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Many proteins are still routinely expressed prokaryotically in Escherichia coli , some because they are toxic to eukaryotes. Immunotoxins, which are fusion proteins of a targeting moiety and a truncated Pseudomonas exotoxin A, kill target cells by arresting protein synthesis. Thus, immunotoxins must be expressed in E. coli . Proteins expressed in E. coli are contaminated by endotoxin (also called lipopolysaccharides (LPS)). LPS binds to toll-like receptors, inducing up to life-threatening systemic inflammation in mammals. Therefore, accepted LPS limits for therapeutics as well as for substances used in immunological studies in animals are very low. Here, we report the use of Triton X-114 and polyamine-based wash strategies, which only in combination achieved LPS-contamination well below FDA limits. Resulting LPS-reduced immunotoxins were purer and up to 2.4-fold more active in vitro. Increased activity was associated with a 2.4-fold increase in affinity on cell surface expressed target antigen. The combination method maintained enzymatic function, protein stability, and in vivo efficacy and was effective for Fab as well as dsFv formats. With some modifications, the principle of this novel combination may be applied to any chromatography-based purification process.

Multiple sclerosis (MS) is an autoimmune disorder manifested via chronic inflammation, demyelination, and neurodegeneration inside the central nervous system. The progressive phase of MS is characterized by neurodegeneration, but unlike classical neurodegenerative diseases, amyloid-like aggregation of self-proteins has not been documented. There is evidence that citrullination protects an immunodominant peptide of human myelin oligodendrocyte glycoprotein (MOG 34-56 ) against destructive processing in Epstein-Barr virus-infected B-lymphocytes (EBV-BLCs) in marmosets and causes exacerbation of ongoing MS-like encephalopathies in mice. Here we collected evidence that citrullination of MOG can also lead to amyloid-like behavior shifting the disease pathogenesis toward neurodegeneration. We observed that an immunodominant MOG peptide, MOG 35-55 , displays amyloid-like behavior upon site-specific citrullination at positions 41, 46, and/or 52. These amyloid aggregates are shown to be toxic to the EBV-BLCs and to dendritic cells at concentrations favored for antigen presentation, suggesting a role of amyloid-like aggregation in the pathogenesis of progressive MS.

New biomimetic magnetite nanoparticles (hereafter BMNPs) with sizes larger than most common superparamagnetic nanoparticles were produced in the presence of the recombinant MamC protein from Magnetococcus marinus MC-1 and functionalized with doxorubicin (DOXO) intended as potential drug nanocarriers. Unlike inorganic magnetite nanoparticles, in BMNPs the MamC protein controls their size and morphology, providing them with magnetic properties consistent with a large magnetic moment per particle; moreover, it provides the nanoparticles with novel surface properties. BMNPs display the isoelectric point at pH 4.4, being strongly negatively charged at physiological pH (pH 7.4). This allows both (i) their functionalization with DOXO, which is positively charged at pH 7.4, and (ii) the stability of the DOXO-surface bond and DOXO release to be pH dependent and governed by electrostatic interactions. DOXO adsorption follows a Langmuir-Freundlich model, and the coupling of DOXO to BMNPs (binary biomimetic nanoparticles) is very stable at physiological pH (maximum release of 5% of the drug adsorbed). Conversely, when pH decreases, these electrostatic interactions weaken, and at pH 5, DOXO is released up to ∼35% of the amount initially adsorbed. The DOXO-BMNPs display cytotoxicity on the GTL-16 human gastric carcinoma cell line in a dose-dependent manner, reaching about ∼70% of mortality at the maximum amount tested, while the nonloaded BMNPs are fully cytocompatible. The present data suggest that BMNPs could be useful as potential drug nanocarriers with a drug adsorption-release governed by changes in local pH values.

Fluorescent nanodiamonds are gaining increasing attention as fluorescent labels in biology in view of the fact that they are essentially nontoxic, do not bleach, and can be used as nanoscale sensors for various physical and chemical properties. To fully realize the nanosensing potential of nanodiamonds in biological applications, two problems need to be addressed: their limited colloidal stability, especially in the presence of salts, and their limited ability to be taken up by cells. We show that the physical adsorption of a suitably designed recombinant polypeptide can address both the colloidal stability problem and the problem of the limited uptake of nanodiamonds by cells in a very straightforward way, while preserving both their spectroscopic properties and their excellent biocompatibility.

