Allurin, a 21-kDa sperm chemoattractant from Xenopus egg jelly, is related to mammalian sperm-binding proteins.

Previously, we demonstrated that a protein from Xenopus egg jelly exhibits sperm chemoattractant activity when assayed by either video microscopy or by sperm passage across a porous filter. Here we describe the isolation and purification of allurin, the protein responsible for this activity. Freshly oviposited jellied eggs were soaked in buffer, and the conditioned medium was loaded onto an anion exchange column and eluted with an NaCl gradient. The active fraction was purified further by RP-HPLC, the chemoattractant protein appearing as a single sharp peak. The amino acid sequence of the protein, determined by direct sequencing and cloning of cDNAs coding for the protein, consisted of 184 amino acids having a molecular mass of 21,073 Da. The protein shares homology with the mammalian cysteine-rich secretory protein (CRISP) family that includes testes-specific spermatocyte protein 1, a cell adhesion protein which links spermatocytes to Seritoli cells, and acidic epididymal glycoproteins that bind to sperm and have been implicated in sperm-egg fusion. Phylogenetic analysis suggests that allurin evolved from the ancestral protein that gave rise to the mammalian CRISP family. Addition of allurin to this family portends that the CRISP family represents a group of "sperm escort" proteins, which bind to sperm at various steps in their life history, facilitating passage from one functional stage to the next. Allurin stands out in this regard, representing both the first vertebrate sperm chemoattractant to be purified and sequenced and the first member of the CRISP family to be found in the female reproductive tract.

Characterization of the two myotoxin a isomers from the prairie rattlesnake (Crotalus viridis viridis) by capillary zone electrophoresis and fluorescence quenching studies.

The two myotoxin a isomers from the venom of the prairie rattlesnake Crotalus viridis viridis have different isoelectric points, as determined by capillary zone electrophoresis. The pI values are 10.50 and 10.57, respectively, and both are higher than the previously reported pI value for myotoxin a. The difference in the isoelectric points between the two isomers is attributed to altered surface charge as a result of the conformational change in myotoxin a. Both isomers exist in crude venom, discounting the possibility that they are artifacts formed during the purification process. Fluorescence quenching of myotoxin a reveals heterogeneity of the tryptophans, possibly due to different environments. The fraction of the total tryptophan fluorescence quenched by iodide is 81% and is attributed to solvent-accessible tryptophan residues at the protein surface. The 19% non-quenchable tryptophans probably represent residues that are shielded from the solvent exposure. The ratio of buried to exposed tryptophans is similar to the ratio of isomers seen by capillary zone electrophoresis and reverse-phase high-performance liquid chromatography (c. 1 : 4).

The role of Pro20 in the isomerization of myotoxin a from Crotalus viridis viridis: folding and structural characterization of synthetic myotoxin a and its Pro20Gly homolog.

Biochemical characterization has established the presence of two conformational forms of myotoxin a. To test the hypothesis that this may be due to cis-trans isomerization at Pro20, synthetic versions of myotoxin a and its Pro20-->Gly structural homolog were folded, then purified using a two-step cation-exchange/reverse-phase perfusion chromatography method. The disulfide bond configuration for the folded proteins was found to be the same as that of native myotoxin a. CE and RPHPLC revealed that folded synthetic myotoxin a exists in two conformations while the Pro20-->Gly homolog exists in only one, supporting the hypothesis that cis-trans isomerization at Pro20 is the source of the myotoxin a conformational heterogeneity.

Evidence for isomerization in myotoxin a from the prairie rattlesnake (Crotalus viridis viridis).

