Vladimir N. Kokryakov

Circular minidefensins and posttranslational generation of molecular diversity.

We purified two new minidefensins (RTD‐2 and RTD‐3) from the bone marrow of rhesus monkeys. Both were circular octadecapeptides that contained three intramolecular disulfide bonds and were homologous to RTD‐1, a circular (θ) defensin previously described by Tang et al. (Science, 286, 498–502, 1999). However, whereas the 18 residues of RTD‐1 represent spliced nonapeptide fragments derived from two different demidefensin precursors, RTD‐2 and ‐3 comprise tandem nonapeptide repeats derived from only one of the RTD‐1 precursors. Thus, circular minidefensins are products of a novel posttranslational system that generates effector molecule diversity without commensurate genome expansion. A system wherein two demidefensin genes can produce three circular minidefensins might allow n such genes to produce (n/2)(n+1) peptides.

Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina

Two novel 21‐residue antimicrobial peptides, arenicin‐1 and arenicin‐2, exhibiting activity against Gram‐positive and Gram‐negative bacteria and fungi, were purified from coelomocytes of marine polychaeta Arenicola marina (lugworm) by preparative gel electrophoresis and RP‐HPLC. Molecular masses (2758.3 and 2772.3 Da) and complete amino acid sequences (RWCVYAYVRVRGVLVRYRRCW and RWCVYAYVRIRGVLVRYRRCW) 1 were determined for each isoform. Each arenicin has one disulfide bond (Cys3‐Cys20). The total RNA was isolated from the lugworm coelomocytes, RT‐PCR and cloning were performed, and cDNA was sequenced. A 202‐residue preproarenicin contains a putative signal peptide (25 amino acids) and a long prodomain. Arenicins have no structure similarity to any previously identified antimicrobial peptides.

Prophenin-1, an exceptionally proline-rich antimicrobial peptide from porcine leukocytes

We purified and characterized an unusual antimicrobial peptide, prophenin‐1 (PF‐1), from porcine leukocytes. The peptide had a mass of 8,683 and contained 79 residues, including 42 (53.2%) prolines and 15 (19.0%) phenylalanines. Its N‐terminal 60 residues consisted of three perfect and three nearly perfect repeats of a decamer, FPPPNFPGPR. Prophenin‐1 was encoded on a cathelin‐containing precursor and showed substantially more activity against E. coli, a Gram‐negative bacterium, than against Listeria monocytogenes, a Gram‐positive organism, in vitro.

Isolation, purification and de novo sequencing of TBD-1, the first beta-defensin from leukocytes of reptiles

A novel peptide with antimicrobial activity was isolated from leukocytes of the European pond turtle Emys orbicularis and purified to homogeneity by preparative gel electrophoresis followed by reversed phase chromatography. It was highly active in vitro against Escherichia coli, Listeria monocytogenes, methicillin‐resistant Staphylococcus aureus, and Candida albicans. The isolated peptide was sequenced de novo by tandem mass spectrometry using both collision‐induced and electron‐transfer dissociation in combination with different chemical derivatization techniques. The 40‐residue peptide, called TBD‐1 (turtle β‐defensin 1), represents the first defensin isolated from reptilian leukocytes. It contains three disulfide bonds and shows high structural similarities to β‐defensins isolated from birds and mammals.

De novo sequencing of two new cyclic θ‐defensins from baboon (Papio hamadryas) leukocytes by matrix‐assisted laser desorption/ionization mass spectrometry

Two cyclic theta-defensin peptides were isolated from leukocytes of the hamadryas baboon, Papio hamadryas, and purified to homogeneity by gel electrophoresis and reversed-phase high-performance liquid chromatography. Both peptides had high in vitro activity against Escherichia coli, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. Here, we report their de novo sequencing by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). This was accomplished by combining conventional enzymatic digestion with N-terminal derivatization by 2-sulfobenzoic acid cyclic anhydride (SACA) or 4-sulfophenylisothiocyanate (SPITC) to facilitate the interpretation of fragment ion spectra. In addition to the two cyclic theta-defensins (PhTDs) we also sequenced a novel Papio hamadryas alpha-defensin, PhD-4, which showed high sequence homology to rhesus alpha-defensin RMAD-1 and human alpha-defensin HNP-1.

ChBac3.4: A Novel Proline-Rich Antimicrobial Peptide from Goat Leukocytes

We isolated a new proline-rich peptide, ChBac3.4, from leukocytes of the goat (Capra hirca) and determined its amino acid sequence by Edman degradation and mass spectrometry. ChBac3.4 (RFRLPFRRPPIRIHP PPFYPPFRRFL–NH2) had over 50% sequence identity to the Bac5 peptides found in the leukocytes of goats, sheep and cattle. ChBac3.4 exhibited broadspectrum antimicrobial activity, especially under low salt conditions. Since E. coli ML35p treated with ChBac3.4 manifested increased outer and inner membrane permeability and a rapid and extensive loss of cytoplasmic potassium, the antimicrobial properties of this peptide may depend, in part, on its ability to damage microbial membranes. Nevertheless, even high concentrations of ChBac3.4 were not significantly hemolytic for human erythrocytes. In vitro, ChBac3.4 was selectively cytotoxic, damaging human K562 erythroleukemia cells and human U937 hystiocytic lymphoma cells, but not other human target cells. ChBac3.4 appears to differ from other proline-rich cathelicidins in virtue of its increased ability to damage microbial membranes. This novel antimicrobial peptide warrants further study, especially with respect to its various effects on microbial and mammalian cells.