Amyloid-beta (Aβ) oligomers are thought to be causative for the development and progression of Alzheimer's disease (AD). Starting from the Aβ oligomer eliminating d-enantiomeric peptide D3, we developed and applied a two-step procedure based on peptide microarrays to identify D3 derivatives with increased binding affinity and specificity for monomeric Aβ(1-42) to further enhance the Aβ oligomer elimination efficacy. Out of more than 1000 D3 derivatives, we selected seven novel d-peptides, named ANK1 to ANK7, and characterized them in more detail in vitro. All ANK peptides bound to monomeric Aβ(1-42), eliminated Aβ(1-42) oligomers, inhibited Aβ(1-42) fibril formation, and reduced Aβ(1-42)-induced cytotoxicity more efficiently than D3. Additionally, ANK6 completely inhibited the prion-like propagation of preformed Aβ(1-42) seeds and showed a nonsignificant tendency for improving memory performance of tg-APPSwDI mice after i.p. application for 4 weeks. This supports the hypothesis that stabilization of Aβ monomers and thereby induced elimination of Aβ oligomers is a suitable therapeutic strategy.

Phenol-soluble modulins (PSMs) are peptide virulence factors produced by staphylococci. These peptides contribute to the overall pathogenicity of these bacteria, eliciting multiple immune responses from host cells. Many of the α-type PSMs exhibit cytolytic properties and are able to lyse particular eukaryotic cells, including erythrocytes, neutrophils, and leukocytes. In addition, they also appear to contribute to the protection of the bacterial cell from the host immune response through biofilm formation and detachment. In this study, three of these peptide toxins, PSMs α1, α3, and β2, normally produced by Staphylococcus aureus, have been synthesized using solid-supported peptide synthesis (SPPS) (PSMα1 and PSMα3) or made by heterologous expression in Escherichia coli (PSMβ2). Their three-dimensional structures were elucidated using nuclear magnetic resonance spectroscopy. PSMα1 and PSMα3 each consist of a single amphipathic helix with a slight bend near the N- and C-termini, respectively. PSMβ2 contains three amphipathic helices, which fold to produce a "v-like" shape between α-helix 2 and α-helix 3, with α-helix 1 folded over such that it is perpendicular to α-helix 3. The availability of three-dimensional structures permits spatial analysis of features and residues proposed to control the biological activity of these peptide toxins.

The scorpion toxin tamapin displays the most potent and selective blockage against KCa2.2 channels known to date. In this work, we report the biosynthesis, three-dimensional structure, and cytotoxicity on cancer cell lines (Jurkat E6-1 and human mammary breast cancer MDA-MB-231) of recombinant tamapin and five related peptides bearing mutations on residues (R6A,R7A, R13A, R6A-R7A, and GS-tamapin) that were previously suggested to be important for tamapin's activity. The indicated cell lines were used as they constitutively express KCa2.2 channels. The studied toxin-like peptides displayed lethal responses on Jurkat T cells and breast cancer cells; their effect is dose- and time-dependent with IC50 values in the nanomolar range. The order of potency is r-tamapin>GS-tamapin>R6A>R13A>R6A-R7A>R7A for Jurkat T cells and r-tamapin>R7A for MDA-MB-231 breast cancer cells. Our structural determination by NMR demonstrated that r-tamapin preserves the folding of the αKTx5 subfamily and that neither single nor double alanine mutations affect the three-dimensional structure of the wild-type peptide. In contrast, our activity assays show that changes in cytotoxicity are related to the chemical nature of certain residues. Our results suggest that the toxic activity of r-tamapin on Jurkat and breast cancer cells could be mediated by the interaction of charged residues in tamapin with KCa2.2 channels via the apoptotic cell death pathway.

Recombinant proteins are promising tools for tissue engineering and drug delivery applications. Protein-based biomaterials have several advantages over natural and synthetic polymers, including precise control over amino acid composition and molecular weight, modular swapping of functional domains, and tunable mechanical and physical properties. In this work, we describe recombinant proteins based on abductin, an elastomeric protein that is found in the inner hinge of bivalves and functions as a coil spring to keep shells open. We illustrate, for the first time, the design, cloning, expression, and purification of a recombinant protein based on consensus abductin sequences derived from Argopecten irradians . The molecular weight of the protein was confirmed by mass spectrometry, and the protein was 94% pure. Circular dichroism studies showed that the dominant structures of abductin-based proteins were polyproline II helix structures in aqueous solution and type II β-turns in trifluoroethanol. Dynamic light scattering studies illustrated that the abductin-based proteins exhibit reversible upper critical solution temperature behavior and irreversible aggregation behavior at high temperatures. A LIVE/DEAD assay revealed that human umbilical vein endothelial cells had a viability of 98 ± 4% after being cultured for two days on the abductin-based protein. Initial cell spreading on the abductin-based protein was similar to that on bovine serum albumin. These studies thus demonstrate the potential of abductin-based proteins in tissue engineering and drug delivery applications due to the cytocompatibility and its response to temperature.

