Increasing the potency of a cytotoxin with an arginine graft

Variants and homologs of bovine pancreatic ribonuclease (RNase A) can exhibit cytotoxic activity. This toxicity relies on cellular internalization of the enzyme. Residues Glu49 and Asp53 form an anionic patch on the surface of RNase A. We find that replacing these two residues with arginine does not affect catalytic activity or affinity for the cytosolic ribonuclease inhibitor (RI) protein. This 'arginine graft' does, however, increase toxicity towards human cancer cells. Appending a nonaarginine domain to this cationic variant results in an additional increase in cytotoxicity, providing one of the most cytotoxic known variants of RNase A. These findings correlate the potency of a ribonuclease with its deliverance of ribonucleolytic activity to the cytosol, and indicate a rational means to enhance the efficacy of ribonucleases and other cytotoxic proteins.     

Inhibition of cell growth by a fused protein of human ribonuclease 1 and human basic fibroblast growth factor

Pancreatic-type RNases are considered to have cytotoxic potentialdue to their ability to degrade RNA molecules when they enterthe cytosol. However, most of these RNases show little cytotoxicitybecause cells have no active uptake mechanism for these RNasesand because the ubiquitous cytoplasmic RNase inhibitor is consideredto play a protective role against the endocytotic leak of RNasesfrom the outside of cells. To study the cytotoxic potentialof RNase toward malignant cells targeting growth factor receptors,the C-terminus of human RNase 1 was fused to the N-terminusof human basic fibroblast growth factor (bFGF). This RNase–FGFfused protein effectively inhibited the growth of mouse melanomacell line B16/BL6 with high levels of cell surface FGF receptor.This effect appeared to result from prolongation of the overallcell cycle rather than the killing of cells or specific arrestin a particular phase of the cell cycle. Thus, human RNase 1fused to a ligand of cell surface molecules, such as the FGFreceptor, is shown to be an effective candidate for a selectivecell targeting agent with low toxic effects on normal cell types.     

Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI

Three mutants of Escherichia coli ribonuclease HI, in whichan invariant acidic residue Asp134 was replaced, were crystallized,and their three-dimensional structures were determined by X-raycrystallography. The D134A mutant is completely inactive, whereasthe other two mutants, D134H and D134N, retain 59 and 90% activitiesrelative to the wild-type, respectively. The overall structuresof these three mutant proteins are identical with that of thewild-type enzyme, except for local conformational changes ofthe flexible loops. The ribonuclease H family has a common activesite, which is composed of four invariant acidic residues (Asp10,G1u48, Asp70 and Asp134 in E.coli ribonuclease HI), and theirrelative positions in the mutants, even including the side-chainatoms, are almost the same as those in the wild-type. The positionsof the -polar atoms at residue 134 in the wild-type, as wellas D134H and D134N, coincide well with each other. They arelocated near the imidazole side chain of His124, which is assumedto participate in the catalytic reaction, in addition to thefour invariant acidic residues. Combined with the pH profilesof the enzymatic activities of the two other mutants, H124Aand H124A/D134N, the crystallographic results allow us to proposea new catalytic mechanism of ribonuclease H, which includesthe roles for Asp134 and His124.     

Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (V(H) and V(L)) domains of two different specificities connected by three linkers. When assembled in the order V(H)(A)-linker(1)-V(L)(B)-linker(2)-V(H)(B)-linker(3)-V(L)(A), the single-chain molecule either folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody, or forms a homodimer that is twice as large, a so-called tandem diabody. The formation of the tandem diabody is determined by the association of complementary V(H) and V(L) domains located on different polypeptide chains, and depends on the length and probably the amino acid composition of the three linkers joining the variable domains. We generated a number of single-chain constructs using four V(H) and V(L) domains specific either for human CD3, a component of T-cell receptor (TCR) complex, or for CD19, a human B-cell antigen, separated by different rationally designed peptide linkers of 6-27 amino acid residues. The generated bispecific constructs were expressed in bacterial periplasm and their molecular forms, antigen-binding properties, stability, and T-cell proliferative and anti-tumor activities were compared. Using peripheral blood mononuclear cell cultures from patients suffering from B-cell chronic lymphocytic leukemia, we demonstrated that the tandab-mediated activation of autologous T cells and depletion of malignant cells correlates with the stability of the recombinant molecule and with the distance between the CD19 and CD3 binding sites.     