Myotoxin a, from the venom of the prairie rattlesnake, Crotalus viridis viridis, exists as a temperature-dependent equilibrium of two interconverting forms. Reverse-phase high-performance liquid chromatography (RP-HPLC) shows that the two forms interconvert slowly enough at 25 degrees C to be seen as two separate peaks with a molar ratio of c. 1:4. Each peak can be isolated and individually injected to give the same two peaks in the same ratio of areas. The two peaks merge during chromatography at elevated temperatures, indicating an increase in the rate of interconversion. At low temperature, c. 5 degrees C, the individual peaks can be isolated and maintained for several days without reaching equilibrium. Mass analysis by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry shows that myotoxin a is present in both RP-HPLC peaks, suggesting that the two resolved forms are conformational isomers. Capillary zone electrophoresis (CZE) also shows two resolved, but interconvertible peaks over a range of pH values. Furthermore, RP-HPLC chromatograms of myotoxin a at concentrations from 0.013 mM to 0.41 mM maintain a consistent ratio of peak areas, without evidence of dimerization. Two-dimensional 1H-NMR nuclear Overhauser enhancement spectroscopy indicates the presence of a cis-proline peptide bond, consistent with an equilibrium mixture of cis-trans isomers; however, addition of peptidyl-prolyl cis-trans isomerase (PPI) does not enhance the rate of equilibration of the RP-HPLC peaks isolated at c. 5 degrees C.

Mass spectrometric immunoassay.

A new, general method of immunoassay is demonstrated. The approach is based on the microscale immunoaffinity capture of target antigens followed by mass-specific identification and quantitation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Immunoaffinity capture of antigens effectively overcomes signal suppression effects typically encountered during traditional matrix-assisted laser desorption/ionization analysis of complex biological mixtures while simultaneously concentrating the analyte into a small volume. Mass spectrometric detection of antigens is unambiguous, as antigen signals are observed at characteristic mass-to-charge values in the mass spectrum, offering a high level of immunity to artifacts due to nonbiospecific retention of mixture components. However, the most important aspect of such mass-specific detection is the ability to use a single assay to screen biological systems for the presence of multiple, mass-resolved antigens. Analyte quantitation is possible by using a single antibody to capture both the antigen and an antigen variant which has been chemically modified to have a different mass. With proper calibration, the relative signal intensities of the two species in the mass spectrum can be used to determine the antigen concentration. Sample incubation and processing methods were such that a typical analysis could be performed in less than 1 h while subnanomolar sensitivities were maintained. The technique has been used for the rapid, selective, and quantitative screening of human blood for the presence of myotoxin a, and Mojave toxin form the venoms of the prairie rattlesnakes, Crotalus viridis viridis, and and the Mojave rattlesnake, Crotalus scutulatus scutulatus.

1H-NMR study of neurotoxin B-IV from the marine worm Cerebratulus lacteus. Solution properties, sequence-specific resonance assignments, secondary structure and global fold.

Sequence-specific resonance assignments are reported for the 500-MHz 1H-NMR spectrum of the 55-residue neurotoxin B-IV, isolated from the heteronemertine worm Cerebratulus lacteus. A range of two-dimensional homonuclear correlated and NOE spectra was used in making these assignments, which include NH, C alpha H and C beta H resonances, as well as most resonances from longer-chain spin systems, with the exception of the ten Lys residues, where spectral overlap prevented complete, unambiguous assignments. The secondary structure of B-IV was identified from the pattern of sequential (i, i + 1) and medium range (i, i + 2/3/4) NOE connectivities and the location of slowly exchanging backbone amide protons. Two helices are present, incorporating residues 13-26 and 33-49, and the C-terminal five residues form a helix-like structure. A type-I reverse turn, involving residues 28-31 is present in a small loop linking the two major helices, and the N-terminus appears to be unordered at 27 degrees C, although it may adopt a more ordered conformation at lower temperatures. These elements of secondary structure, together with the four disulfide bonds in the protein, provide sufficient information to define the global fold of the molecule in solution. The pH and temperature dependence of the toxin have been investigated by 1H-NMR and the pKa values of several ionisable sidechains determined.

Mojave toxin affects fusion of myoblasts and viability of myotubes in cell cultures.