The transthyretin amyloidoses are diseases of protein misfolding characterized by the extracellular deposition of fibrils and other aggregates of the homotetrameric protein transthyretin (TTR) in peripheral nerves, heart, and other tissues. Age is the major risk factor for the development of these diseases. We hypothesized that an age-associated increase in the level of protein oxidation could be involved in the onset of the senile forms of the TTR amyloidoses. To test this hypothesis, we have produced and characterized relevant age-related oxidative modifications of the wild type (WT) and the Val122Ile (V122I) TTR variant, both involved in cardiac TTR deposition in the elderly. Our studies show that methionine/cysteine-oxidized TTR and carbonylated TTR from either the WT or the V122I variant are thermodynamically less stable than their nonoxidized counterparts. Moreover, carbonylated WT and carbonylated V122I TTR have a stronger propensity to form aggregates and fibrils than WT and V122I TTR, respectively, at physiologically attainable pH values. It is well-known that TTR tetramer dissociation, the limiting step for aggregation and amyloid fibril formation, can be prevented by small molecules that bind the TTR tetramer interface. Here, we report that carbonylated WT TTR is less amenable to resveratrol-mediated tetramer stabilization than WT TTR. All the oxidized forms of TTR tested are cytotoxic to a human cardiomyocyte cell line known to be a target for cardiac-specific TTR variants. Overall, these studies demonstrate that age-related oxidative modifications of TTR can contribute to the onset of the senile forms of the TTR amyloidoses.

The growing interest in membrane interactions of amyloidogenic proteins indicates that lipid binding and the regulation of membrane potential are critical to the onset and progression of neurodegenerative diseases such as Parkinson's (PD), Alzheimer's (AD), and prion diseases. Advancing the understanding of this field requires the application of varied biophysical and biological techniques designed to probe the characteristics and underlying mechanisms of membrane-peptide interactions. Therefore, the development of a rapid cytotoxicity evaluation system using a membrane potential-sensitive bis-oxonol fluorescent dye, DiBAC4(3) is reported here. The exposure of C-terminal truncated α-synuclein 119 (α-Syn119) and amyloid-β(1-42) (Aβ(1-42)) to U2-OS cell cultures resulted in an immediate, significant, and concentration-dependent increase in fluorescence response of DiBAC4(3). This response was strongly correlated with the cytotoxicity of α-Syn119 and Aβ(1-42) as determined by conventional CC8 and ATP assays. Furthermore, the capacity of well-defined polyphenolic antioxidants (i.e., pyrroloquinoline quinone (PQQ), baicalein, (-)-epigallocatechin-3-gallate (EGCG), and myricetin) to mitigate amyloid-induced cytotoxicity was evaluated using the developed biosensing system. We envisage that this work would accelerate the development of a rapid and cost-effective high-throughput screening platform in drug discovery for AD and PD.

The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species. The heterogeneities observed in the RP-HPLC have been determined to arise from unpaired cysteines (Cys-22 and Cys-96) that are buried in the V(H) domain. The Fab containing free thiols (referred to as "free-thiol Fab") and the Fab containing the disulfide (referred to as "intact Fab") of mAb A were generated through limited Lys-C digestion and purified with an ion exchange chromatography method. The binding of free-thiol Fab and intact Fab to its antigen was measured in a cell-based binding assay and an enzyme linked immunosorbent assay. The unpaired cysteines in the Fab of mAb A were found to have no significant impact on the binding to its target. Consistent with these Fab binding data, the enriched intact mAb A containing free thiols was determined to be fully active in a potency assay. The data reported here demonstrate that the redox status of cysteines is potentially a major source of heterogeneity for an antibody.

Vibrio cholerae accessory cholera enterotoxin (Ace) is the third toxin, along with cholera toxin (CT) and zonula occludens toxin (Zot), that causes the endemic disease cholera. Structural characterization of Ace has been restricted because of the limited production of this toxic protein by V. cholerae. We have cloned, overexpressed, and purified Ace from V. cholerae strain O395 in Escherichia coli to homogeneity and determined its biological activity. The unfolding of the purified protein was investigated using circular dichroism and intrinsic tryptophan fluorescence. Because Ace is predominantly a hydrophobic protein, the degree of exposure of hydrophobic regions was identified from the spectral changes of the environment-sensitive fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) that quenches the fluorescence of tryptophan residues of Ace in a concentration-dependent manner. Results showed that bis-ANS binds one monomeric unit of Ace with a 1:1 stoichiometry and a K' of 0.72 μM. Ace exists as a dimer, with higher oligomeric forms appearing upon glutaraldehyde cross-linking. This study also reports the binding of virstatin, a small molecule that inhibits virulence regulation in V. cholerae, to Ace. The binding constant (K=9×10(4) M(-1)) and the standard free energy change (ΔG°=-12 kcal mol(-1)) of Ace-virstatin interaction have been evaluated by the fluorescence quenching method. The binding does not affect the oligomeric status of Ace. A cell viability assay of the antibacterial activity of Ace has been performed using various microbial strains. A homology model of Ace, consistent with the experimental results, has been constructed.

