Igor Križaj

Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.

The amino acid sequence of a novel inhibitor of cathepsin D from potato.

The amino acid sequence of a cathepsin D inhibitor isolated from potato is described. It was determined by analysis of peptides generated by use of the glycine‐specific proteinase PPIV. The order of the peptides was established by examination of tryptic peptides derived from the two cyanogen bromide peptides. The inhibitor comprises 187 amino acid residues, and has a calculated M r of 20 450.

Equinatoxins, pore-forming proteins from the sea anemone Actinia equina, belong to a multigene family.

The multigene family of equinatoxins, pore-forming proteins from sea anemone Actinia equina, has been studied at the protein and gene levels. We report the cDNA sequence of a new, sphingomyelin inhibited equinatoxin, EqtIV. The N-terminal sequences of natural Eqt I and III were also determined, confirming two isoforms of EqtI, differing at position 13. The number of Eqt genes determined by Southern blot hybridization was found to be more than five, indicating that Eqts belong to a multigene family.

Primary structure of ammodytoxin C further reveals the toxic site of ammodytoxin

The sequence of ammodytoxin C, a presynaptically toxic, basic phospholipase A2 of Vipera ammodytes ammodytes venom was determined. The toxin differs only in two amino acid residues from the most toxic isotoxin ammodytoxin A and is 18-times less lethal. Ammodytoxin B which is 30-times less lethal than ammodytoxin A differs from it only in three amino acid residues. From the three-dimensional model of ammodytoxin A, it can be seen that mutated regions of ammodytoxin B and ammodytoxin C are on the surface, and relatively distant from each other. The observed decrease in toxicity of ammodytoxin C could be a consequence of changed charge in position 128 where a Lys is exchanged for Glu. The resulting change in electrostatic properties of the molecule which influences the orientation of the molecule during the approach to the charged nerve-terminal membrane might be responsible for the observed decrease in toxicity.

Cathepsin D inactivates cysteine proteinase inhibitors cystatins

The formation of inactive complexes in excess molar amounts of human cathepsins H and L with their protein inhibitors human stefin A, human stefin B and chicken cystatin at pH 5.6 has been shown by measurement of enzyme activity coupled with reverse-phase HPLC not to involve covalent cleavage of the inhibitors. Inhibition must be the direct result of binding. On the contrary the interaction of cystatins with aspartic proteinase cathepsin D at pH 3.5 for 60 min followed by HPLC resulted in their inactivation accompanied by peptide bond cleavage at several sites, preferentially those involving hydrophobic amino acid residues. The released peptides do not inhibit papain and cathepsin L. These results explain reported elevated levels of cysteine proteinases and lead to the proposal that cathepsin D exerts an important function, through inactivation of cystatins, in the increased activities of cysteine proteinases in human diseases including muscular distrophy.

The neurotoxic phospholipase A2 associates, through a non-phosphorylated binding motif, with 14-3-3 protein γ and ε isoforms

Two novel acceptors for ammodytoxin C, a presynaptically neurotoxic phospholipase A2 from snake venom, have been purified from porcine cerebral cortex by a toxin-affinity-based procedure. Usingtandem mass spectrometry, the isolated acceptors were identified as 14-3-3c and e isoforms, highly conserved cytoplasmic proteins involved in the regulation of numerous physiological processes. The interaction between ammodytoxin C and 14-3-3 proteins is direct and not mediated by calmodulin, a high-affinity acceptor for both ammodytoxin C and 14-3-3 proteins, as demonstrated in pull-down experiments and by surface plasmon resonance. The latter technique gave an apparent dissociation constant of 1:0 � 0:2lM for the interaction between chip-immobilized 143-3 and ammodytoxin C. 14-3-3 usually interacts with proteins through specific phospho-Ser/Thr motifs. Ammodytoxin C is not a phospho-protein, therefore the interaction must occur through a non-phosphorylated binding site, most probably the KEESEK sequence at its C-terminal end. The interaction we describe suggests an explanation for the pathophysiological effects evoked by some secreted phospholipases A2, such as the inhibition of protein phosphorylation, of terminal ion currents, and of neurotransmission, as well as the initiation of neuronal cell death, all processes regulated by 14-3-3 proteins. 2003 Elsevier Science (USA). All rights reserved.

