Lucia Kuhn-Nentwig

Evolution of a regulatory enzyme: cytochrome-c oxidase (complex IV)

Publisher Summary This chapter provides an overview of evolution of cytochrome-c oxidase. A large variety of oxygen-linked enzymes evolved, but only cytochrome-c oxidase couples the reduction of oxygen to water with the production of adenosine triphosphate (ATP). Cytochrome-c oxidase occurs in all eukaryotic organisms and in some aerobic bacteria. Studies on the mechanism of electron transfer from cytochrome c to oxygen could not detect basic functional differences between the enzyme from prokaryotes, unicellular eukaryotes, and animal tissues. With the improvement of separation methods, it was possible, however, to demonstrate differences in the protein composition of cytochrome-c oxidase from lower and higher developed organisms. A variable number of subunits, ranging from 2 to 13, have been identified in the enzyme complex from prokaryotes and mammalian tissues, respectively. The chapter reviews the information available on the structure and function of multiple and variable amounts of subunits in cytochrome-c oxidase from different organisms. It explains the role of cytochrome-c oxidase in energy metabolism.

Antimicrobial and cytolytic peptides of venomous arthropods

As a response to invading microorganisms, the innate immune system of arthropods has evolved a complex arrangement of constitutive and inducible antimicrobial peptides that immediately destroy a large variety of pathogens. At the same time, venomous arthropods have developed an additional offensive system in their venom glands to subdue their prey items. In this complex venom system, several enzymes, low-molecular-mass compounds, neurotoxins, antimicrobial and cytolytic peptides interact together, resulting in extremely rapid immobilization and/or killing of prey or aggressors. This review provides an overview of antimicrobial peptides identified in the hemolymph of venomous arthropods, and especially of cytolytic peptides in their venom. For these peptides a dual role is proposed: acting as antimicrobials as well as increasing the potency of the venom by influencing excitable cells.

[4] Isozymes of cytochrome-c oxidase: Characterization and isolation from different tissues

Publisher Summary This chapter discusses the procedure for the isolation of cytochrome- c oxidase from mitochondria of different vertebrate tissues, involving extraction of matrix proteins and most other membrane proteins with nonionic detergents, chromatography on DEAE-cellulose in the presence of Triton X-100, and ammonium sulfate fractionation in the presence of sodium cholate. Cytochrome- c oxidase, the terminal enzyme of the respiratory chain of most aerobic organisms, is a membrane-bound protein complex composed of a variable number of subunits, depending on the evolutionary stage of the organism. Tissue-specific isozymes of mammalian cytochrome- c oxidase are detected, based on differences in (1) apparent molecular weight after SDS-gel electrophoresis, (2) N-terminal amino acid sequence, (3) immunological reactivity of nuclear encoded subunits, (4) kinetic properties, and (5) reactivity of carboxylic groups in the presence and absence of cytochrome- c . The mitochondrially encoded subunits of cytochrome- c oxidase from different tissues of the same organism are suggested to be identical. The methods for the identification of cytochrome-c oxidase isozymes always include separation of the 13 subunits by SDS-gel electrophoresis, representing the most difficult step, as no other method is available for the complete separation of all subunits in one step.

Cupiennin 1, a New Family of Highly Basic Antimicrobial Peptides in the Venom of the Spider Cupiennius salei(Ctenidae)

A new family of antimicrobial peptides was isolated from the venom of Cupiennius salei. The peptides were purified to homogeneity, and the sequence of cupiennin 1a was determined by Edman degradation: GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH2. The amino acid sequences of cupiennin 1b, c, and d were obtained by a combination of sequence analysis and mass spectrometric measurements of comparative tryptic peptide mapping. All peptides consist of 35 amino acid residues and are characterized by a more hydrophobic N-terminal chain region and a C terminus composed preferentially of polar and charged residues. The total charge of all cupiennins calculated under physiological conditions is +8, and their C terminus, formed by a glutamic acid residue, is amidated. Conformational studies of the peptides revealed a high helix forming potential. Antimicrobial assays on bacteria with cupiennin 1a, 1d, and synthesized cupiennins 1a* and 1d* showed minimal inhibitory concentrations for bacteria in the submicromolar range. Their lytic effect on human red blood cells was lower by a factor of 8 to 14 than the highly hemolytic melittin. Cupiennin 1a, 1b, 1d, 1a*, and 1d* showed pronounced insecticidal activity. The immediate biological effects and the structural properties of the isolated cupiennins indicate a membrane-destroying mode of action on prokaryotic as well as eukaryotic cells.

Venom Composition and Strategies in Spiders: Is Everything Possible?