Magnetic resonance imaging is a noninvasive imaging modality with high spatial and temporal resolution. Contrast agents (CAs) are frequently used to increase the contrast between tissues of interest. To increase the effectiveness of MR agents, small molecule CAs have been attached to macromolecules. We have created a family of biodegradable, macromolecular CAs based on protein polymers, allowing control over the CA properties. The protein polymers are monodisperse, random coil, and contain evenly spaced lysines that serve as reactive sites for Gd(III) chelates. The exact sequence and length of the protein can be specified, enabling controlled variation in lysine spacing and molecular weight. Relaxivity could be modulated by changing protein polymer length and lysine spacing. Relaxivities of up to approximately 14 mM(-1) s(-1) per Gd(III) and approximately 461 mM(-1) s(-1) per conjugate were observed. These CAs are biodegradable by incubation with plasmin, such that they can be easily excreted after use. They do not reduce cell viability, a prerequisite for future in vivo studies. The protein polymer CAs can be customized for different clinical diagnostic applications, including biomaterial tracking, as a balanced agent with high relaxivity and appropriate molar mass.

We prepared a series of cryptogein mutants, an elicitor from Phytophthora cryptogea, with altered abilities to bind sterols and fatty acids. The induction of the early events, i.e., synthesis of active oxygen species and pH changes, in suspension tobacco cells by these mutated proteins was proportional to their ability to bind sterols but not fatty acids. Although the cryptogein-sterol complex was suggested to be a form triggering a defense reaction in tobacco, some proteins unable to bind sterols induced the synthesis of active oxygen species and pH changes. The modeling experiments showed that conformational changes after the introduction of bulky residues into the omega loop of cryptogein resemble those induced by sterol binding. These changes may be necessary for the ability to trigger the early events by elicitins. However, the ability to stimulate necrosis in suspension tobacco cells and the expression of defense proteins in tobacco plants were linked neither to the lipid binding capacity nor to the capacity to provoke the early events. On the basis of these experiments and previous results, we propose that elicitins could stimulate two signal pathways. The first one induces necroses and the expression of pathogen-related proteins, includes tyrosine protein kinases and mitogen-activated protein kinases, and depends on the overall structure and charge distribution. The second type of interaction is mediated by phospholipase C and protein kinase C. It triggers the synthesis of active oxygen species and pH changes. This interaction depends on the ability of elicitins to bind sterols.

Helix 3 of the Cry1Aa toxin from Bacillus thuringiensis possesses eight charged amino acids. These residues, with the exception of those involved in intramolecular salt bridges (E90, R93, E112, and R115), were mutated individually either to a neutral or to an oppositely charged amino acid. The mutated genes were expressed, and the resultant, trypsin-activated toxins were assessed for their toxicity to Manduca sexta larvae and their ability to permeabilize M. sexta larval midgut brush border membrane vesicles to KCl, sucrose, raffinose, potassium gluconate, and N-methyl-D-glucamine hydrochloride with a light-scattering assay based on osmotic swelling. Most mutants were considerably less toxic than Cry1Aa. Replacing either E101, E116, E118, or D120 by cysteine, glutamine, or lysine residues had only minor effects on the properties of the pores formed by the modified toxins. However, half of these mutants (E101C, E101Q, E101K, E116K, E118C, and D120K) had a significantly slower rate of pore formation than Cry1Aa. Mutations at R99 (R99C, R99E, and R99Y) resulted in an almost complete loss of pore-forming ability. These results are consistent with a model in which alpha-helix 3 plays an important role in the mechanism of pore formation without being directly involved in determining the properties of the pores.

Four peptide inhibitors of small-conductance Ca(2+)-activated, apamin-sensitive K(+) channels (SK(Ca)) have been isolated from the venom of the Chinese scorpion Buthus martensi, named BmP01, BmP02, BmP03, and BmP05, respectively [Romi-Lebrun, R. (1997) Eur. J. Biochem. 245, 457-464]. Among them BmP05 with 31 amino acid residues has been intensively studied due to its most potent toxicity. To investigate the structure-function relationship of BmP05, its wild type and seven mutants (their C-termini unamidated) were successfully expressed in the yeast secretion system and purified with a high yield over 8 mg/L. Their toxicity to mice and electrophysiological activity on the K(+) currents (SK(Ca) and Kv) in rat adrenal chromaffin cells were measured and compared. The results indicated the following: (1) As a selective antagonist against SK(Ca), 1 microM rBmP05 is equivalent to 0.2 microM apamin, and its IC(50) is 0.92 microM. (2) The basic residues Lys and Arg located at positions 6 and 13 in the N-terminal alpha-helix region are essential and synergetic in the interaction of the toxin with SK(Ca). (3) Disruption of the alpha-helix by mutation of Gln at position 9 with Pro results in almost total loss of toxicity. (4) The C-terminal residue His31 plays an auxiliary role in the interaction of the toxin with SK(Ca). (5) The beta-turn connecting two beta-sheets near the C-terminal part is responsible for the specificity of the toxin to the different subtypes of K(+) channels.

