Solid phase synthesis of two cholera toxin B subunit antigens

The 30-50 and 50-75 sequences of the cholera toxin beta chain including the amino-acids that are thought to be involved in toxin-receptor binding have been synthesized using the solid phase method. They were then purified by gel permeation and ion exchange chromatography. Both these free peptides induced serum antibodies recognising the native toxin after oral or intraperitoneal administration. Only the antibodies raised against the 50-75 peptide, however, were able to neutralize toxin activity.     

Peptidomic identification and biological validation of neuroendocrine regulatory peptide-1 and -2

Recent advances in peptidomics have enabled the identification of previously uncharacterized peptides. However, sequence information alone does not allow us to identify candidates for bioactive peptides. To increase an opportunity to discover bioactive peptides, we have focused on C-terminal amidation, a post-translational modification shared by many bioactive peptides. We analyzed peptides secreted from human medullary thyroid carcinoma TT cells that produce amidated peptides, and we identified two novel amidated peptides, designated neuroendocrine regulatory peptide (NERP)-1 and NERP-2. NERPs are derived from distinct regions of the neurosecretory protein that was originally identified as a product of a nerve growth factor-responsive gene in PC12 cells. Mass spectrometric analysis of the immunoprecipitate using specific antibodies as well as reversed phase-high performance liquid chromatography coupled with radioimmunoassay analysis of brain extract demonstrated the endogenous presence of NERP-1 and NERP-2 in the rat. NERPs are abundant in the paraventricular and supraoptic nuclei of the rat hypothalamus and colocalized frequently with vasopressin but rarely with oxytocin. NERPs dose-dependently suppressed vasopressin release induced by intracerebroventricular injection of hypertonic NaCl or angiotensin II in vivo. NERPs also suppressed basal and angiotensin II-induced vasopressin secretion from hypothalamic explants in vitro. Bioactivity of NERPs required C-terminal amidation. Anti-NERP IgGs canceled plasma vasopressin reduction in response to water loading, indicating that NERPs could be potent endogenous suppressors of vasopressin release. These findings suggest that NERPs are novel modulators in body fluid homeostasis.     

The amino acid sequence of toxin V II 2, a cytotoxin homologue from banded Egyptian cobra (Naja haje annulifera) venom.

Toxin V II 2 comprises 60 amino acid residues and is cross-linked by four disulphide bridges. The complete amino acid sequence of this toxin was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides were purified by ion-exchange chromatography and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequenator or by manual manipulation, was employed to obtain the sequence of the intact toxin and the pure peptides. The chymotryptic digest provided the necessary overlapping peptides which allowed the alignment of tryptic peptides. The amino acid sequence of Naja haje annulifera toxin V II 2 shows a high degree of homology with cytotoxin V II 1 of the same venom.     

Influence of dietary omega-3 fatty acids on transmembrane signalling in rat submandibular salivary gland

We have previously shown the incorporation of dietary omega-3 and omega-6 fatty acids from menhaden oil and corn oil, respectively, into membrane phospholipids of submandibular salivary gland (SMSG) of rat [Alam S. Q. and Alam B. S. (1988) Arch. Oral Biol. 33, 295-299]. We now demonstrate the influence of such incorporation on the regulation of G proteins and adenylate cyclase activity. Cholera toxin ribosylated three protein peptides (Mr 42,000, 44,000 and 46,000) to different extents in the two groups. We found 4.9- and 2.6-fold higher and 0.4-fold lower ribosylation of Mr 42,000, 44,000 and 46,000 peptides, respectively, in SMSG membranes of rats fed a diet containing 10% menhaden oil (group II) compared to those fed 10% corn oil (group I). Functional distinctions between different forms of these peptides are not known. Cholera toxin also exhibited radiolabelling of three peptides in the SMSG membranes from normal or fasting rats. In these membranes inhibitory G proteins were not detected by pertussis toxin dependent ADP ribosylation or by a low concentration of guanylyl 5-imidodiphosphate (10(-8) M), which selectively activates inhibitory G proteins which inhibit forskolin stimulated activity of adenylate cyclase. In group II membranes both basal and fluoride stimulated activities of adenylate cyclase were found to be significantly higher than the corresponding values in group I (P less than 0.02). In cholera toxin dependent ribosylated membranes of group I, basal and fluoride stimulated activities of adenylate cyclase were significantly higher than those obtained in the absence of cholera toxin (P less than 0.02). Surprisingly, corresponding values were found to be lower in ribosylated membranes of group II. This could be due either to conformational changes in heavily ribosylated G proteins, which alters coupling with the catalytic subunit of adenylate cyclase, or due to dissociation of excessive inhibitory beta gamma complex from alpha beta gamma complex upon the activation of G proteins.     

