Modulation of biorecognition of glucoamylases with Concanavalin A by glycosylation via recombinant expression.

Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 x 10(-6) M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl-alpha-d-mannopyranoside.

Manipulation of host cytokine network by ticks: a potential gateway for pathogen transmission.

Ticks are obligatory blood-feeding arthropods that secrete various immunomodulatory molecules to antagonize host inflammatory and immune responses. Cytokines play an important role in regulating these responses. We investigated the extent to which ticks interact with the sophisticated cytokine network by comparing the effect of salivary gland extracts (SGE) of 3 ixodid tick species, Dermacentor reticulatus, Amblyomma variegatum and Ixodes ricinus, all of which are important vectors of tick-borne pathogens. Using specific ELISAs, anti-cytokine activity was demonstrated with 7 cytokines: IL-8, MCP-1, MIP-1alpha, RANTES, eotaxin, IL-2 and IL-4. The results varied between species, and between adult males and females of the same species. Relatively high activity levels were detected in saliva of female D. reticulatus, confirming that the observed anti-cytokine activities are an integral part of tick saliva secreted into the host. Results with fractionated SGE indicated that from 2 to 6 putative cytokine binding molecules are produced, depending on species and sex. Binding ability of SGE molecules was verified by cross-linking with radio-isotope labelled MIP-1alpha. By targeting different cytokines, ixodid ticks can manipulate the cytokine network, which will greatly facilitate blood-feeding and provide a gateway for tick-borne pathogens that helps explain why ticks are such efficient and effective disease vectors.

Vesicular stomatitis virus nucleocapsid protein production in cells treated with selected fast protein liquid chromatography fractions of tick salivary gland extracts.

A salivary gland extract (SGE) prepared from 5-days-fed Dermacentor reticulatus female ticks was fractionated by fast protein liquid chromatography (FPLC). The effect of three FPLC fractions selected on the basis of anti-interleukin 8 (anti-IL-8) activity on vesicular stomatitis virus (VSV) nucleocapsid (N) protein formation in mouse L-cells was determined. Infected 14C-labeled cells treated with the FPLC fractions were analyzed by two-dimensional (2D) electrophoresis. The yields of VSV N protein were evaluated by Imagemaster software analysis. Most noticeable was an increase in the N protein production after treatment with the fraction 39 corresponding to the major peak of the anti-IL-8 activity. The nature of the substance in SGE that was responsible for this effect remains unclear.

Anti-interleukin-8 activity of tick salivary gland extracts.

Interleukin-8 (IL-8) is one of many mammalian chemokines (chemotactic cytokines) that direct mammalian inflammatory and immune cells to sites of injury and infection. Chemokines are produced locally and act on leucocytes through selective receptors. The principal role of IL-8 is to control the movement and activity of neutrophils. To date, several tick species have been shown to modulate the production or activity of certain cytokines but none of these are chemokines. Using an IL-8 specific ELISA, we showed that salivary gland extracts (SGE) from several ixodid tick species (Dermacentor reticulatus, Amblyomma variegatum, Rhipicephalus appendiculatus, Haemaphysalis inermis and Ixodes ricinus) reduced the level of detectable IL-8. Analyses of fractionated SGE revealed one similar peak of activity for D. reticulatus, A. variegatum and R. appendiculatus; a second peak, observed for D. reticulatus and A. variegatum, differed between the two species. Using radiolabelled IL-8, SGE and peak activity fractions of D. reticulatus were shown to bind the chemokine, and to inhibit binding of IL-8 to its receptors on human granuolocytes enriched for neutrophils. The biological significance of these observations was demonstrated by the ability of SGE to inhibit IL-8 induced chemotaxis of human blood granulocytes. Future isolation and characterization of the active molecules will enable determination of their functional roles in bloodfeeding and effect on tick-borne pathogen transmission.

Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes.

The mutation Gly467-->Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the kcat towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (Ka congruent with 10(13).M(-1)) than the Glu enzyme (Ka congruent with 10(12).M(-1)). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (Ka congruent with 10(4).M(-1)) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.

Structure of glucoamylase from Saccharomycopsis fibuligera at 1.7 A resolution.

The yeast Saccharomycopsis fibuligera produces a glucoamylase which belongs to sequence family 15 of glycosyl hydrolases. The structure of the non-glycosyl-ated recombinant enzyme has been determined by molecular replacement and refined against 1.7 A resolution synchrotron data to an R factor of 14.6%. This is the first report of the three-dimensional structure of a yeast family 15 glucoamylase. The refinement from the initial molecular-replacement model was not straightforward. It involved the use of an unrestrained automated refinement procedure (uARP) in combination with the maximum-likelihood refinement program REFMAC. The enzyme consists of 492 amino-acid residues and has 14 alpha-helices, 12 of which form an (alpha/alpha)6 barrel. It contains a single catalytic domain but no starch-binding domain. The fold of the molecule and the active site are compared to the known structure of the catalytic domain of a fungal family 15 glucoamylase and are shown to be closely similar. The active- and specificity-site residues are especially highly conserved. The model of the acarbose inhibitor from the analysis of the fungal enzyme fits tightly into the present structure. The active-site topology is a pocket and hydrolysis proceeds with inversion of the configuration at the anomeric carbon. The enzyme acts as an exo-glycosyl hydrolase. There is a Tris [2-amino-2-(hydroxymethyl)-1,3-propanediol] molecule acting as an inhibitor in the active-site pocket.

Crystallization and preliminary X-ray analysis of the Saccharomycopsis fibuligera glucoamylase expressed from the GLU1 gene in Escherichia coli.

The active non-glycosylated glucoamylase, overexpressed from the Saccharomycopsis fibuligera GLU1 gene in Escherichia coli BL21(DE3), has been purified from the solubilized inclusion bodies and then renatured in vitro. Crystals of the recombinant glucoamylase were obtained by vapour diffusion using PEG as precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell dimensions of a = 58.1, b = 87.8 and c = 99.9 A, and diffract to 1.7 A resolution. This is the first report of the crystallization of the full-length glucoamylase corresponding to the mature enzyme.

High-yield production of Saccharomycopsis fibuligera glucoamylase in Escherichia coli, refolding, and comparison of the nonglycosylated and glycosylated enzyme forms.

The truncated GLA1 gene encoding the mature form of glucoamylase from the yeast Saccharomycopsis fibuligera has been over-expressed in Escherichia coli using the IPTG inducible pET system. Over-expression has led to the accumulation of insoluble glucoamylase in inclusion bodies from which an electrophoretically homogeneous active enzyme has been prepared yielding 30 mg per litre medium. This protein represents an N-terminus Met-free, non-glycosylated product which displays the identical specific activity of 45 units/mg and reduced thermal stability when compared to glycosylated enzymes isolated from Saccharomyces cerevisiae carrying the GLA1 gene. These data suggest that S. cerevisiae glycosylation of S. fibuligera glucoamylase does not play a critical role in enzymatic activity but that it does contribute to its thermal stability.

Purification and characterization of the cell-wall-associated and extracellular alpha-glucosidases from Saccharomycopsis fibuligera.

Cell-wall-associated and extracellular alpha-glucosidases were purified to homogeneity from Saccharomycopsis fibuligera KZ growing on a medium containing cellobiose as the sole source of carbon; this substrate has the greatest inducing effect on the production of both forms of the enzyme. Depending on the source of carbon, 75-90% of the enzyme is associated with cell wall, from which it can be completely released by 1% Triton X-100 at 25 degrees C in 2 h. Both enzymes are glycoproteins in monomeric form with an apparent molecular mass of 132 kDa estimated by SDS/PAGE and 135 kDa estimated by gel filtration. N-linked carbohydrate accounts for 12% of the total mass. Both forms exhibited optimum activity at pH 5.5 and seem to be stable in the pH range 4.0-8.0 on incubation at 4 degrees C for 24 h. The cell-wall-associated form had an optimum activity at 42.5 degrees C and was stable in the absence of substrate up to 30 degrees C, while the extracellular form had optimal activity at 52.5 degrees C and was stable up to 40 degrees C. Both forms are unable to renature after thermal inactivation. The cell-wall-associated and extracellular alpha-glucosidases cleaved the same kind of substrates, from maltose to maltoheptaose, isomaltase and panose, although showing different rates of hydrolysis, and had little or no activity with polysaccharides. The extracellular form cross-reacts with antibody raised against the cell-wall-associated form, and both forms show the same peptide pattern after cleavage with chymotrypsin. The amino acid sequences of six peptides from both forms show marked similarity to those of Schwanniomyces occidentalis glucoamylase.

Pseudorabies virus growth factor can be resolved into two active components.

Pseudorabies virus (PRV) growth factor (PRGF) which induces a transformed phenotype in normal MK-2 cells and represses the transformed phenotype of Hela cells was partially purified and resolved into two components (M(r) < 300 and < 180). Each of the PRGF components retained the transforming activity of the original factor in MK-2 cells but lost its transformation-repressing activity in Hela cells. The latter activity of PRGF could be reconstituted by simultaneous application of its two components. Two monoclonal antibodies against gII glycoprotein of PRV were able to neutralize both PRGF activities, thus supporting the previously suggested hypothesis that the PRV gene for glycoprotein gII might be involved in PRGF synthesis.

The nucleotide sequence of the glucoamylase gene GLA1 from Saccharomycopsis fibuligera KZ.

The nucleotide sequence of the 2544-bp PstI fragment carrying the glucoamylase gene of Saccharomycopsis fibuligera KZ, designated as GLA1, has been determined. When compared with the nucleotide sequence of the GLU1 gene one nucleotide substitution was found in the 321- bp of the 5'-flanking region: 24 nucleotides were altered within the 1557 bp of the structural gene causing the deduced protein products of both genes to differ in three amino acids in the signal-peptide region and in eight amino acids of the mature protein. Six nucleotide insertions and 27 substitutions were in the 663 bp of the 3'-flanking region. The gene product expressed and secreted in Saccharomyces cerevisiae into the functional enzyme was not homogeneous. In situ detection of the enzyme in a polyacrylamide gel revealed two dominant and three minor bands.