Bovine seminal RNase (BS-RNase) is a homodimeric enzyme with a cytotoxic activity selective for tumor cells. In this study, the relationships of its cytotoxic activity to its dimeric structure and its resistance to the cytosolic RNase inhibitor (cRI) are investigated systematically by site-directed mutagenesis. The results show that (1) the dimericity of BS-RNase is essential for its full cytotoxic action; (2) the role of the dimeric structure in the antitumor activity is that of making the enzyme insensitive to the cytosolic RNase inhibitor; (3) a RNase may not be completely insensitive to cRI to exploit a full cytotoxic potential.

The single tryptophan residue (Trp187) of human recombinant annexin V, containing 320 residues and 5328 atoms, was replaced with three different isosteric analogues where hydrogen atoms at positions 4, 5, and 6 in the indole ring were exchanged with fluorine. Such single atom exchanges of H --> F represent atomic mutations that result in slightly increased covalent bond lengths and inverted polarities in the residue side-chain structure. These minimal changes in the local geometry do not affect the secondary and tertiary structures of the mutants, which were identical to those of wild-type protein in the crystal form. But the mutants exhibit significant differences in stability, folding cooperativity, biological activity, and fluorescence properties if compared to the wild-type protein. These rather large global effects, resulting from the minimal local changes, have to be attributed either to the relatively strong changes in polar interactions of the indole ring or to differences in the van der Waals radii or to a combination of both facts. The changes in local geometry that are below resolution of protein X-ray crystallographic studies are probably of secondary importance in comparison to the strong electronegativity introduced by the fluorine atom. Correspondingly, these types of mutations provide an interesting approach to study cooperative functions of integrated residues and modulation of particular physicochemical properties, in the present case of electronegativity, in a uniquely structured and hierarchically organized protein molecule.

Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the group of ribonucleolytic toxins called ribotoxins. It specifically cleaves a single phosphodiester bond in a conserved stem and loop structure in the 28S rRNA of large ribosomal subunit and potently inhibits eukaryotic protein synthesis. Restrictocin contains 149 amino acid residues and includes four cysteines at positions 5, 75, 131, and 147. These cysteine residues are involved in the formation of two disulfide bonds, one between Cys 5 and Cys 147 and another between Cys 75 and Cys 131. In the current study, all four cysteine residues were changed to alanine individually and in different combinations by site-directed mutagenesis so as to remove one or both the disulfides. The mutants were expressed and purified from Escherichia coli. Removal of any cysteine or any one of the disulfide bonds individually did not affect the ability of the toxin to specifically cleave the 28S rRNA or to inhibit protein synthesis in vitro. However, the toxin without both disulfide bonds completely lost both ribonucleolytic and protein synthesis inhibition activities. The active mutants, containing only one disulfide bond, exhibited relatively high susceptibility to trypsin digestion. Thus, none of the four cysteine residues is directly involved in restrictocin catalysis; however, the presence of any one of the two disulfide bonds is absolutely essential and sufficient to maintain the enzymatically active conformation of restrictocin. For maintenance of the unique stability displayed by the native toxin, both disulfide bonds are required.

Toxicological evaluation of xylanase X(22) from Aspergillus nidulans expressed in a wine yeast strain was carried out. The safety of the X(22) intake was assessed by digestibility, bioinformatic, and mouse short-term repeated dosing studies, although X(22) shows resistance to proteolytic degradation in the gastrointestinal system, is a minority protein component (<0.5 10(-)(6) %) of the produced wine, and shows no significant amino acid sequence homology to any known food allergens. The 4-week oral toxicity study was performed in Swiss mice at a dose level of 0.01, 0.1, or 1 mg/kg/day (these dosages correlate to 8, 80, and 800 times, respectively, the enzyme amount contained in 250 mL of wine). Body weight, food and fluid intake, urinalysis, and hematology data were obtained. Postmortem examinations and histopathology by both light and electron microscopy were performed. According to the results of this study, no adverse effects were detected by oral administration of X(22).

