Juraj Gašperík

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.

Molecular cloning and 3D structure prediction of the first raw-starch-degrading glucoamylase without a separate starch-binding domain.

Raw-starch-degrading glucoamylases have been known as multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain (SBD) by an O-glycosylated linker region. A molecular genetics approach has been chosen to find structural differences between two related glucoamylases, raw-starch-degrading Glm and nondegrading Glu, from the yeasts Saccharomycopsis fibuligera IFO 0111 and HUT 7212, respectively. We have found that Glm and Glu show a high primary (77%) and tertiary structure similarity. Glm, although possessing a good ability for raw starch degradation, did not show consensus amino acid residues to any SBD found in glucoamylases or other amylolytic enzymes. Raw starch binding and digestion by Glm must thus depend on the existence of a site(s) lying within the intact protein which lacks a separate SBD. The enzyme represents a structurally new type of raw-starch-degrading glucoamylase.

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.

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.

Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain

Most glucoamylases (α‐1,4‐d‐glucan glucohydrolase, EC 3.2.1.3) have structures consisting of both a catalytic and a starch binding domain. The structure of a glucoamylase from Saccharomycopsis fibuligera HUT 7212 (Glu), determined a few years ago, consists of a single catalytic domain. The structure of this enzyme with the resolution extended to 1.1 Å and that of the enzyme–acarbose complex at 1.6 Å resolution are presented here. The structure at atomic resolution, besides its high accuracy, shows clearly the influence of cryo‐cooling, which is manifested in shrinkage of the molecule and lowering the volume of the unit cell. In the structure of the complex, two acarbose molecules are bound, one at the active site and the second at a site remote from the active site, curved around Tyr464 which resembles the inhibitor molecule in the ‘sugar tongs’ surface binding site in the structure of barley α‐amylase isozyme 1 complexed with a thiomalto‐oligosaccharide. Based on the close similarity in sequence of glucoamylase Glu, which does not degrade raw starch, to that of glucoamylase (Glm) from S. fibuligera IFO 0111, a raw starch‐degrading enzyme, it is reasonable to expect the presence of the remote starch binding site at structurally equivalent positions in both enzymes. We propose the role of this site is to fix the enzyme onto the surface of a starch granule while the active site degrades the polysaccharide. This hypothesis is verified here by the preparation of mutants of glucoamylases Glu and Glm.

Glucoamylases encoded by variantSaccharomycopsis fibuligera genes: Structure and properties

The genes of two variant glucoamylases (GLA1 and GLU1) ofSaccharomycopsis fibuligera were expressed inSaccharomyces cerevisiae, and biochemical properties of the secreted enzymes were compared. It was found that three amino acid alterations in the signal peptide and N-terminal regions of the variants had no effect on the levels of the secreted enzymes. Amino acid alterations in the C-terminal region of the mature proteins influenced their specific activity, substrate specificity, as well as temperature and pH optima. Because of the glycosylation heterogeneity, the glucoamylases of each gene variant were isolated and purified in two forms (A and B), which were essentially similar in catalytic and physicochemical properties but differed in their thermal stability and ability to renaturate after thermal denaturation.

Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias.

X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix.

Purification and characterization of the amylolytic enzymes of Saccharomycopsis fibuligera

Abstract 1. 1. A complex of extracellular amylolytic enzymes produced by Saccharomycopsis fibuligera KZ , grown on fine fibre (waste product from corn starch production) and corn-steep liquor, has been studied. 2. 2. α-Amylases and glucoamylases, as the main representatives of this complex, were separated by hydrophobic chromatography on Spheron 300 LC. 3. 3. Individual isoenzymes of one type were separated on FPLC-Mono Q. 4. 4. The relative molecular weight of α-amylases is 54,000, glucoamylases 62,000, maximal activity is reached by both enzymes between pH 5.0 and 6.2 at a temperature of 40–50°C. 5. 5. Glucoamylases have a higher stability of the native structure than α-amylases, they retain 55% of their original activity, even after 10 min of incubation at 100°C.

Yeast glucoamylases: molecular-genetic and structural characterization

Glucoamylase is an extracellular enzyme produced mainly by microorganisms. It belongs to the commercially frequently exploited biocatalysts. The major application of glucoamylase is in the starch bioprocessing to produce glucose and in alcoholic fermentations of starchy materials. Filamentous fungi have been the source of glucoamylases for industrial purposes as well as an object of numerous research studies. Some yeasts also secrete a large amount of glucoamylase with biochemical characteristics slightly different from those of filamentous fungi. Modern biotechnological applications require glucoamylases of certain properties optimal for a given process. Novel biocatalysts can be prepared from already existing enzymes using techniques of protein engineering or directed evolution. Tailoring of a commercial glucoamylase requires knowledge, on a molecular level, of structure/function relationships of enzymes originating from various sources and having different catalytic properties. Sequences of the cloned genes, their recombinant expression and the tertiary structure determination of glucoamylase are prerequisite to obtain such information. The presented review focuses on molecular-genetic and structural aspects of yeast glucoamylases, supplemented with the basic biochemical characterization of the given enzymes.

Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2

We report the domain analysis of the N-terminal region (residues 1–759) of the human cardiac ryanodine receptor (RyR2) that encompasses one of the discrete RyR2 mutation clusters associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1) and arrhythmogenic right ventricular dysplasia (ARVD2). Our strategy utilizes a bioinformatics approach complemented by protein expression, solubility analysis and limited proteolytic digestion. Based on the bioinformatics analysis, we designed a series of specific RyR2 N-terminal fragments for cloning and overexpression in Escherichia coli. High yields of soluble proteins were achieved for fragments RyR21–606·His6, RyR2391–606·His6, RyR2409–606·His6, Trx·RyR2384–606·His6, Trx·RyR2391-606·His6 and Trx·RyR2409–606·His6. The folding of RyR21–606·His6 was analyzed by circular dichroism spectroscopy resulting in α-helix and β-sheet content of ∼23% and ∼29%, respectively, at temperatures up to 35 °C, which is in agreement with sequence based secondary structure predictions. Tryptic digestion of the largest recombinant protein, RyR21–606·His6, resulted in the appearance of two specific subfragments of ∼40 and 25 kDa. The 25 kDa fragment exhibited greater stability. Hybridization with anti-His6·Tag antibody indicated that RyR21–606·His6 is cleaved from the N-terminus and amino acid sequencing of the proteolytic fragments revealed that digestion occurred after residues 259 and 384, respectively.