This review on all spider venom components known by the end of 2010 bases on 1618 records for venom compounds from 174 spider species (= 0.41% of all known species) belonging to 32 families (= 29% of all existing spider families). Spiders investigated for venom research are either big (many mygalomorph species, Nephilidae, Ctenidae and Sparassidae) or medically important for humans (e.g. Loxosceles or Latrodectus species). Venom research widely ignored so far the two most species-rich families (Salticidae and Linyphiidae) and strongly neglected several other very abundant families (Araneidae, Lycosidae, Theridiidae, Thomisidae and Gnaphosidae). We grouped the known 1618 records for venom compounds into six categories: low molecular mass compounds (16 % of all compounds), acylpolyamines (11 %), linear peptides (6 %), cysteine-knotted mini-proteins (60 %), neurotoxic proteins (1 %) and enzymes (6 %). Low molecular mass compounds are known from many spider families and contain organic acids, nucleosides, nucleotides, amino acids, amines, polyamines, and some further substances, many of them acting as neurotransmitters. Acylpolyamines contain amino acids (Araneidae and Nephilidae) or not (several other families) and show a very high diversity within one species. Linear peptides, also called cytolytic, membranolytic or antimicrobial, exert a highly specific structure and are so far only known from Ctenidae, Lycosidae, Oxyopidae and Zodariidae. Cysteine-knotted mini-proteins represent the majority of venom compounds because research so far focused on them. They probably occur in most but not all spider families. Neurotoxic proteins so far are only known from theridiid spiders. Enzymes had been neglected for some time but meanwhile it becomes obvious that they play an important role in spider venoms. Sixteen enzymes either cleave polymers in the extracellular matrix or target phospholipids and related compounds in membranes. The overall structure of these compounds is given and the function, as far as it is known, is described. Since several of these component groups are presented in one average spider venom, we discuss the known interactions and synergisms and give reasons for such a functional redundancy. We also discuss main evolutionary pathways for spider venom compounds such as high variability among components of one group, synergistic interactions between cysteine-knotted mini-proteins and other components (low molecular mass compounds and linear peptides), change of function from ion-channel acting mini-proteins to cytolytic effects and replacement of mini-proteins by linear peptides, acylpolyamines, large proteins or enzymes. We also add first phylogenetic considerations.

The venom optimisation hypothesis: a spider injects large venom quantities only into difficult prey types.

The spider Cupiennius salei needs 0.01-10 microl venom to kill a prey item. Since its venom glands contain only 10 microl and regeneration requires 8-16 days C. salei should use its venom very economically. By a monoclonal antibody we measured, for the first time, the amounts of venom injected by a spider into different prey types. Crickets and stick insects, as victims without special defence mechanism, received only the minimum amount of venom which is not significantly different from the LD(50). Blowflies and ground beetles received considerably more venom because they are difficult to overwhelm or even endanger the spider by their defence behaviour. These results support our venom optimisation hypothesis which supposes that spiders use their venom as economically as possible.

Purification of toxic peptides and the amino acid sequence of CSTX-1 from the multicomponent venom of Cupiennius salei (Araneae:Ctenidae)

The venom of the wandering spider Cupiennius salei was analysed biochemically by gel filtration, cation exchange chromatography, RP-HPLC, IEF, SDS-PAGE and TLC-electrophoresis. The native venom contains high levels of Na+, K+, Ca2+, histamine and taurine. It shows considerable activity of hyaluronidase, but not proteolytic activity. Thirteen peptides (CSTX-1 to CSTX-13) with an apparent mol. wt between 2.6 and 12.5 kDa causing differently strong toxic, effects were purified. Toxicity data of the crude venom (insects and mouse) are given and compared with the toxicity of CSTX-1, which causes most of the crude venoms toxicity. CSTX-1 has a mol. wt of 8352.6 and its amino acid sequence of 74 amino acids is given.

Characterisation of antibacterial activity of peptides isolated from the venom of the spider Cupiennius salei (Araneae: Ctenidae)

The characterisation of the antimicrobial activity of five antibacterial peptides, isolated from the venom of the neotropical wandering spider Cupiennius salei is reported here. The peptides have a molecular mass, determined by electrospray ionisation-mass spectrometry, between 3-4 kDa. Minimal inhibitory concentrations against five different bacteria species were determined by a liquid growth inhibition assay. All five peptides showed minimal inhibitory concentrations that are comparable to those of other known antibacterial peptides, like insect defensins and cecropins, found in the last years in a large diversity of animals. The peptides are supposed to lyse the cells by formation of either distinct channels or pores, but their mode of action is not yet revealed.

Quantity and quality of venom released by a spider (Cupiennius salei, Ctenidae)

The amount of venom injected by the spider Cupiennius salei depended on the efficiency of the mechanical defence of the prey species. Spiders were milked for the first venom (i.e. the first microlitre of venom emitted) versus remaining venom, and for venom regenerated from emptied glands. HPLC gel filtration and IEF electrophoresis showed that the protein content of the first venom was only half as compared to that of the remaining venom, and that this was due to a dilution of all proteins. Venom regeneration came in two speeds. The amount of venom was regenerated more rapidly than the protein concentration. Newly regenerated venom as compared to older venom was characterized by a lower concentration of all proteins and by a higher total concentration of free amino acids, whereas histamine and taurine did not follow this trend. K+ concentration and pH remained similar during venom regeneration. Crickets injected with the venoms showed less acute symptoms when the protein concentration was lower, namely with the first venom and with newly regenerated venom. Consequently, a spider which modulates the quantity of venom injected into a prey also directly changes the venom quality. The ecological consequences of this are discussed. This paper also discusses which region of a gland (ampulla, extracellular and intracellular parts of the glandular sac) is involved in the changes of the venom quality.

Solution structure and interaction of cupiennin 1a, a spider venom peptide, with phospholipid bilayers

The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix-hinge-helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.