Several immunotoxins (ITs) were synthesized by the attachment of clavin, a recombinant toxic protein derived from Aspergillus clavatus, to the monoclonal antibody Mgr6 that recognizes an epitope of the gp185(HER-2) extracellular domain expressed on breast and ovarian carcinoma cells. Conjugation and purification parameters were analyzed in an effort to optimize the antitumor activity and stability of the ITs in vivo. To modulate the in vitro and in vivo properties of the immunotoxins, different coupling procedures were used and both disulfide and thioether linkages were obtained. Unhindered and hindered disulfide with a methyl group linkage ethyl S-acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT) or ethyl S-acetyl 3-mercaptobutyrothioimidate ester hydrochloride (M-AMPT) were obtained by reaction with recombinant clavin, while the monoclonal antibody Mgr6 was derivatized with ethyl 3-[(4-carboxamidophenyl)dithio]propionthioimidate ester hydrochloride (CDPT). To achieve higher hindrance (a disulfide bond with a geminal dimethyl group), Mgr6 was derivatized with the N-hydroxysuccinimidyl 3-methyl-3-(acetylthio)butanoate (SAMBA) and clavin with CDPT. To evaluate the relevance of the disulfide bond in the potency and pharmacokinetic behavior of the ITs, a conjugate consisting of a stable thioether bond was also prepared by derivatizing Mgr6 with the N-hydroxysuccinimidyl ester of iodoacetic acid (SIA) and clavin with AMPT. The immunotoxins were purified and characterized using a single-step chromatographic procedure. Specificity and cytotoxicity were assayed on target and unrelated cell lines. The data indicate that the introduction of a hindered disulfide linkage into ITs has little or no effect on antitumor activity and suggest that disulfide cleavage is essential for activity; indeed, the intracellularly unbreakable thioether linkage produced an inactive IT. Analysis of IT stability in vitro showed that the release of mAb by incubation with glutathione is proportional to the presence of methyl groups and increases exponentially with the increase in steric hindrance. Analysis of the pharmacokinetic behavior of ITs in Balb/c mice given intravenous bolus injections indicated that ITs with higher in vitro stability were eliminated more slowly; i.e., the disulfide bearing a methyl group doubled the beta-phase half-life (from 3.5 to 7.1 h) compared with that of the unhindered, while a geminal dimethyl protection increased the elimination phase to 24 h. The thioether linkage showed its intrinsic stability with a beta-phase half-life of 46 h. The thioether linkage also increased the distribution phase from 17 to 32 min. The in vitro characteristics and in vivo stability of Mgr6-clavin conjugates composed of a methyl and dimethyl steric hindered disulfide suggest clinical usefulness.

During the intoxication process, Pseudomonas exotoxin (PE) and immunotoxins containing PE internalize into the target cell and become processed into two fragments, and the carboxyl fragment translocates into the cytosol where it inactivates elongation factor 2. We have proposed that after internalization into cells the carboxyl-terminal fragment of PE (amino acids 280-613), which ends in REDLK, binds to the KDEL receptor (ERD2) which carries it to the endoplasmic reticulum, from which the PE fragment translocates to the cytosol. Earlier experiments showing that REDL but not REDLK binds to the KDEL receptor suggested that the terminal lysine is removed sometime during the intoxication process. To determine if and where this occurs, we exposed a peptide ending in REDLK to malignant cells in culture and found that binding to the KDEL receptor was restored. Restoration of receptor binding also occurred if a peptide or toxin ending in REDLK at its carboxyl terminus was incubated with plasma, indicating that the terminal lysine is removed prior to entry of the toxin into the cell. We conclude that plasma carboxypeptidase(s) cleave(s) the lysine residue from the carboxyl terminus of PE and PE-containing immunotoxins as an early and essential step in their cellular intoxication pathway.

Bryonia dioica (Cucurbitaceae family) produces at least two type I ribosome-inactivating proteins, bryodin 1 (BD1) and bryodin 2 (BD2). A cDNA sequence encoding BD1 was isolated from B. dioica leaf mRNA using degenerative oligonucleotides and codes for a 22 amino acid signal peptide followed by a protein of 267 residues. Expression of two recombinant BD1 (rBD1) forms in Escherichia coli yielded proteins of 267 (to the natural stop codon) and 247 amino acids (to the putative cleavage site yielding the mature protein) that had identical protein synthesis inhibition activity as compared to native BD1. The substitution of Lys for Glu at position 189 near the active site reduced the ability of rBD1 to inhibit protein synthesis by 10-fold. Toxicologic analysis showed that rBD1 was well tolerated in rodents with LD50 values of 40 mg/kg in mice and >25 mg/kg in rats. A crystal of mature rBD1 protein was used to collect X-ray diffraction data to 2.1 A resolution. The rBD1 crystal structure was solved and showed extensive homology with other type I RIPs and A chains of type II RIPs. The studies described here demonstrate that rBD1 retains full biologic activity and serve as a guide for using this potent, yet nontoxic, RIP in the construction of single-chain immunotoxin fusion proteins.