Mojave toxin is a neurotoxic, heterodimeric phospholipase isolated from the venom of Crotalus scutulatus scutulatus. Responses of primary rat muscle cell cultures and clonal muscle cell lines to treatment with Mojave toxin and its constituent subunits were examined. Continuous exposure of cells to 0.5 microM or 1.0 microM Mojave toxin or the basic subunit, added 24 hr after plating, prevented fusion of primary myoblasts and C2 myoblasts to multinucleate myotubes. Under the same experimental conditions, some myotube formation was observed when RMo cells were used, but the number and size of the myotubes were reduced substantially compared to untreated controls. The addition of Mojave toxin to established myotubes that arose from differentiation of primary myoblasts or C2 myoblasts essentially led to total disappearance of the myotubes from the cell layer within 48 hr. Myotubes from RMo cells treated in the same manner, however, did not disappear, but they were smaller and less numerous than comparable controls. Similar results were generated by exposure of myotubes to the basic subunit of Mojave toxin under the same conditions. The underlying layer of mononucleate cells was retained in both instances. Toxin-free cultures continued to develop in the usual manner. Treatment with 1.0 microM concentrations of the acidic subunit, pancreatic phospholipase A2 or a non-neurotoxic phospholipase from Naja naja atra gave results indistinguishable from untreated control cultures.

Effects of myotoxin alpha on fusion and contractile activity in myoblast-myotube cell cultures.

Cultured myoblasts and moytubes were used to study the effects of purified myotoxins from rattlesnake venoms. Standard cell culture techniques were used to establish and maintain primary cultures derived from neonatal rat tissue and two clonal cell lines, rat RMo cells and mouse C2 cells. Toxin concentrations, ranging from 0.04 to 1.0 microM, were added to the cultures at various times under distinct, well-defined conditions. Addition of myotoxin alpha to primary myoblast cultures did not appear to affect the fusion process, whereas similar experiments with two clonal cell lines produced larger myotubes when contrasted with untreated control cultures, particularly with RMo cells. The myotubes derived from primary cell cultures twitched spontaneously but the twitching ceased when the medium was replaced with a serum-free chemically defined incubation medium. Addition of myotoxin alpha to the primary myotubes incubated with serum-free defined medium caused the myotubes to twitch again. Derivatives of myotoxin alpha were prepared by reactions with tetranitromethane and with iodoacetic acid, the latter under reducing and non-reducing conditions. The resulting products, purified but not chemically characterized, were nearly devoid of activity when primary cultures were used to test activity.

The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom.

A new myotoxic phospholipase A2 homologue, having a serine residue in position 49 instead of highly conserved aspartic acid, was found in the venom of Vipera ammodytes. The primary structure revealed additional mutations in the positions important for enzymatic activity. Tyr28 is exchanged for a histidine and Gly33 for asparagine. These changes render earlier-reported weak enzymatic activity unlikely. The role of this rather abundant venom fraction is apparently in myotoxicity, which was confirmed in the muscle-cell culture from neonatal rats. The muscle-cell culture proved to be a good tool to investigate the effects of various myotoxins on muscle cells.

Interaction of the Bacillus sphaericus mosquito larvicidal proteins.

Genes for 51.4- and 41.9-kDa insecticidal proteins of Bacillus sphaericus were separately cloned and expressed in Escherichia coli. Both proteins were required for toxicity. Approximately equal numbers of cells containing the 51.4- and 41.9-kDa proteins produced the greatest toxicity; excess 41.9-kDa protein did not affect toxicity, whereas excess 51.4-kDa protein reduced activity. Larvae were killed when 41.9-kDa protein was fed up to 24 h after the 51.4-kDa protein, but not when the order of feeding was reversed. Radiolabelled toxins bound in approximately equal amounts to the gastric caecum and posterior midgut of Culex quinquefasciatus larvae. Radiolabelled 51.4-kDa protein was rapidly degraded by ca. 12-13 kDa in the larval gut, while 41.9-kDa protein was degraded by 1-2 kDa. Nonreduced toxin extracted from B. sphaericus produced a band on SDS-PAGE of ca. 68-74 kDa that contained both 51.4- and 41.9-kDa proteins based on sequence analysis, and a band of ca. 51 kDa that contained primarily 41.9-kDa protein. Escherichia coli containing 51.4-kDa protein enhanced toxicity of the latter eluted SDS-PAGE band. These proteins may associate very strongly, and trace amounts of 51.4-kDa protein in preparations of 41.9-kDa protein from B. sphaericus may be responsible for the previously reported toxicity of the latter.

The complete sequence of the acidic subunit from Mojave toxin determined by Edman degradation and mass spectrometry.

