characterization of fructose bisphosphate aldolase from the ground squirrel,Spermophilus lateralis: enzyme role in mammalian hibernation

Previous work has reported the production of an Escherichia coli branching enzyme with a 112-residue deletion at the amino terminal by limited proteolysis. Here, we study the chain transfer pattern of this enzyme. Gel-permeation chromatography of in vitro branched amylose shows that the truncated branching enzyme transfers fewer short chains (degree of polymerization [d.p.] <20) and a greater proportion of intermediate size chains (d.p. 30-90) than the native enzyme. High-performance anion-exchange chromatography (HPAEC) of the branching limited alpha-glucan product indicates that the truncated branching enzyme transfers a smaller proportion of chains with d.p. 4-11 and more chains longer than d.p. 12. Also, the genes encoding native or truncated branching enzyme were individually expressed in a branching enzyme-deficient mutant, AC71 (glgB(-)). By HPAEC analysis of the purified alpha-glucans we find that truncated branching enzyme transfers fewer chains of d.p. 5-11 and more chains longer than d.p. 12 relative to the full-length enzyme. These observations allow us to conclude that truncation of the amino-terminal domain has altered the branching pattern of the enzyme. Our results are consistent with the construction of hybrid branching enzymes from the maize isoforms.     

Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans

External signals induce the switch from a yeast to a hyphal growth form in the fungal pathogen Candida albicans. We demonstrate here that the catalytic subunit of a protein kinase A (PKA) isoform encoded by TPK2 is required for internal signalling leading to hyphal differentiation. TPK2 complements the growth defect of a Saccharomyces cerevisiae tpk1-3 mutant and Tpk2p is able to phosphorylate an established PKA-acceptor peptide (kemptide). Deletion of TPK2 blocks morphogenesis and partially reduces virulence, whereas TPK2 overexpression induces hyphal formation and stimulates agar invasion. The defective tpk2 phenotype is suppressed by overproduction of known signalling components, including Efg1p and Cek1p, whereas TPK2 overexpression reconstitutes the cek1 but not the efg1 phenotype. The results indicate that PKA activity of Tpk2p is an important contributing factor in regulating dimorphism of C. albicans.     

Degradation of human subendothelial extracellular matrix by proteinase-secreting Candida albicans

Candida albicans infections in severely immunocompromized patients are not confined to mucosal surfaces; instead the fungus can invade through epithelial and endothelial layers into the bloodstream and spread to other organs, causing disseminated infections with often fatal outcome. We investigated whether secretion of the C. albicans acid proteinase facilitates invasion into deeper tissues by degrading the subendothelial basement membrane. After cultivation under conditions that induce the secretion of the acid proteinase, C. albicans degraded radioactively metabolically labeled extracellular matrix proteins from a human endothelial cell line. The degradation was inhibited in the presence of pepstatin A, an inhibitor of acid proteinases. Pepstatin A-sensitive degradation of the soluble and immobilized extracellular matrix proteins fibronectin and laminin by proteinase-producing C. albicans was also detected, whereas no degradation was observed when the expression of the acid proteinase was repressed. Our results demonstrate that the C. albicans acid proteinase degrades human subendothelial extracellular matrix; this may be of importance in the penetration of C. albicans into circulation and deep organs.     

Preparation of target antigens specifically recognized by human natural killer cells

In an attempt to identify target cell membrane molecules recognized by natural killer (NK) cells, artificial membranes were prepared from detergent-solubilized plasma membranes of NK target cells and synthetic lipids. Such reconstituted membranes from human and rat NK target cells were shown to inhibit both human and rat NK-target cell conjugates in a species-specific fashion; these reconstituted membranes failed to inhibit NK cytotoxicity. The detergent-solubilized material from the human NK target K562 was subjected to various procedures prior to reconstitution and the conjugate inhibition assay. Conjugate inhibitory activity was lost upon trypsin digestion and incubation at 65 degrees C. This inhibition activity was absorbed to concanavalin A agarose and could subsequently be eluted with alpha-methylmannoside, resulting in approximately 20-fold purification. Gel filtration of this material on an AcA-34 column in detergent gave a broad activity peak with maximal activity in the molecular weight range of 30,000-165,000. Gel electrophoresis of purified membranes demonstrated multiple molecular weight bands in lipid membranes. The K562 membrane material, purified by concanavalin A agarose and gel filtration, inhibited conjugates between human NK cells and any of four human target cells, but not of conjugates with (1) human large granular lymphocytes and antibody-coated mouse tumor cells nor (2) rat NK cells and their target cells. Thus the purified glycoproteins from K562 retain the property of specific inhibition of human NK-target conjugates.