Independent selection by I-Ak molecules of two epitopes found in tandem in an extended polypeptide antigen

The protein hen egg white lysozyme (HEL) contains two segments, in tandem, from which two families of peptides are selected by the class II molecule I-Ak, during processing. These encompass peptides primarily from residues 31-47 and 48-63. Mutant HEL proteins were created with changes in residues 52 and 55, resulting in a lack of binding and selection of the 48-63 peptides to I-Ak molecules. Such mutant HEL proteins donated the same amount of 31-47 peptide as did the unmodified protein. Other mutant HEL molecules containing proline residues at residue 46, 47, or 48 resulted in extensions of the selected 31-47 or 48-62 families to their overlapping regions (in the carboxyl or amino termini, respectively). However, the amount of each family of peptide selected was not changed. We conclude that the presence or absence of the major peptide from HEL does not influence the selection of other epitopes, and that these two families are selected independently of each other.     

The primary structures of two types of the Ulex europeus seed lectin

The complete amino acid sequences of the Ulex europeus anti-H(O) lectins I and II were determined by using a protein sequencer. After digestion with endoproteinases Lys-C and Asp-N of the lectins reduced with 2-mercaptoethanol and modified with iodoacetamide, the resulting peptides were purified by reversed-phase high-performance liquid chromatography and subjected to sequence analysis. The complete primary structures of these two Ulex lectins I and II were compared with those of nine lectins already determined, including that of Lotus tetragonolobus anti-H(O) lectin which we have determined. Extensive homologies were found among them.     

Omega-agatoxins: novel calcium channel antagonists of two subtypes from funnel web spider (Agelenopsis aperta) venom

A new series of polypeptide presynaptic antagonists ("omega-agatoxins") was purified from venom of the funnel web spider Agelenopsis aperta. Physiological data indicate that all of these peptides are antagonists of voltage-sensitive calcium channels. Although all three omega-agatoxins (Aga) described here (omega-Aga-IA, omega-Aga-IB, and omega-Aga-IIA) block insect neuromuscular transmission presynaptically, biochemical data permit their subclassification as Type I and Type II toxins. Type I toxins (omega-Aga-IA and -IB) are 7.5 kDa, have closely related amino acid sequences, and exhibit characteristic tryptophan-like UV absorbance spectra. Complete Edman sequencing of omega-Aga-IA reveals it to be a 66-amino acid polypeptide containing 9 cysteines and 5 tryptophan residues. omega-Aga-IIA, a Type II toxin, is 11 kDa, shows limited amino acid sequence similarity to the Type I toxins, and exhibits mixed tryptophan- and tyrosine-like absorbance. Nanomolar concentrations of omega-Aga-IIA inhibit the specific binding of 125I-labeled omega-conotoxin GVIA to chick synaptosomal membranes while omega-Aga-IA and -IB have no effect under identical conditions. The omega-agatoxins thus are defined as two subtypes of neuronal calcium channel toxins with different structural characteristics and calcium channel binding specificities.     

Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

Cholera toxin catalyzes transfer of radiolabel from [32P]NAD+ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of Mr = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (Mr = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and [32P]NAD+ caused radiolabeling of purified microtubule and intermediate filament proteins.     

Immunochemistry of sea anemone toxins: structure-antigenicity relationships and toxin-receptor interactions probed by antibodies specific for one antigenic region

Two antibody subpopulations directed against Anemonia sulcata toxin I or II have been purified by immunoaffinity chromatography. These antibodies are specific for a single antigenic region and were used in a structure-antigenicity relationship study using homologous toxins and chemically modified derivatives of A. sulcata toxin II. Asp-7 and/or Asp-9 and Gln-47 of toxin II were found to be implicated in the antigenic region recognized by the two antibody subpopulations. On the contrary, Arg-14, Lys-35, -36, and -46, and alpha-NH2 of the glycine residue of A. sulcata toxin II are not involved in the corresponding antigenic region. When assayed for interaction with the sodium channel, the antigenic region of toxin II, including Asp-9 and Gln-47, appeared fully accessible to its specific antibodies, suggesting that it is not involved in the binding of the toxin to its receptor.     