Mojave toxin, a heterodimeric, neurotoxic phospholipase complex from Crotalus scutulatus scutulatus, is one of a group of closely related rattlesnake toxins for which much structural information is still lacking. The complete amino-acid sequence of the acidic subunit from Mojave toxin was determined. The three individual peptide chains, derived from the acidic subunit by reductive alkylation, were separated by high-performance liquid chromatography. Fragmentations of the A and B chains were done using specific proteinases and the resulting peptide mixtures were fractionated by reverse-phase high-performance liquid chromatography. Sequence analyses on the intact chains and the fragments from digests were done by automated Edman degradation, carboxypeptidase Y degradation and triple-quadrupole and tandem-quadrupole Fourier-transform mass spectrometry. The sequence for each acidic subunit chain is very similar to the corresponding chain from the related neurotoxin complex, crotoxin, and overall the sequence is similar to the sequences of group I and II phospholipases A2. The N-terminus of the B chain is blocked by pyroglutamic acid. The existence of two distinct and closely related C chains was established. It is unlikely that the small sequence difference can account for the isoforms that are present in purified Mojave toxin and in unfractionated venom.

Mojave toxin: rapid purification, heterogeneity and resistance to denaturation by urea.

This report establishes that purified Mojave toxin prepared from the snake venom of Crotalus scutulatus scutulatus contains multiple heterogeneous dimers (isoforms) differing slightly in isoelectric points. This conclusion is based upon chromatographic, immunological, sodium dodecyl sulfate--polyacrylamide gel electrophoretic and polyacrylamide isoelectric focusing experiments. The Mojave toxin-related proteins were rapidly purified from venom via a single chromatography step. Generation of Mojave toxin-related proteins from isolated subunits and immunoblots of these proteins subsequent to electrophoretic separation demonstrate that each of the proteins consists of acidic and phospholipase basic subunits. The analysis of venom in narrow range polyacrylamide isoelectric focusing gels at varying concentrations of urea, in conjunction with immunoblots utilizing antibodies specific to the basic subunit, demonstrates that the isoforms of Mojave toxin are native and not artifacts from isolation procedures. Analyses of venoms from Crotalus scutulatus scutulatus individuals indicate that each snake produces multiple isoforms of the neurotoxin. Additionally, the same predominant isoform of Mojave toxin is present in both individual and commercial venoms. The heterogeneity of the Mojave toxin-related proteins is largely due to differences in the acidic subunits and some of the forms may reflect post-translational processing of the protein. The Mojave toxin-related proteins demonstrate a resistance to urea denaturation by characteristically entering and focusing in polyacrylamide isoelectric focusing gels containing 0-6 M urea, but dissociating to constituent subunits in 8 M urea. Experimental evidence suggest that salt bridges may be important in stabilization of the Mojave toxin complex.

Binding of the Bacillus sphaericus mosquito larvicidal toxin to cultured insect cells.

Using both fluorescent labelled toxin and antibody--secondary antibody techniques, the Bacillus sphaericus toxin was found to bind strongly to susceptible Culex quinquefasciatus cells, but far less strongly to cells of insensitive insects. An insensitive clone of the C. quinquefasciatus cell line was discovered which bound toxin efficiently. The toxin was bound in the cold to sensitive cells and these cells could be rescued from cytotoxicity for ca. 15 min after warming, by which time toxin appeared to be internalized. Binding was saturable. This toxin is apparently internalized by receptor-mediated endocytosis, probably involving a glycoprotein receptor containing N-acetyl-D-glucosamine. Evidence for toxin binding to lipids was not found. Antibody appeared to detect internalized toxin, and high concentrations of sugars inhibited cytotoxicity; these results along with evidence from a recent ultrastructural study suggest that this toxin may form pores in the cell membrane.

Assignment of the aromatic 1H-NMR resonances of myotoxin a isolated from the venom of Crotalus viridis viridis.

