Urs Kämpfer

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.

Biochemical properties of histone-like proteins of procyclic Trypanosoma brucei brucei

Four histone-like proteins a, b, c, d were extracted with 0.2 M H2SO4 from soluble nuclear chromatin of Trypanosoma brucei brucei procyclic culture forms and purified by FPLC reversed phase chromatography. The amino acid composition of these proteins and their electrophoretic mobilities in three different gel systems strongly indicated their core histone nature. Similarities were found between a, b, c and d with the core histones H3, H2A, H2B and H4 of higher eukaryotes, respectively. On the other hand, these proteins also showed differences as compared to higher eukaryotes; proteins a and d clearly differed from their counterparts H3 and H4 on the basis of their hydrophobic properties. The results indicate the occurrence of core histone variants in T.b. brucei which may influence DNA-histone and histone-histone interactions as well as the chromatin compaction in the nucleus of this protozoan parasite.

A lysine rich C-terminal tail is directly involved in the toxicity of CSTX-1, a neurotoxic peptide from the venom of the spider Cupiennius salei

CSTX-1 (74 amino acids, 8,352.62 Da) is a potent neurotoxin from the venom of Cupiennius salei. With the monoclonal antibody 9H3 against CSTX-1, we identified two similar peptides by Western blot analysis. These two peptides were purified by RP-HPLC: CSTX-2a (61 amino acids, 6865.75 Da) and CSTX-2b (60 amino acids, 6709.57 Da). Using ESI-MS analysis and sequencing we verified that CSTX-2a is a truncated version of CSTX-1. CSTX-2b differs from CSTX-2a by the absence of Arg61. Toxicity of CSTX-1, CSTX-2a, and CSTX-2b to Drosophila melanogaster showed that the absence of the last 13 amino acids of CSTX-1 results in a seven-fold activity loss. CSTX-2b, which lacks Arg61 is 190-fold less toxic. We conclude that the C-terminal part of CSTX-1, especially Arg61, is essential for the expression of toxicity. CSTX-1 is degraded to CSTX-2a and CSTX-2b by proteases that are released from venom gland cells by apocrine secretion.

Biochemical and functional characterization of histone H1-like proteins in procyclic Trypanosoma brucei brucei

Four variants and/or posttranslational modifications of histone H1-like proteins ofTrypanosoma brucei brucei procyclic culture forms were extracted with 0.25N HCl from isolated nuclei and analyzed by two-dimensional gel electrophoresis. The amino acid composition of these proteins, their ability to space nucleosomes regularly and to induce salt-dependent condensation of the chromatin indicated their histone H1 nature. On the other hand, the histone H1-like proteins clearly differed from their higher-eukaryote counterparts by their weak interaction with DNA under low-salt conditions. As a consequence, intact nucleosome filaments were prepared according to a new preparation protocol especially adapted to the unstable chromatin ofT. b. brucei. Our results indicate that the biochemical properties of the histone H1-like proteins contribute to the structural and functional differences between the chromatin of procyclicT. b. brucei and that of higher eukaryotes.

Multicomponent venom of the spider Cupiennius salei: a bioanalytical investigation applying different strategies

The multicomponent venom of the spider Cupiennius salei was separated by three different chromatographic strategies to facilitate subsequent analysis of peptidic venom components by tandem mass spectrometry (MALDI‐TOF‐MS and ESI‐MS), Edman degradation and amino acid analysis: (a) desalting of the crude venom by RP‐HPLC only, (b) chromatographic separation of the crude venom into 42 fractions by RP‐HPLC, and (c) multidimensional purification of the crude venom by size exclusion and cation exchange chromatography and RP‐HPLC. A total of 286 components were identified in the venom of C. salei by mass spectrometry and the sequence of 49 new peptides was determined de novo by Edman degradation and tandem mass spectrometry; 30 were C‐terminally amidated. The novel peptides were assigned to two main groups: (a) short cationic peptides and (b) Cys‐containing peptides with the inhibitor cystine knot motif. Bioinformatics revealed a limited number of substantial similarities, namely with the peptides CpTx1 from the spider Cheiracantium punctorium and U3‐ctenitoxin‐Asp1a from the South American fishing spider (Ancylometes sp.) and with sequences from a Lycosa singoriensis venom gland transcriptome analysis. The results clearly indicate that the quality of the data is strongly dependent on the chosen separation strategy. The combination of orthogonal analytical methods efficiently excludes alkali ion and matrix adducts, provides indispensable information for an unambiguous identification of isomasses, and results in the most comprehensive repertoire of peptides identified in the venom of C. salei so far.

CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure.