Molecular basis for Bacillus thuringiensis Cry1Ab toxin specificity: two structural determinants in the Manduca sexta Bt-R1 receptor interact with loops alpha-8 and 2 in domain II of Cy1Ab toxin

The identification of epitopes involved in Cry toxin-receptor interaction could provide insights into the molecular basis of insect specificity and for designing new toxins to overcome the potential problem of insect resistance. In previous works, we determined that the Manduca sexta Cry1A cadherin-like receptor (Bt-R(1)) interacts with Cry1A toxins through epitope (865)NITIHITDTNN(875) and by loop 2 of domain II in the toxin (Gomez, I., Miranda-Rios, J., Rudi?o-Pi?era, E., Oltean, D. I., Gill, S. S., Bravo, A., and Soberón, M. (2002) J. Biol. Chem. 277, 30137-30143.). In this work, we narrowed to 12 amino acids a previously identified Bt-R(1) 66 amino acids epitope (Dorsch, J. A., Candas, M., Griko, N. B., Maaty, W. S. A., Midbo, E. G., Vadlamudi, R. K., and Bulla, L. A., Jr. (2002) Insect Biochem. Mol. Biol. 32, 1025-1036) and identified loop alpha-8 of Cry1Ab domain II as its cognate binding epitope. Two amino acid Bt-R(1) toxin binding regions of 70 residues, one comprised of residues 831-900 containing the (865)NITIHITDTNN(875) epitope (TBR1) and the other comprised of residues 1291-1360 (TBR2) were cloned by RT-PCR and produced in Escherichia coli. Cry1A toxins bind with the two TBR regions in contrast with the nontoxic Cry3A toxin. The loop 2 synthetic peptide competed with the binding of Cry1Ab toxin to both TBR regions in contrast to the alpha-8 synthetic peptide that only competed with Cry1Ab binding to TBR2. Western blots and competition ELISA analysis showed that the Cry1Ab loop 2 RR368-9EE mutant did not show observable binding to TBR1 but still bound the TBR2 peptide. This result suggests that loop alpha-8 interacts with the TBR2 region. Competition ELISA analysis of Cry1Ab binding to the two TBR peptides revealed that the toxin binds the TBR1 region with 6-fold higher affinity than the TBR2 region. The amino acid sequence of TBR2 involved on Cry1Ab interaction was narrowed to 12 amino acids, (1331)IPLPASILTVTV(1342), by using synthetic peptides as competitors for Cry1Ab binding to Bt-R(1). Our results show that the specificity of Cry1A involves at least two structural determinants on both molecules.     

Isolation of two biologically active peptides, erythrotropin I and erythrotropin II from fetal calf intestine

A bioassay based on the measurement of thymidine incorporation into trichloroacetic acid-insoluble materials in erythroid cell suspensions from fetal calf liver was used as the assay for purification of two small peptides (erythrotropins I and II) from fetal calf intestine. The peptides were purified using reversed-phase and gel permeation high performance liquid chromatography (HPLC). The two peptides have very similar amino acid compositions and a molecular weight of about 3500 daltons. Erythrotropin II stimulated thymidine incorporation and potentiated the action of erythropoietin in cultures of erythroid cells from fetal rat liver.     

Activation of Clostridium botulinum type E toxin purified by two different procedures

Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods. The specific activity [2 X 10(4) mouse LD50 (mg protein)-1] of the toxin purified from cell extract under slightly acidic conditions was lower than that [3 X 10(5) LD50 (mg protein)-1] of the toxin purified from culture supernate under slightly alkaline conditions. Both toxin preparations were activated by trypsin treatment, but to different extents, the degree of activation of the toxin from cell extract being about 30-fold higher than that of the toxin from culture supernate. The two toxin preparations had the same electrophoretic mobility on SDS-polyacrylamide gels and antigenic specificity as revealed by agar gel double-immunodiffusion tests. The antigenic specificity of the two toxin preparations was unaltered by trypsin treatment. In SDS-polyacrylamide gel electrophoresis, a single band of Mr 144,000 was demonstrated before trypsin treatment and two bands of Mr 100,000 and 55,000 appeared after trypsin treatment. The two toxin preparations were labelled with 125I and chymotryptic peptide maps were obtained before and after trypsin treatment. The two toxin preparations without trypsin treatment demonstrated many differences in their peptide maps, but the preparations after trypsin activation had similar peptide maps. These results indicate that the toxin obtained from culture fluid was a partially activated form, and that its molecular conformation was different from that of the toxin from cell extract. Differences in specific activity and activation ratio by trypsin treatment may be due to differences in the conformation of the toxin molecules.     

Use of antibodies specific to defined regions of scorpion alpha-toxin to study its interaction with its receptor site on the sodium channel

Five antibody populations selected by immunoaffinity chromatography for their specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the 125I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to their antibodies when the toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only alpha-helix region (residues 23-32) and a beta-turn structure (residues 32-35) are accessible to their respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.     