The 400 MHz 1H-NMR spectrum of myotoxin a from the venom of Crotalus viridis viridis is described. The identification of spin systems in the aromatic region corresponding to the six aromatic residues of myotoxin a was completed using both one- and two-dimensional NMR spectroscopy and the pH dependence of chemical shifts. Assignments of these spin systems to specific residues was possible for the singly occurring amino acids Tyr-1 and Phe-12. Resonances from Tyr-1, His-5 and His-10 were shifted significantly from their random coil values in a pH-dependent manner. These shift perturbations were deemed evidence of a helical arrangement of the amino terminal region which placed these residues in close proximity to each other.

Reductive methylation as a tool for the identification of the amino groups in alpha-bungarotoxin interacting with nicotinic acetylcholine receptor.

alpha-Bungarotoxin (alpha Bgt) is a postsynaptic neurotoxin which blocks cholinergic transmission at the neuromuscular junction by binding tightly to the acetylcholine receptor (AcChR). The number of methylation sites in alpha Bgt has been shown to decrease significantly upon binding of the toxin to the AcChR [Soler, G., Farach, M. C., Farach, H. A., Mattingly, J. R., & Martinez-Carrion, M. (1983) Arch. Biochem. Biophys. 225, 872-878]. We have compared the chemical reactivities of amino groups in free and AcChR-bound alpha Bgt in an attempt to identify the regions in the alpha Bgt molecule that become masked upon binding to the AcChR. Free alpha Bgt and AcChR-bound alpha Bgt were reductively methylated with formaldehyde and sodium cyanoborohydride, and the rate of modification of each one of the available amino groups was followed by cleaving the methylated toxin with V8 protease and resolving the resulting peptides by reversed-phase, high-performance liquid chromatography. Under conditions of limited reagent availability, five of seven amino groups in free alpha Bgt reacted readily, whereas two other amino groups, probably those corresponding to Lys-51 and Lys-70, displayed lower reactivity. Upon binding to the AcChR, the rates of reductive methylation of residues Ile-1, Lys-26, and Lys-38 were considerably reduced (although to differing extents). The degree of protection was most pronounced for Lys-26. The rates of methylation of the amino groups in all other positions remained unchanged. These results allow further definition of the minimal binding surface of a representative neurotoxin.

Amino acid sequences of myotoxins from Crotalus viridis concolor venom.

Myotoxins I and II were isolated from the venom of Crotalus viridis concolor. Complete sequences were derived for each reduced, alkylated toxin with data obtained by a single run on a gas phase sequencer and from fragments derived by cyanogen bromide cleavage. The results demonstrate that microheterogeneity is present in myotoxin II. The newly established sequences were compared with 3447 protein sequences in the Protein Information Resource database. The only homologous proteins found were other known myotoxins from rattlesnake venoms, namely myotoxin a, crotamine and peptide C.

The effects of myotoxin from midget faded rattlesnake (Crotalus viridis concolor) venom on neonatal rat myotubes in cell culture.

Neonatal rat myoblasts were isolated and grown in culture until they fused into multinucleated myotubes. A small percentage of the myotubes showed spontaneous contractions when maintained in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum. Incubation of mature myotubes (at least 3 days after fusion) with myotoxin II from Crotalus viridis concolor venom at a concentration as low as 18.5 nM caused a marked increase in the number of myotubes demonstrating contractile activity. The increase was apparent within 24 hr of myotoxin application. The response of the myotubes appeared to be specific since, of the proteins tested, only native myotoxins caused the increase in contractile activity. This tissue culture system offers a rapid screening assay that requires less time and fewer animals than the assays currently in use for determining myotoxic activity.

Antigenic relationships between Mojave toxin subunits, Mojave toxin and some crotalid venoms.

Immunochemical responses of a number of pit viper venoms to antibodies derived separately from the acidic and basic subunits were investigated by enzyme linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion. The polyclonal antisera to the basic subunit were generated in rabbits, whereas mouse hybridoma cell cultures were used to produce antibodies to the acidic subunit. The immunochemical response of a venom correlated well with published values for LD50 dose for the test venom. Many venoms that elicited a positive response with antiserum to the basic subunit also reacted strongly with the hybridoma derived antibodies to the acidic subunit. The data support the conclusion that crotalid venoms which are more lethal have in common a potent venom component that is immunochemically related to Mojave toxin.