Anton Štalc

Human granulocyte colony stimulating factor (hG-CSF) expressed by methylotrophic yeast Pichia pastoris

Abstract Human granulocyte colony stimulating factor (hG-CSF) was expressed in the methylotrophic yeast Pichia pastoris, using two different constructs which resulted in proteins with different N-terminal sequences. In the first construct, a hexa-histidine tag and enterokinase cleavage site were added to the N-terminus of the protein to achieve one-step separation and exact processing. In the second construct, the gene was fused to the α-MF prepro leader at the Lys-Arg processing site (without Glu-Ala spacer). The PCR products were cloned in pPIC9 commercial vector and integrated into the alcohol oxidase region of the host genome. Transformation was done by electroporation or spheroplasting. Selection of good producing clones was performed by immunoblot analyses of the supernatants from shake-flask fermentation. Proper processing of the products was confirmed by N-terminal sequencing of the secreted proteins. With both plasmid constructs, the target proteins, bearing the histidine tag or not, represented majority of the secreted proteins. Although the proteins were present in the soluble form, they were highly aggregated, which interfered with purification. The most efficient way to obtain monomeric, biologically active protein was complete denaturation by guanidine-HCl or urea and subsequent renaturation during gel filtration chromatography.

A contribution to the mechanism of action of SAD-128

SAD-128 was found to be an effective protector of acetylcholinesterase against inhibition by soman, due to its ability to function as a reversible inhibitor and allosteric modifier of the AChE active site. It also attenuated aging of the soman-inhibited enzyme. In order to study the connection between some of these effects of SAD-128 and structural changes in acetylcholinesterase and/or the membrane to which the enzyme is bound, the influences of SAD-128 on the EPR spectra of the spin labelled enzyme and of the membrane were studied under various conditions and the results correlated with the kinetic parameters. SAD-128 increases the fluidity of human erythrocyte membranes but not that of the Torpedo marmorata electric organ. Similarly, the binding properties of membrane acetylcholinesterase for SAD-128, expressed in terms of the Hill coefficient, differ for the two preparations. Some structural changes in the enzyme active site were also observed in the presence of SAD-128. The high protective effect of SAD-128 against AChE inhibition was confirmed by the EPR method regardless of the organophosphorus inhibitor tested. On the other hand, the effect of SAD-128 on the retardation of irreversible inhibition of the enzyme essentially depends on the inhibitor used. From present results it can be concluded that the protective effects of SAD-128 against inhibition of m-AChE are related to the structural changes of the active site and can be additionally moderated by the microviscosity changes of the membrane.

On the location of active serines of membrane acetylcholinesterase studied by the ESR method

1. An attempt was made to find out the causes of the discrepancy between the ESR spectra of membrane acetylcholinesterase (EC 3.1.1.7) obtained by Morrisett and co-workers and those obtained by the present authors. 2. In order to see whether the discrepancy was due to the different spin-labeling procedures, the same membrane acetylcholinesterase preparations were spin-labeled with the same compound, using the two different spin-labeling procedures. The enzyme activity was determined with pH-static titration and the ESR spectra recorded. 3. It was found that after spin-labeling according to Morrisett and co-workers, there were from 10-100 times more spin-label molecules bound to the enzyme preparations than there were active serines in them. 4. Using the method of Morrisett and co-workers, the majority of spin-label molecules was found to be bound to sites outside the active serines whereas the spin-labeling procedures of the present authors proved to be selective for active serines; the discrepancy in ESR spectra is explained.

An ESR study of the influence of some physico-chemical factors on the conformation of a postsynaptic acetylcholinesterase

1. In a previous ESR study of a membrane acetylcholinesterase (EC 3.1.1.7) we found, contrary to observations by other authors, spectra indicating that the active serine might be located in a pocket of the enzyme surface. In order to inquire into this possibility, ESR spectra were studied under the influence of different physico-chemical factors known to cause an unfolding of proteins. 2. The active serine of the postsynaptic membrane acetylcholinesterase of Torpedo marmorata electric organ was spin labeled using 1-oxyl-2, 2, 6, 6-tetramethyl-4-piperidinyletoxyphosphonofluoridate. 3. The effect of the chosen physico-chemical factors was an increase in the rotational freedom of spin labels; this result corroborates the suggestion that the active center of our acetylcholinesterase preparation is located in a pocket.

Different effects of two peripheral anionic site-binding ligands on acetylcholinesterase active-site gorge topography revealed by electron paramagnetic resonance.