Anthrax Lethal Factor as an Immune Target in Humans and Transgenic Mice and the Impact of HLA Polymorphism on CD4+ T Cell Immunity

Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4⁺ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified.     

Multiple forms of copper (II) co-ordination occur throughout the disordered N-terminal region of the prion protein at pH 7.4

Although the physiological function of the prion protein remains unknown, in vitro experiments suggest that the protein may bind copper (II) ions and play a role in copper transport or homoeostasis in vivo. The unstructured N-terminal region of the prion protein has been shown to bind up to six copper (II) ions, with each of these ions co-ordinated by a single histidine imidazole and nearby backbone amide nitrogen atoms. Individually, these sites have micromolar affinities, which is weaker than would be expected of a true cuproprotein. In the present study, we show that with subsaturating levels of copper, different forms of co-ordination will occur, which have higher affinity. We have investigated the copper-binding properties of two peptides representing the known copper-binding regions of the prion protein: residues 57-91, which contains four tandem repeats of the octapeptide GGGWGQPH, and residues 91-115. Using equilibrium dialysis and spectroscopic methods, we unambiguously demonstrate that the mode of copper co-ordination in both of these peptides depends on the number of copper ions bound and that, at low copper occupancy, copper ions are co-ordinated with sub-micromolar affinity by multiple histidine imidazole groups. At pH 7.4, three different modes of copper co-ordination are accessible within the octapeptide repeats and two within the peptide comprising residues 91-115. The highest affinity copper (II)-binding modes cause self-association of both peptides, suggesting a role for copper (II) in controlling prion protein self-association in vivo.     

Quantitative comparison of the efficiency of antibodies against S1 and S2 subunit of SARS coronavirus spike protein in virus neutralization and blocking of receptor binding: implications for the functional roles of S2 subunit

Neutralizing effects of antibodies targeting the C-terminal stalk (S2) subunit of the spike protein of severe acute respiratory syndrome coronavirus have previously been reported, although its mechanism remained elusive. In this study, high titered mouse antisera against the N-terminal globular (S1) and S2 subunits of the S protein were generated and total immunoglobulin G (IgG) was purified from these antisera. The efficiency of these purified IgGs in virus neutralization and blocking of receptor binding were compared quantitatively using virus neutralization assay and a previously developed cell-based receptor binding assay, respectively. We demonstrated that anti-S1 IgG neutralizes the virus and binds to the membrane associated S protein more efficiently than anti-S2 IgG does. Moreover, both anti-S1 and anti-S2 IgGs were able to abolish the binding between S protein and its cellular receptor(s), although anti-S1 IgG showed a significantly higher blocking efficiency. The unexpected blocking ability of anti-S2 IgG towards the receptor binding implied a possible role of the S2 subunit in virus docking process and argues against the current hypothesis of viral entry. On the other hand, the functional roles of the previously reported neutralizing epitopes within S2 subunit were investigated using an antigen specific antibody depletion assay. Depletion of antibodies against these regions significantly diminished, though not completely abolished, the neutralizing effects of anti-S2 IgG. It suggests the absence of a major neutralizing domain on S2 protein. The possible ways of anti-S2 IgGs to abolish the receptor binding and the factors restricting anti-S2 IgGs to neutralize the virus are discussed.     

Purification of pertussis toxin substrates from bovine heart membranes

Pertussis toxin substrates were purified from bovine heart membranes by means of a sequential column chromatographic techniques. The elution pattern from the final ion-exchange chromatography and relative mobilities of these peptides on SDS-polyacrylamide gel electrophoresis revealed that the bovine heart membranes contained two distinct pertussis toxin substrates: Gi2 and Go.     

Purification and characterization of two forms of rat interleukin-2

Rat IL-2 produced by spleen cells in culture with concanavalin A was purified using gel filtration, hydrophobic chromatography, and ion-exchange chromatography. At least two forms of rat IL-2 were found to be separable by ion-exchange chromatography. These two forms have been designated form I and form II. Form I of rat IL-2 was purified by a factor of 1297 and found to have a pI of 6.4. Form II was purified by a factor of 669 and found to have a pI between 5.4 and 6.1. Lectin chromatography was used to demonstrate that these two forms most likely differ in the extent of glycosylation. In the presence of tunicamycin the production of form II was significantly reduced. The two forms of rat IL-2 differ in their abilities to promote a mixed-lymphocyte reaction. Their differences in glycosylation may be the reason for these differences in activity.