Purification and characterisation of two hemorrhagic metalloproteinases from the venom of the long-nosed viper, Vipera ammodytes ammodytes

Two hemorrhagic proteins, VaH1 and VaH2, have been purified from Vipera ammodytes ammodytes venom. They are monomeric glycoproteins of an apparent molecular mass of 70kDa and multiple isoelectric points around pH 5.5. Both molecules are proteolytically active against azocasein as substrate. VaH1, which was characterised in detail, showed maximum activity at pH 7.5. Ethylenediaminetetraacetic acid eliminated the proteolytic as well as the hemorrhagic activity of VaH1 while iodoacetamide, phenylmethylsulfonyl fluoride and pepstatin A, inhibitors of cysteine, serine and aspartic proteinases respectively, had no effect. VaH1 is therefore a metalloproteinase whose hemorrhagic activity is very likely the result of its proteolytic activity. VaH1 is a fibrinogenase, hydrolysing exclusively the Aalpha-chain of fibrinogen. In the B-chain of insulin it cleaved with a high preference the bond between Ala(14) and Leu(15). Based on its molecular mass, VaH1 (as well as VaH2) is a Class P-III metalloproteinase. Partial amino acid sequences of its CNBr fragments demonstrated a high level of identity with the reprolysin subfamily of zinc-metalloproteinases.

Neuronal receptors for phospholipases A2 and β-neurotoxicity

Abstract Some phospholipases A 2 interrupt neuromuscular communication by blocking the release of neurotransmitter into the synaptic cleft. Despite numerous studies, the molecular mechanism of their action is still largely obscure. In this review the best-characterized receptors for β-neurotoxins are presented. We propose a model which could be useful in investigating the apparent inconsistency between the observed heterogeneity in the neuronal binding of β-neurotoxins and the very similar pathomorphological and electrophysiological effects which they produce in the intoxicated tissue. We assume that β-neurotoxins enter the nerve ending to exert their toxic effect. The model involves different pathways for phospholipase A 2 neurotoxins to reach the site of action inside the neuron, their respective extra- and intracellular neuronal receptors being key features of the pathway. Once in the nerve cell, β-neurotoxins impair the function of the synaptic vesicles by phospholipid hydrolysis of the inner leaflet of the vesicle bilayer. The proportion of the products of the phospholipid hydrolysis, lysophospholipids and phospholipids in the membrane, has been demonstrated to be very important for the shaping of the membrane, affecting its fusogenic properties. Due to the same final step in the action of β-neurotoxins, phospholipid hydrolysis, the consequences of their poisoning are practically identical.

R25 is an intracellular membrane receptor for a snake venom secretory phospholipase A21

Ammodytoxin is a presynaptically neurotoxic (β‐neurotoxic) snake venom secretory phospholipase A2 (sPLA2). We detected a 25 kDa protein which binds the toxin with very high affinity (R25) in porcine cerebral cortex [Vučemilo et al., Biochem. Biophys. Res. Commun. 251 (1998) 209–212]. Here we show that R25 is an integral membrane protein with intracellular localisation. It is the first sPLA2 receptor known to date that localises to intracellular membranes. Centrifugation on sucrose gradients was used to fractionate porcine cerebral cortex. The subcellular composition of the fractions was determined by following the distribution of organelle‐specific markers. The distribution of R25 in the fractions matched the distribution of the mitochondrial marker succinate dehydrogenase, but not the markers for plasma membrane, lysosomes, endoplasmic reticulum, synaptic and secretory vesicles. R25 most likely resides in mitochondria, which are known to be targets for sPLA2 neurotoxins in the nerve ending and are potentially implicated in the process of β‐neurotoxicity.

Membrane cholesterol and sphingomyelin, and ostreolysin A are obligatory for pore-formation by a MACPF/CDC-like pore-forming protein, pleurotolysin B

The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin.