Ctenidins: antimicrobial glycine-rich peptides from the hemocytes of the spider Cupiennius salei

Three novel glycine-rich peptides, named ctenidin 1-3, with activity against the Gram-negative bacterium E. coli, were isolated and characterized from hemocytes of the spider Cupiennius salei. Ctenidins have a high glycine content (>70%), similarly to other glycine-rich peptides, the acanthoscurrins, from another spider, Acanthoscurria gomesiana. A combination of mass spectrometry, Edman degradation, and cDNA cloning revealed the presence of three isoforms of ctenidin, at least two of them originating from simple, intronless genes. The full-length sequences of the ctenidins consist of a 19 amino acid residues signal peptide followed by the mature peptides of 109, 119, or 120 amino acid residues. The mature peptides are post-translationally modified by the cleavage of one or two C-terminal cationic amino acid residue(s) and amidation of the newly created mature C-terminus. Tissue expression analysis revealed that ctenidins are constitutively expressed in hemocytes and to a small extent also in the subesophageal nerve mas

Defensin Levels in Spider Hemolymph

Spiders, like all arthropods, exclusively rely on an innate immune system localized in the hemocytes to protect against pathogen invasion. In the hemocytes of the wandering spider Cupiennius salei (C. salei), defensin expression was found to be constitutive. Defensins belong to the group of antimicrobial peptides, which appear in most taxonomic groups, and play an essential role in innate immunity. It has further been reported that during the primary immune answer of C. salei, the peptide content of hemocytes changes markedly, which may indicate the release of defensins from the hemocytes. However, no data on the peptide levels in C. salei hemolymph has so far been published. Formerly, the involvement in the primary immune answer was considered the only function of defensins. However, recent findings strongly suggest that the importance of defensins goes far beyond. There is evidence for defensins contributing to the adaptive immune response, to angiogenesis, and furthermore to tissue repair, i.e. to a variety of essential processes in living organisms. To date, only very little is known about the identity of C. salei defensins and their detailed mode of action. The goal of the work presented herein is the identification of hitherto unknown C. salei defensins in hemocytes and the hemolymph. Moreover, the levels of defensin expression under differential conditions are compared by the means of liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Neurotoxic Peptides in the Multicomponent Venom of the Spider Cupiennius Salei Part II. Elucidation of the Disulphide-Bridge Pattern of the Neurotoxic Peptide CSTX-9 by Tandem Mass Spectrometry

The disulphide-bridge pattern of the CSTX-9 polypeptide present in the multicomponent venom of the spider Cupiennius salei was determined de novo by nano-electrospray tandem mass spectrometry. Cleavage of native CSTX-9 with immobilized trypsin resulted in four disulphide-linked peptides with a mass of 2953.43 Da, which were subjected to low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight (QqTOF) mass spectrometer. The product ion spectra not only provided sequence information of the peptides, the occurrence of characteristic fragment ions generated by cleavage of the peptide bonds adjacent to the bridged cysteines also allowed the disulphide-bridge pattern to be identified unambiguously. CSTX-9 was found to be a member of the neurotoxic polypeptide family incorporating the inhibitor cystine knot (ICK) structural motif. The results demonstrate the potential of modern analytical instrumentation for elucidation of complex molecular structures

Annexin VI participates in the formation of a reversible, membrane- cytoskeleton complex in smooth muscle cells

The plasmalemma of smooth muscle cells is periodically banded. This arrangement ensures efficient transmission of contractile activity, via the firm, actin-anchoring regions, while the more elastic caveolae-containing 'hinge' regions facilitate rapid cellular adaptation to changes in cell length. Since cellular mechanics are undoubtedly regulated by components of the membrane and cytoskeleton, we have investigated the potential role played by annexins (a family of phospholipid- and actin-binding, Ca2+-regulated proteins) in regulating sarcolemmal organization. Stimulation of smooth muscle cells elicited a relocation of annexin VI from the cytoplasm to the plasmalemma. In smooth, but not in striated muscle extracts, annexins II and VI coprecipitated with actomyosin and the caveolar fraction of the sarcolemma at elevated Ca2+ concentrations. Recombination of actomyosin, annexins, and caveolar lipids in the presence of Ca2+ led to formation of a structured precipitate. Participation of all 3 components was required, indicating that a Ca2+-dependent, cytoskeleton-membrane complex had been generated. This association, which occurred at physiological Ca2+ concentrations, corroborates our biochemical fractionation and immunohistochemical findings and suggests that annexins play a role in regulating sarcolemmal organization during smooth muscle contraction