Bioactive IL7-diphtheria fusion toxin secreted by mammalian cells

A number of targeted cytotoxic agents have been developed that selectively kill malignant or otherwise pathological cells. These engineered proteins consist of a potent cytotoxic element connected to a ligand domain that binds to specific molecules on the surface of the target cell. Several of these agents have shown promise in clinical trials and one is currently administered to patients. A significant technical obstacle that has impeded the development of some of these toxins is the difficulty of preparing certain recombinant proteins in properly folded forms. These fusion proteins have generally been produced in bacteria requiring them to be denatured and renatured in vitro. For some proteins this is an efficient process whereas for others it is not. We describe here a system to produce fusion toxins rapidly and efficiently by engineering mammalian cells to secrete them as properly folded molecules which can be purified in native form from cell culture medium. We have used this system to produce highly active preparations of DAB(389)-IL7, a molecule consisting of the catalytic and transmembrane domains of diphtheria toxin fused to interleukin 7. This system is generalizable and can be used to produce and evaluate rapidly fusion toxins incorporating novel or uncharacterized ligands.     

Anti-angiogenic effect of an insertional fusion protein of human basic fibroblast growth factor and ribonuclease-1

Human pancreatic ribonuclease-1 (RNase1) does not exhibit its cytotoxicity unless it is artificially internalized into the cytosol. Furthermore, once it encounters the cytosolic RNase inhibitor (RI), the activity of RNase1 is seriously reduced. To achieve the cellular targeting of RNase1 and the blocking of RI binding simultaneously, the basic fibroblast growth factor (bFGF) sequence was inserted into RNase1 at the RI binding site using a gene fusion technique. The effect of this fusion protein, CL-RFN89, on the angiogenesis, which was accelerated by FGF-FGF receptor interaction, was investigated. It was shown by using fluorescein-labeled CL-RFN89, that the binding to human umbilical vein endothelial cells (HUVECs) was dependent on the existence of the FGF receptors. In addition, CL-RFN89 inhibited the cellular growth of HUVECs in vitro and also inhibited the tube formation, using a three-dimensional tube formation assay. Furthermore, this fusion protein was shown to prevent in vivo tumor cell-induced angiogenesis, using the mouse dorsal air sac assay. These results demonstrated that CL-RFN89 inhibits angiogenesis in vitro and in vivo and that it can be expected to be a potent antiangiogenic agent.     

Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv

Using molecular modeling technology, we have recently identifiedtwo positions in conserved framework regions of antibody Fvfragments (Fvs) that are distant from CDRs, and potentiallycan be used to make recombinant Fv fragments in which the unstableV_H and V_L heterodimer is stabilized by an interchain disulfidebond inserted between structurally conserved framework positions.A disulfide bond has been introduced at one of these positions,V_H44-V_L105, and shown to stabilize various Fvs that retain fullbinding and specificity. Recombinant immunotoxias, e.g. B3(dsFv)-PE38KDELin which this disulfide-stabilized Fv moiety is connected toa truncated form of Pseudomonas exotoxin (PE; PE38KDEL) whichcontains the translocation and ADP ribosylation domains, areindistinguishable in binding and specificity from its single-chainimmunotoxin counterparts. We have now analyzed the alternativeposition, (V_H111-V_L48), predicted by the modeling methodology,for disulfide stabilization of mAb B3(Fv) by producing a recombinantimmunotoxin with such disulfide-stabilized (ds) Fv. This immunotoxinwas also very active and retained full specificity to B3 antigen-positivecells. However, it was 2- to 3-fold less active than the V_H44-V_L105dsFv-molecule. We also tested various biochemical features ofV_H44-V_L105 and V_H111-V_L48 dsFv immunotoxins and compared themwith the corresponding single-chain immunotoxin. We found thedsFv immunotoxins were more stable in human serum and more resistantto thermal and chemical denaturation than the single chain (sc)Fv immunotoxin. Because dsFv immunotoxins and dsFvs have fullactivity and specificity and improved stability, they may bemore useful than scFv immunotoxins as therapeutic and diagnosticagents.     