The solution structure of a recombinant active alpha-neurotoxin from Leiurus quinquestriatus hebraeus, Lqh(alpha)IT, was determined by proton two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). This toxin is the most insecticidal among scorpion alpha-neurotoxins and, therefore, serves as a model for clarifying the structural basis for their biological activity and selective toxicity. A set of 29 structures was generated without constraint violations exceeding 0.4 A. These structures had root mean square deviations of 0.49 and 1.00 A with respect to the average structure for backbone atoms and all heavy atoms, respectively. Similarly to other scorpion toxins, the structure of Lqh(alpha)IT consists of an alpha-helix, a three-strand antiparallel beta-sheet, three type I tight turns, a five-residue turn, and a hydrophobic patch that includes tyrosine and tryptophan rings in a "herringbone" arrangement. Positive phi angles were found for Ala50 and Asn11, suggesting their proximity to functionally important regions of the molecule. The sample exhibited conformational heterogeneity over a wide range of experimental conditions, and two conformations were observed for the majority of protein residues. The ratio between these conformations was temperature-dependent, and the rate of their interconversions was estimated to be on the order of 1-5 s(-1) at 308 K. The conformation of the polypeptide backbone of Lqh(alpha)IT is very similar to that of the most active antimammalian scorpion alpha-toxin, AaHII, from Androctonus australis Hector (60% amino acid sequence homology). Yet, several important differences were observed at the 5-residue turn comprising residues Lys8-Cys12, the C-terminal segment, and the mutual disposition of these two regions. 2D NMR studies of the R64H mutant, which is 3 times more toxic than the unmodified Lqh(alpha)IT, demonstrated the importance of the spatial orientation of the last residue side chain for toxicity of Lqh(alpha)IT.

We demonstrate that post-translational bromination of a tryptophan residue occurs in the biologically active octapeptide bromocontryphan, purified and characterized from Conus radiatus venom. Clones encoding bromocontryphan were identified from a cDNA library made from C. radiatus venom ducts. The mRNA sequence obtained predicts a prepropeptide which has the mature peptide sequence at the C-terminal end, with the L-6-bromotryptophan residue encoded by UGG, the Trp codon. These data provide the first direct evidence for post-translational bromination of a polypeptide which is translated through the normal cellular machinery. In addition to bromination, the peptide, which induces a "stiff tail" syndrome in mice, has several other modifications as shown by the sequence [Formula: See Text] in which Hyp = hydroxyproline. Asterisks indicate post-translational modifications (left to right): proteolytic cleavage at the N-terminus; hydroxylation of Pro3; epimerization of Trp4; bromination of Trp7, and C-terminal amidation. Bromocontryphan appears to have the highest density of post-translational modifications known among gene-encoded polypeptides. The overall result is a molecule which closely resembles marine natural products produced through specialized biosynthetic pathways comprising many enzyme-catalyzed steps.

Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor bean seeds, consists of a cell binding lectin subunit (RTB) disulfide linked to an rRNA N-glycosidase protein synthesis inactivating subunit (RTA). While X-ray crystallography and equilibrium dialysis suggested two sugar-combining sites located in subdomains 1 alpha and 2 gamma, biochemical and mutational analyses suggested the existence of a third lectin site. We performed oligonucleotide-directed mutagenesis on RTB cDNA to create mutants with modifications in subdomains 1 alpha, 2 gamma, and either 1 beta or 2 alpha. The triple-site mutant RTBs were expressed in insect cells. Partially purified recombinant proteins obtained from infected cell extracts and cell supernatants were characterized for asialofetuin and cell binding, immunoreactivites, ability to reassociate with RTA, and recombinant heterodimer cell cytotoxicity. Yields of both triple-site mutants were similar to the parent double-site mutant. Both mutants showed immunoreactivity with a panel of anti-RTB monoclonal and polyclonal antibodies. The triple-site mutant with modification of amino acid residues in subdomains 1 alpha, 2 alpha, and 2 gamma bound asialofetuin and cells similarly to the parent 1 alpha, 2 gamma, subdomain mutant. In contrast, the 1 alpha, 1 beta, 2 gamma subdomain triple-site mutant had a one and one-half log decrease in asialofetuin and cell binding relative to the parent double-site mutant. The 1 alpha, 2 alpha, 2 gamma triple-site mutant and 1 alpha, 2 gamma parent protein had sugar binding which was inhibited by 3-27-fold by lactose and asialofetuin. Both triple-site mutants reassociated well with RTA. The 1 alpha, 2 alpha, 2 gamma triple-site mutant-RTA was equally cytotoxic to mammalian cells as the double-site mutant-RTA heterodimer. In contrast, the 1 alpha, 1 beta, 2 gamma triple-site mutant-RTA was 25 times less toxic than the double mutant and 20 times more toxic than RTA alone. These data support a model for at least three lectin-binding subdomains in RTB.

