Igor Križaj - Academia.edu (original) (raw)

Papers by Igor Križaj

European journal of biochemistry, Dec 1, 2002

Nucleic Acids Research, Aug 11, 1990

Thrombosis Research, Sep 1, 2023

Acta Chimica Slovenica, Sep 10, 2017

Biochimie, Oct 1, 2021

Secreted phospholipases A2 (sPLA2s) form a widespread group of structurally-related enzymes that ... more Secreted phospholipases A2 (sPLA2s) form a widespread group of structurally-related enzymes that catalyse the hydrolysis of the sn-2 ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. In humans, nine catalytically active and two inactive sPLA2 proteins have been identified. These enzymes play diverse biological roles, including host defence against bacteria, parasites and viruses. Several of these endogenous sPLA2s may play a defensive role in viral infections, as they display in vitro antiviral activity by both direct and indirect mechanisms. However, endogenous sPLA2s may also exert an offensive and negative role, dampening the antiviral response or promoting inflammation in animal models of viral infection. Similarly, several exogenous sPLA2s, most of them from snake venoms and other animal venoms, possess in vitro antiviral activities. Thus, both endogenous and exogenous sPLA2s may be exploited for the development of new antiviral substances or as therapeutic targets for antagonistic drugs that may promote a more robust antiviral response. In this review, the antiviral versus proviral role of both endogenous and exogenous sPLA2s against various viruses including coronaviruses is presented. Based on the highlighted developments in this area of research, possible directions of future investigation are envisaged. One of them is also a possibility of exploiting sPLA2s as biological markers of the severity of the Covid-19 pandemic caused by SARS-CoV-2 infection.

Journal of Colloid and Interface Science, Dec 1, 2017

European journal of biochemistry, Apr 15, 2001

Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine o... more Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine or arginine. This has led to the conclusion that a basic residue at this position, which interacts with the active-site aspartates, is essential for folding and activation of the zymogen. Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at 36p. To investigate the effect of this natural mutation which appears to contradict the proposed role of this residue, calf and lamb prochymosins and their two reciprocal mutants, K36pE and E36pK, respectively, were expressed in Escherichia coli, refolded in vitro, and autoactivated at pH 2 and 4.7. All four zymogens could be activated to active chymosin and, at both pH values, the two proteins with Glu36p showed higher activation rates than the two Lys36p forms. Glu36p was also demonstrated in natural prochymosin isolated from the fourth stomach of lamb, as well as being encoded in the genomes of sheep, goat and mouflon, which belong to the subfamily Caprinae. A conserved basic residue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation. The apparently contradictory results for porcine pepsinogen A [Richter, C., Tanaka, T., Koseki, T. &amp;amp;amp;amp; Yada, R.Y. (1999) Eur. J. Biochem. 261, 746-752] can be reconciled with those for prochymosin. Lys/Arg36p is involved in stabilizing the propeptide-enzyme interaction, along with residues nearer the N-terminus of the propeptide, the sequence of which varies between species. The relative contribution of residue 36p to stability differs between pepsinogen and prochymosin, being larger in the former.

Journal of Proteome Research, Sep 12, 2012

For some decades, cone snail venoms have been providing peptides, generally termed conopeptides, ... more For some decades, cone snail venoms have been providing peptides, generally termed conopeptides, that exhibit a large diversity of pharmacological properties. However, little attention has been devoted to the high molecular mass (HMM) proteins in venoms of mollusks. In order to shed more light on cone snail venom HMM components, the proteins of dissected and injected venom of a fish-hunting cone snail, Conus consors, were extensively assessed. HMM venom proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by mass spectrometry (MS). The MS data were interpreted using UniProt database, EST libraries from C. consors venom duct and salivary gland, and their genomic information. Numerous protein families were discovered in the lumen of the venom duct and assigned a biological function, thus pointing to their potential role in venom production and maturation. Interestingly, the study also revealed original proteins defining new families of unknown function. Only two groups of HMM proteins passing the venom selection process, echotoxins and hyaluronidases, were clearly present in the injected venom. They are suggested to contribute to the envenomation process. This newly devised integrated HMM proteomic analysis is a big step toward identification of the protein arsenal used in a cone snail venom apparatus for venom production, maturation, and function.