Effect of mutations on the dimer stability and the pH optimum of the human foamy virus protease

To explore the role of residues being close to the catalytic aspartates in the higher pH optimum and in the lower dimer stability of human foamy virus (HFV) protease (PR) in comparison with human immunodeficiency virus type 1 (HIV-1) protease, single (Q8R, H22L, S25T, T28D) and double (Q8R-T28D, H22L-T28D) mutants were created based on sequence alignments and on the molecular model of HFV PR. The wild-type and mutant enzymes were expressed in fusion with maltose binding protein in Escherichia coli and the fusion proteins were purified by affinity chromatography. Specificity constant of most mutants was lower, but the value of Q8R-T28D double mutant enzyme was higher than that of the wild-type HFV PR. Furthermore, urea denaturation at two pH values and pH optimum values showed an increased stability and pH optimum for most mutants. These results suggest that the mutated residues may not be responsible for the higher pH optimum of HFV PR, but they may contribute to the lower dimer stability as compared with that of HIV-1 PR.     

Conformation and stability of barley chymotrypsin inhibitor-2 (CI-2) mutants containing multiple lysine substitutions

A major goal of agricultural biotechnology is to increase thenutritional value of maize seed through the expression of heterologousproteins enriched in lysine. One promising candidate is barleychymotrypsin inhibitor-2 (CI-2), a plant protein that has beenextensively characterized with respect to structure and function.Based on the tertiary structure of wild-type (WT) CI-2, fivemutants with lysine contents ranging from 20 to 25 mol percentwere designed, expressed in Escherichia coli and purified byion exchange and gel permeation chromatography. Inasmuch asprevious transgenic experiments suggested that proper foldingand stability may be essential for in vivo accumulation of theengineered proteins in plant cells, we first undertook an invitro study of the conformation and thermodynamic stabilityof the CI-2 mutants in order to select an ideal candidate forplant expression. Mutant and WT CI-2 proteins had similar circulardichroism spectra, suggesting similar secondary structures.However, differences in the accessibility of the sole tryptophanresidue, Trp24, indicated that the local conformation differedamong the mutants. The thermodynamic stability of the mutantsranged from <2 to 4.9 kcal/mol compared with ~7 kcal/molfor the wild-type protein. In conjunction with proteolytic stabilitystudies, we have identified one mutant that has the potentialto be expressed in a stable manner in plant cells.     

Escherichia coli aspartate carbamoyltransferase: the probing of crystal structure analysis via site-specific mutagenesis

Crystal structures are known for aspartate carbamoyltransferase(ATCase) in the T and R states, with and without the allostericactivator adenosine triphosphate (ATP) or inhibitor cytidinetriphosphate (CTP). Visual inspection of X-ray crystal structuresdoes not provide all of the information necessary for the determinationof structure-function relationships in protein molecules. Thisproblem is compounded because the crystalline states of themolecule may introduce effects due to crystal packing, restrictedflexibility and less than optimum enzymatic conditions. Therefore,alternative techniques are required to test mechanisms conjecturedfrom three-dimensional crystal structures of proteins. The techniqueof site-specific mutagenesis allows the researcher to test structure- function models based on threedimensional structures and toobtain further insight into characteristics of the enzyme. Site-specificmutagenesis has been used to probe residues believed to be criticalin the structure and function of ATCase. Selection of residuesto be mutated has depended extensively on three-dimensionalcrystal structures of the enzyme. To date, 48 site-specificmutations at 37 different amino acid sites have been published.Although a total of 118 mutants at 58 different sites has beencommunicated to our laboratory, only published mutants willbe considered in this review. In this paper, we compile forthe first time, review, and analyze the site-specific mutantsof ATCase. Site-specific mutagenesis of proteins has becomea powerful technique in modern-day molecular biology, especiallyin studying a molecule as large as aspartate carbamoyltransferase.In this review, the role of site-specific mutagenesis of ATCaseis discussed and improvements in the analysis are suggested.     