Abstract. CSTX-9 (68 residues, 7530.9 Da) is one of the most abundant toxic polypeptides in the venom of the wandering spider Cupiennius salei. The amino acid sequence was determined by Edman degradation using reduced and alkylated CSTX-9 and peptides generated by cleavages with endoproteinase Asp-N and trypsin, respectively. Sequence comparison with CSTX-1, the most abundant and the most toxic polypeptide in the crude spider venom, revealed a high degree of similarity (53% identity). By means of limited proteolysis with immobilised trypsin and RP-HPLC, the cystine-containing peptides of CSTX-9 were isolated and the disulphide bridges were assigned by amino acid analysis, Edman degradation and nanospray tandem mass spectrometry. The four disulphide bonds present in CSTX-9 are arranged in the following pattern: 1-4, 2-5, 3-8 and 6-7 (Cys6-Cys21, Cys13-Cys30, Cys20-Cys48, Cys32-Cys46). Sequence comparison of CSTX-1 with CSTX-9 clearly indicates the same disulphide bridge pattern, which is also found in other spider polypeptide toxins, e.g. agatoxins (ω-AGA-IVA, ω-AGA-IVB, μ-AGA-I and μ-AGA-VI) from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxins (CT-I, CT-II and CT-III) from Hololena curta. CSTX-1/CSTX-9 belong to the family of ion channel toxins containing the inhibitor cystine knot structural motif. CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhibits a ninefold lower toxicity to Drosophila melanogaster than CSTX-1. This is in accordance with previous observations of CSTX-2a and CSTX-2b, two truncated forms of CSTX-1 which, like CSTX-9, also lack the C-terminal lysine-rich tail.

Partial amino acid sequence and functional aspects of histone H1 proteins in Trypanosoma brucei brucei

Summary— Trypanosoma brucei brucei, a protozoan parasite of wild and domestic animals in Africa, is related to the pathogenic agent of human sleeping sickness. Four H1 histone proteins were isolated from nuclei of procyclic culture forms and cleaved with proteases. Amino acid sequence analysis of purified fragments indicated the presence of variants which displayed sequence identities as compared to the C‐terminal domain of human H1. Substitutions of amino acids and posttranslational modifications of the histones in iT b brucei H1 may influence protein conformation and histone‐histone as well as histone‐DNA interactions in the chromatin of the parasite. Digestion of soluble chromatin with immobilized trypsin at low and high ionic strengths indicated an internal localization of H1 in the condensed chromatin. The influence of histone H1 of T b brucei on the compaction pattern of the chromatin was investigated by dissociation and reconstitution experiments. Electron microscopy revealed that trypanosome H1 was able to induce condensation of the chromatin of the parasite and of rat liver into dense tangles. After dephosphorylation of H1, 30 nm fibers were induced in rat liver chromatin, while the resulting fibers were distinctly thinner in T b brucei. It can be concluded that the absence of 30 nm fibers in T b brucei chromatin cannot be explained by the divergent variants and posttranslational phosphorylations of H1 only but rather by the influence of both, the divergent core histones, previously described, and H1 properties.

Amino acid sequence of the heavy chain of porcine plasmin. Comparison of the carbohydrate attachment sites with the human and bovine species

Abstract The amino acid sequence of the heavy chain of porcine plasmin (560 residues) was determined. Selected fragments were produced by cleavage with cyanogen bromide, elastase, Staphylococcus aureus V8 protease, trypsin or clostripain or with combinations thereof. The fragments were aligned with overlapping sequences. Sequence comparison with the human and bovine species indicated that the porcine molecule contains the same structural and functional domains as the two others. In particular, the characteristic arrangement of the disulfide bonds clearly indicates the five kringle structures. The heavy chain of porcine plasmin has a sequence homology of 84% with the bovine and of 78% with the human molecule. The average degree of identity among the kringles is over 80%. In the connecting strands between the kringles the identity decreases drastically (to 42% in the connecting strand between kringles 4 and 5) except for the strand between kringles 2 and 3 (over 80%). Porcine plasminogen contains two carbohydrate moieties. The N-glycosidic site at Asn289 is only partially glycosylated as in the human and bovine species and corresponds to Asn288 (human) and Asn289. (bovine), respectively. The O-glycosidic site is located at Thr249 in the connecting strand between kringles 2 and 3, whereas in the human and bovine molecule it occurs in the connecting strand between kringles 3 and 4, at Thr345 and Ser339, respectively.

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.

Structure of Cupiennius salei venom hyaluronidase Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40–60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. Function of venom hyaluronidases Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.

Elucidation of the disulfide bridge pattern of the recombinant human growth and differentiation factor 5 dimer and the interchain Cys/Ala mutant monomer

Growth and differentiation factor 5 (GDF5) is involved in many developmental processes such as chondrogenesis and joint and bone formation. A recombinant monomeric human GDF5 mutant rGDF5(C84A) is in vitro as potent as the dimeric native form, and clinical investigations of rGDF5(C84A) are in progress. Native homodimeric GDF5 belongs to the transforming growth factor beta (TGF-beta) superfamily; each monomer contains a cystine knot formed by three intrachain disulfide bridges, and the monomers are connected via an interchain disulfide bridge. The disulfide bridge pattern of recombinant homodimeric rGDF5 was recently elucidated by X-ray diffraction. A combination of proteolytic degradation with thermolysin, separation of the generated fragments by reverse-phase high-performance liquid chromatography (RP-HPLC), and subsequent analyses of the disulfide-linked peptides by electrospray-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, amino acid analysis, and Edman degradation led to the unambiguous identification of the disulfide bridge pattern of the monomeric mutant rGDF5(C84A) and of the homodimeric rGDF5 in solution. The cystine knot of homodimeric rGDF5 exhibits the pattern Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 (three intrachain disulfide bonds), and the monomers are connected by a single interchain disulfide bridge (Cys4-Cys4) in accordance with other members of the TGF-beta superfamily. The monomeric mutant rGDF5(C84A) exhibits the same cystine knot pattern as homodimeric rGDF5.