The alpha neurotoxin Lqh alpha IT is toxic to both insects and mammals but exhibits a bioactivity ratio favoring insects (insect/mammal approximately 2). With the objective of increasing this ratio by genetic manipulation of the amino acid sequence, a cDNA clone encoding Lqh alpha IT was used to produce recombinant variants of the toxin in a high efficiency bacterial expression system. The unmodified recombinant toxin, isolated from inclusion bodies and renatured in vitro, exhibited chemical and biological properties indistinguishable from those of the authentic native toxin. Alteration of the toxin by site-directed mutagenesis led to a substantial reduction in anti-mammalian toxicity (mouse LD50 reduced 6.4-fold) but only a slight reduction (x 1.5) in the insect ED50 value for paralysis. The reduction in anti-mammalian toxicity was correlated with a approximately 2-fold reduction of its potency for slowing of sodium channel inactivation in mammalian neurons, while no change in mutant toxin binding affinity to insect neuronal receptors was registered. These results demonstrate for the first time expression of a recombinant sodium channel neurotoxin in Escherichia coli and the use of site-directed mutagenesis to improve phylogenetic selectivity. This recombinant approach provides a promising strategy for optimizing the selective toxicity of peptide neurotoxins.

DNA encoding ricin B chain was modified by site-directed mutagenesis, and eight separate mutant RTB cDNAs including four novel mutants were ligated into the baculovirus transfer vector, pAcGP67A. Cotransfection of S. frugiperda Sf9 cells with BaculoGold DNA was followed by limiting dilution isolation of recombinant baculoviruses. Infection of Sf9 cells at a multiplicity of infection of 5 in the presence of 25 mM lactose produced 0.05-1 mg/L of soluble, glycosylated 34 kDa proteins immunoreactive with monoclonal and polyclonal antibodies to ricin B chain. Mutant ricin B chains were partially purified by monoclonal antibody immunoaffinity chromatography to 10-50% purity in near milligram quantities. The mutant ricin B chains had decreased lectin binding relative to plant ricin B chain as measured by binding to immobilized lactose and asialofetuin and cell binding immunofluorescence. The mutant ricin B chains reassociated with plant RTA similarly to plant RTB, and the recombinant heterodimers had slightly reduced cell cytotoxicity relative to ricin.

B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv region of monoclonal antibody B3 is connected to a truncated form of Pseudomonas exotoxin. It would be desirable to use the lysine residues of the molecule for chemical modification so that it can be derivatized with poly(ethylene glycol) to achieve reduced immunogenicity or with the Bolton-Hunter reagent for biodistribution studies. We found that derivatizing lysine residues of B3(Fv)-PE38 causes a marked loss of specific target cell cytotoxicity and/or immunoreactivity. Here we show that two lysine residues in the antibody-combining region of B3(Fv)-PE38 can be replaced with arginines, with only a small loss of cytotoxicity and no change in specificity. This mutant molecule is 3-fold more resistant to inactivation by derivatization with succinimidyl 4-(N-maleimidomethyl)cyclohexane 1-carboxylate (SMCC) or Bolton-Hunter reagent.

In a previous study, we have demonstrated that conjugates containing soluble, recombinant human CD4 (rCD4) and the deglycosylated form of ricin A chain (dgA) (rCD4-dgA) effectively kill a human T cell line infected with the human immunodeficiency virus (HIV) in vitro. In contrast, such conjugates are 100-1000-fold less toxic to uninfected cells. In order to use a rCD4-dgA conjugate effectively in vivo, it was important to demonstrate that (1) it binds to and kills HIV-infected, but not uninfected, human cells, (2) it is stable in the circulation, and (3) it has an optimal therapeutic index (toxicity to animals versus toxicity to target cells). A major factor affecting the efficacy of such conjugates in vitro and in vivo is the nature of the cross-linker between the ligand (rCD4) and the toxin (dgA). In this report, we have prepared rCD4-dgA conjugates using three different cross-linkers. Different methods of purification have been compared by determining the optimal yield, purity, and retention of biological activity (i.e., binding to gp120 and dgA chain activity). The structure of these conjugates as well as their cytotoxicity to target cells in vitro has been analyzed. Finally, we have compared their pharmacokinetics, tissue localization, and toxicity in mice.