European journal of biochemistry, Mar 1, 1992

The amino acid sequence of a non-toxic phospholipase A2, ammodytin I2, from the venom of the long... more The amino acid sequence of a non-toxic phospholipase A2, ammodytin I2, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) and its cDNA sequence have been determined. The protein sequence was elucidated by sequencing the peptides generated by CNBr cleavage, mild acid hydrolysis and tryptic digestion of maleylated and non-maleylated protein. Sequencing of the cDNA showed that the protein is synthesized as an 137-amino-acid-residue precursor molecule consisting of a 16-residue signal peptide, followed by a 121-residue mature enzyme. Ammodytin I2 cDNA shows 73% nucleotide and 59% amino acid identities in the mature protein region in comparison to that of ammodytoxin A, the most presynaptically neurotoxic phospholipase A2 from the long-nosed viper. Identities in the signal-peptide region are considerably higher, 96% and 100%, respectively.

Journal of immunology research, 2014

Biochimica Et Biophysica Acta - Proteins And Proteomics, Nov 1, 2004

Journal of Blood Disorders and Transfusion, 2017

FEBS Journal, Oct 26, 2007

Ammodytoxins are neurotoxic secretory phospholipase A(2) molecules, some of the most toxic compon... more Ammodytoxins are neurotoxic secretory phospholipase A(2) molecules, some of the most toxic components of the long-nosed viper (Vipera ammodytes ammodytes) venom. Envenomation by this and by closely related vipers is quite frequent in southern parts of Europe and serotherapy is used in the most severe cases. Because of occasional complications, alternative medical treatment of envenomation is needed. In the present study, ammodytoxin inhibitor was purified from the serum of V. a. ammodytes using two affinity procedures and a gel exclusion chromatography step. The ammodytoxin inhibitor from V. a. ammodytes serum consists of 23- and 25-kDa glycoproteins that form an oligomer, probably a tetramer, of about 100 kDa. N-terminal sequencing and immunological analysis revealed that both types of subunit are very similar to gamma-type secretory phospholipase A(2) inhibitors. The ammodytoxin inhibitor from V. a. ammodytes serum is a potent inhibitor of phospholipase activity and hence probably also the neurotoxicity of ammodytoxins. Discovery of the novel natural inhibitor of these potent secretory phospholipase A(2) toxins opens up prospects for the development of new types of small peptide inhibitors for use in regulating the physiological and pathological activities of secretory phospholipases A(2).

Long-nosed viper (Vipera a. ammodytes) venom has been used for the immunobiological production of... more Long-nosed viper (Vipera a. ammodytes) venom has been used for the immunobiological production of antivenoms against the European poisonous snakes’ bites. Toxicity of the venom is one of its intrinsic characteristics and it seems also to determine the quality of produced antivenoms. The method for assessment the toxicity of the venom requires the use of large number of experimental animals and although painful for the animals, is legally approved, since there has been no in vitro alternative yet. Here we present the novel method that might be useful for predicting the long nosed viper venom toxicity and consequently its potential to induce a high-quality antiserum in immunized animals. The method is based on rapid HPLC separation of the venom using Convective Interaction Media (CIM) and the fact that the content of ammodytoxins (Atx), basic neurotoxic phospholipases A2 (sPLA2), in the venom has been correlated to the ability of the venom to produce highly protective antiserum in rabbits. Selective retention of the venom basic proteins on carboxymethyl (CM) CIM was achieved in 50 mM Tris/HCl buffer, pH 9.0. Bound proteins were separated into five fractions (EF1-5) using discontinuous gradient of NaCl in the range from 0 to 0.5 M. Pure AtxA and AtxC were used to establish their precise elution positions. Chromatographic fractions of the venom were assayed for protein composition by SDS-PAGE, lethality in mice and the presence of Atx by ELISA using anti-AtxA antibodies. AtxA was found concentrated in fraction EF2, in agreement with the retention time of the pure AtxA. Two batches of the long-nosed viper venom different in their biological properties related to AtxA content, i.e. lethality and anti-AtxA-antibody-inducing potential in rabbits, were comparatively analyzed under described experimental conditions. Significantly different height and shape of respective peak EF2 qualify the method for rapid prediction of the potential of a particular venom sample to produce a high-quality antiserum in immunized animals.

FEBS Letters, Nov 8, 1993