Both propidium and monoclonal antibody (mAb) 25B1 bind to the peripheral anionic site region of fetal bovine serum acetylcholinesterase (FBS AChE). Using electron paramagnetic resonance (EPR) with spin-labelled organophosphate specifically bound to the AChE active-site serine, we studied the effects of both ligands on the topography of the AChE active-site gorge. After incubation of FBS AChE with Fab fragments of mAb 25B1, freedom of motion of our spin label became more restricted, suggesting closing of the gorge. Stabilization against heat denaturation was also observed. No alterations in the freedom of motion or protection against heat denaturation could be detected after propidium binding. Our results demonstrate that two ligands binding to the peripheral anionic site region of AChE have different effects, suggesting a complex structure for this region of the molecule that allows various types of interactions with different ligands. We also demonstrate that EPR is a suitable tool for studying microtopographical alterations at the active sites of cholinesterases.

An ESR study of the postsynaptic membrane acetylcholinesterase of torpedo marmorata electric organ

SummaryThe effect of the cholinergic activator, phenyltrimethylammonium, on the ESR spectra of spinlabeled membrane bound acetylcholinesterase was studied; a reduction of maximal hyperfine splitting of the anisotropic ESR spectrum by 2 G was observed. The influence of phenyltrimethylammonium was prevented by the two cholinergic blocking agents d-tubocurarine andα-cobratoxine. The present results indicate that the conformation change of the esteratic site of membrane acetylcholinesterase is triggered by the binding of phenyltrimethylammonium to the cholinoreceptor site.

Searching for new TNF-α analogs having potential application in cancer therapy

Two new TNF-α analogs were prepared and tested for their anti-tumor activity on fibrosarcoma SA-1 tumor modelin vivo. In analog LK-801 two histidines (His107HislO8) were introduced into the surface loop thus enabling efficient purification by metal-affinity chromatography. This analog showed less side effects and can serve as a lead compound to look for other useful mutations. Another analog LK-802 was designed by introduction of additional pair of mutations (Cys95Cys148) into LK-801 in order to prepare disulfide linked TNF trimers. Cytotoxicity on mouse cell line L929 was comparable to TNF-α, but effect on tumor growth was quite reduced. Pharmacokinetic study revealed that serum levels of LK-802 were quite low in comparison to native TNF-α. This at least partially explains why anti-tumor activity of LK-802 is reduced and also illustrates the problems in designing the analogs with desiredin vivo biological properties.

Soluble tumor necrosis factor receptor I (sTNFRI) as a prognostic factor in melanoma patients in Slovene population

Abstract Tumor necrosis factor α (TNF-α) and its receptors (TNFRI and TNFRII) which exist in soluble form as a product of cleavage of the extracellular domain of membrane integrated receptors, still rise debate about their importance. It was reported that TNF-α has numerous actions in diseases such as inflammation, autoimmunity, infectious diseases, septic shock and many types of cancer [1, 2]. Several authors have reported the significance of sTNFRI level in serum of cancer patients [3, 4]. This study was performed in collaboration with the Institute of Oncology of Slovenia.At least two different mouse monoclonal antibodies (MAbs) against human sTNFRI have been prepared to obtain a sensitive and reliable sandwich ELISA. It was compared with commercially available R&D and Endogen ELISAs for the determination of sTNFRI. Groups of patients with different stages of melanoma and epithelial ovarian carcinoma were tested and their clinical records were reexamined. Levels of sTNFRI were measured and compared with the normal serum levels of sTNFRI.

Immunologically active mimetics of muramyl dipeptide

N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide, MDP) is the minimal immunologically active component of the bacterial cell wall peptidoglycan [1]. In last 20 years many research groups have been concerned with the synthesis and immunological studies of derivatives of this highly active glycopeptide in order to obtain molecules with improved and more defined pharmacological profiles. Although structure-activity relationships in MDP and its analogs have been well established, rational design of immunomodulators on the basis of MDP as a lead compound is impeded by the fact that the three-dimensional structure of the receptor binding site has not yet been determined. Therefore, systematic chemical modification of the molecule and development of more constrained analogs with reduced degrees of conformational freedom provide a good opportunity to define the bioactive conformation of MDP and analogs.

Allosteric effects of phenyltrimethylammonium and propidium on acetylcholinesterase active site

Different spin labelled fluorophosphates and fluorophosphonates with different chain length were investigated with respect to their sensitivity to the allosteric changes of acetylcholinesterase active site produced by phenyltrimethylammonium (Pta) or d-tubocurarine (TC); only fluorophosphates were found to be sensitive to these changes. Therefore fluorophosphates were chosen also for the study of allosteric effects of propidium. The addition of Pta and propidium to spin labelled membrane acetylcholinesterase of the Torpedo marmorata electric organ decreased maximal hyperfine splitting of the EPR spectrum, indicating that the microgeography of the acetylcholinesterase active site is usually changed in a way which increases the freedom of motion of the spin labels piperidine ring. TC alone did not change the EPR spectrum, but it prevented the influence of Pta and not that of propidium.