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 mass.

Pseudovacuoles--immobilized by high-pressure freezing--are associated with blebbing in walker carcinosarcoma cells

By applying high pressure freezing and freeze-substitution, we observed large inclusions of homogeneous appearance in the front of locomoting Walker carcinosarcoma cells that have not been described earlier. Live cell imaging revealed that these inclusions were poor in lipids and nucleic acids but had a high lysine (and hence protein) content. Usually one such structure 2-5 mum in size was present at the front of motile Walker cells, predominantly in the immediate vicinity of newly forming blebs. By correlating the lysine-rich areas in fixed and embedded cells with electron microscopic pictures, inclusions could be assigned to confined, faintly stained cytoplasmic areas that lacked a surrounding membrane; they were therefore called pseudovacuoles. After high-pressure freezing and freeze substitution, pseudovacuoles appeared to be filled with 20 nm large electron-transparent patches surrounded by 12 and 15 nm large particles. The heat shock protein Hsp90 was identified by peptide sequencing as a major fluorescent band on SDS-PAGE of lysine-labelled Walker cell extracts. By immunofluorescence, Hsp90 was found to be enriched in pseudovacuoles. Colocalization of the lysine with a potassium-specific dye in living cells revealed that pseudovacuoles act as K+ stores in the vicinity of forming blebs. We propose that pseudovacuoles might support blebbing by locally regulating the intracellular hydrostatic pressure.

The Cytotoxic Mode of Action of the Venom of Cupiennius salei (Ctenidae)

The venom of the ctenid spider Cupiennius salei (Fig.16.1) is rich in components which belong to different functional groups. Besides low molecular mass compounds, the venom contains several disulphide-rich peptides, also called mini-proteins, which act as neurotoxins on ion channels or as enhancers of neurotoxins. Likewise, a variety of small cytolytic peptides, which destroy membranes very efficiently, and enzymes are present in the venom. Neurotoxins with cytolytic activity, cytolytic a-helical small cationic peptides and enzymes most probably attacking connective tissue and phospholipid membranes cause the overall cytotoxic effect of this venom. Synergistic and enhancing interactions between components enable the spider to achieve a maximum of toxicity with a minimum of venom quantity.

Synthetic peptides corresponding to a repetitive sequence of malarial histidine rich protein bind haem and inhibit haemozoin formation in vitro

Synthetic peptides containing a repetitive hexapeptide sequence (Ala-His-His-Ala-Ala-Asp) of malarial histidine-rich protein II were evaluated for binding with haem in vitro. The pattern of haem binding suggested that each repeat unit of this sequence provides one binding site for haem. Chloroquine inhibited the haem-peptide complex formation with preferential formation of a haem chloroquine complex. In vitro studies on haem polymerisation showed that none of the peptides could initiate haemozoin formation. However, they could inhibit haemozoin formation promoted by a malarial parasite extract, possibly by competitively binding free haem. These results indicate this hexapeptide sequence represents the haem binding site of the malarial histidine-rich protein and possibly the site of nucleation for haem polymerisation.

On the terminal homologation of physiologically active peptides as a means of increasing stability in human serum--neurotensin, opiorphin, B27-KK10 epitope, NPY

The terminal homologation by CH(2) insertion into the peptides mentioned in the title is described. This involves replacement of the N-terminal amino acid residue by a β(2) - and of the C-terminal amino acid residue by a β(3) -homo-amino acid moiety (β(2) hXaa and β(3) hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N-terminal to the C-terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1) and its C-terminal fragment NT(8-13) are ligands of the G-protein-coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b-2e, for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5-1.3 vs. 0.6 nM). At the same time, one of the homologated NT analogs, 2c, survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8-13) (Tables 2 and 4, and Fig. 8) reveals that this N-terminal NT fragment folds to a turn in CD(3) OH. - In the case of the human analgesic opiorphin (3a), a pentapeptide, and of the HIV-derived B27-KK10 (4a), a decapeptide, terminal homologation (→3b and 4b, resp.) led to a 7- and 70-fold half-life increase in plasma (Fig. 9). With N-terminally homologated NPY, 5c, we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c, and 5c, were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability-increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed.

Evaluation of chemically modified SLA implants (modSLA) biofunctionalized with integrin (RGD)- and heparin (KRSR)-binding peptides

Enhancing osseointegration through surface immobilization of multiple short peptide sequences that mimic extracellular matrix (ECM) proteins, such as arginine-glycine-aspartic acid (RGD) and lysine-arginine-serine-arginine (KRSR), has not yet been extensively explored. Additionally, the effect of biofunctionalizing chemically modified sandblasted and acid-etched surfaces (modSLA) is unknown. The present study evaluated modSLA implant surfaces modified with RGD and KRSR for potentially enhanced effects on bone apposition and interfacial shear strength during early stages of bone regeneration. Two sets of experimental implants were placed in the maxillae of eight miniature pigs, known for their rapid wound healing kinetics: bone chamber implants creating two circular bone defects for histomorphometric analysis on one side and standard thread configuration implants for removal torque testing on the other side. Three different biofunctionalized modSLA surfaces using poly-L-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) as a carrier minimizing nonspecific protein adsorption [(i) 20 pmol cm⁻² KRSR alone (KRSR); or in combination with RGD in two different concentrations; (ii) 0.05 pmol cm⁻² RGD (KRSR/RGD-1); (iii) 1.26 pmol cm⁻² RGD (KRSR/RGD-2)] were compared with (iv) control modSLA. Animals were sacrificed at 2 weeks. Removal torque values (701.48-780.28 N mm), bone-to-implant contact (BIC) (35.22%-41.49%), and new bone fill (28.58%-30.62%) demonstrated no significant differences among treatments. It may be concluded that biofunctionalizing modSLA surfaces with KRSR and RGD derivatives of PLL-g-PEG polymer does not increase BIC, bone fill, or interfacial shear strength.

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.