Expression pattern of arenicins—the antimicrobial peptides of polychaete Arenicola marina

Immune responses of invertebrate animals are mediated through innate mechanisms, among which production of antimicrobial peptides play an important role. Although evolutionary Polychaetes represent an interesting group closely related to a putative common ancestor of other coelomates, their immune mechanisms still remain scarcely investigated. Previously our group has identified arenicins—new antimicrobial peptides of the lugworm Arenicola marina, since then these peptides were thoroughly characterized in terms of their structure and inhibitory potential. In the present study we addressed the question of the physiological functions of arenicins in the lugworm body. Using molecular and immunocytochemical methods we demonstrated that arencins are expressed in the wide range of the lugworm tissues—coelomocytes, body wall, extravasal tissue and the gut. The expression of arenicins is constitutive and does not depend on stimulation of various infectious stimuli. Most intensively arenicins are produced by mature coelomocytes where they function as killing agents inside the phagolysosome. In the gut and the body wall epithelia arenicins are released from producing cells via secretion as they are found both inside the epithelial cells and in the contents of the cuticle. Collectively our study showed that arenicins are found in different body compartments responsible for providing a first line of defense against infections, which implies their important role as key components of both epithelial and systemic branches of host defense.

Lactoferrin from Canine Neutrophils: Isolation and Physicochemical and Antimicrobial Properties

Lactoferrin has been isolated from canine leukocytes for the first time. Lactoferrin was identified by N-terminal amino acid sequence and by capability to capture ferric cations resulting in a complex with absorbance maximum at 460–470 nm. It is demonstrated that canine lactoferrin resembles the human homolog in some physicochemical properties, i.e. molecular weight, carbohydrate presence, and conditions of protein—iron complex dissociation. Bactericidal activity of dog lactoferrin was demonstrated on the gram-negative bacterium Escherichia coli and gram-positive bacterium Listeria monocytogenes. Bactericidal activity of canine lactoferrin is similar to that of human lactoferrin.

Effects of antimicrobial peptides of neutrophils on tumor and normal host cells in culture

We carried out a study of the effects of two structurally different cationic antimicrobial peptides of cathelicidin family, porcine protegrin 1 (PG1) and caprine Bac5 on selected tumor and normal mammalian cells in vitro. Protegrins are amphiphilic β-hairpin molecules having broad-spectrum antimicrobial activity due to their marked membranolytic effects. Bac5 belongs to a group of proline-rich peptides, which adopt a polyproline type II extended helix and kill microorganisms rather by a nonlytic mechanism. We have shown that while PG1 exerted distinct and fast cytotoxic effects towards most of used tumor cells being in a lesser degree toxic for nontransformed host cells; the proline-rich peptide Bac5 possessed modest cytotoxic activity for all tested cells. The toxic effects of PG1 were partially declined in the presence of 10% fetal calf serum. It was revealed that PG1 was able to interact with proteins of serpin family (as was previously established for human defensins by Panyutich at al., 1995). Pre-incubation of PG1 with α1-antitrypsin caused the decrease of the cytotoxic activity of the peptide and, on the other hand, the antiprotease activity of α1-antitrypsin was reduced after the interaction of the serpin with PG1 (while Bac5 did not affect the antiprotease activity of α1-antitrypsin). We used BODIPY FL-tagged PG1 and Bac5 to study the internalization of the labeled peptides into target cells and their intracellular distribution by confocal microscopy. Bac5-BODIPY (at 5 μ M) was rapidly taken into the cells. PG1-BODIPY at non-toxic concentrations (1—3 μM) was also able to enter the cells without their damaging. By using flow cytometry we showed that lowering a temperature to 4°C caused a significant decrease in the uptake into K562 and U937 cells for both Bac5-BODIPY and PG1-BODIPY. A decline of target cells metabolism also diminished the process of both peptides internalization but for a lesser degree. In the presence of endocytosis inhibitors the penetration of Bac5-BODIPY and PG1-BODIPY into K562 cells was also reduced, but not completely abolished, suggesting that along with endocytosis process some direct penetration of the peptides across cell membranes takes place. The ability of the peptides to internalize into eukaryotic cells may contribute to the idea of participation of AMPs in varied intracellular events, occurring in normal or malignant host cells, for instance, in the modulation of intracellular serpins activity.

Effects of defensin and lactoferrin on functional activity of endothelial cells in vitro.

We studied the effects of antibacterial peptides and proteins (defensins and lactoferrins) on functional activity of endothelial cells in vitro: proliferative activity and adhesion of human endothelial ECV-304 cells to the matrix were evaluated. α-Defensin (NP-2) from rabbit neutrophils, total α-defensin (HNP 1-3) from human neutrophils, and lactoferrins from porcine neutrophils and human milk were studied. Defensins stimulated and lactoferrin in doses of 1–10 µg/ml inhibited proliferation and adhesion of endothelial cell. The stimulatory effect of defensins on proliferation and adhesion was reproduced in fibroblast culture. Lactoferrins did not modify proliferation of fibroblasts, but suppressed their adhesion. These data suggest that antibiotic proteins and peptides are prospective objects for the creation of drugs regulating angiogenesis.