Revisiting the correlation between proteins' thermoresistance and organisms' thermophilicity

The possibility to rationally design protein mutants that remain structured and active at high temperatures strongly depends on a better understanding of the mechanisms of protein thermostability. Studies devoted to this issue often rely on the living temperature (T(env)) of the host organism rather than on the melting temperature (T(m)) of the analyzed protein. To investigate the scale of this approximation, we probed the relationship between T(m) and T(env) on a dataset of 127 proteins, and found a much weaker correlation than previously expected: the correlation coefficient is equal to 0.59 and the regression line is T(m) approximately 42.9 degrees C + 0.62T(env). To illustrate the effect of using T(env) rather than T(m) to analyze protein thermoresistance, we derive statistical distance potentials, describing Glu-Arg and Asp-Arg salt bridges, from protein structure sets with high or low T(m) or T(env). The results show that the more favorable nature of salt bridges, relative to other interactions, at high temperatures is more clear-cut when defining thermoresistance in terms of T(m). The T(env)-based sets nevertheless remain informative.     

Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF

The molecular basis for the voltage and pH dependence of the Escherichia coli OmpF porin activity remains unknown. The L3 loop was previously shown not be involved in voltage dependence. Here we used seven OmpF mutants where single extracellular loops, except L3, were deleted one at a time. The proteins are expressed at levels comparable to wild-type and purified as trimers. Wild-type and mutant proteins were inserted into planar lipid bilayers for electrophysiological measurement of their activity. Current-voltage relationships show the typical porin channel closure at voltages greater than the critical voltage. Measurements of critical voltages for the seven deletion mutants showed no significant differences relative to wild-type, hence eliminating the role of single loops in voltage sensitivity. However, deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH. Wild-type channels close more readily at acidic pH and their open probability is decreased by approximately 60% at pH 4.0 relative to pH 7.0. For mutants lacking loop L1, L7 or L8, the channel open probability was found not to be significantly different at pH 4.0 than at pH 7.0. The other deletion mutants retained a pH sensitivity similar to the wild-type channel. Possible mechanistic scenarios for the voltage- and pH dependence of E.coli OmpF porin are discussed based on these results.     

Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis

One approach to the analysis of leucocyte cell surface proteinsis to express their domains with part of another protein asa carrier. We report the use of two immunoglobulin superfamily(IgSF) domains from rat CD4 (CD4d3+4) in producing domains fromvarious superfamilies as chimeric proteins in Chinese hamsterovary cell lines. Four types of construct were successfullyexpressed containing: (i) the two IgSF domains of CD48; (ii)the IgSF domain of mb-1 which is part of the B cell antigenrecognition complex; (iii) a T cell receptor V domain; and (iv)the N-terminal domain of CD5 which belongs to the scavengerreceptor superfamily. This CD5 chimeric protein was antigenkfor a panel of CD5 mAbs showing that mAbs with functional effectsreacted with the N-terminal domain of CD5. The CD48 chimericprotein has been used both as multivalent complexes producedby crosslinking with mAbs recognizing CD4 and in a monomericform to analyse the kinetics of the interaction between CD48and CD2 [van der Merwe et al. (1993) EMBO J., 12, 4945–4954].     

Mapping of residues involved in the interaction between the Bacillus subtilis xylanase A and proteinaceous wheat xylanase inhibitors

The Bacillus subtilis xylanase A was subjected to site-directed mutagenesis, aimed at changing the interaction with Triticum aestivum xylanase inhibitor, the only wheat endogenous proteinaceous xylanase inhibitor interacting with this xylanase. The published structure of Bacillus circulans XynA was used to target amino acids surrounding the active site cleft of B.subtilis XynA for mutation. Twenty-two residues were mutated, resulting in 62 different variants. The catalytic activity of active mutants ranged from 563 to 5635 XU/mg and the interaction with T.aestivum xylanase inhibitor showed a similar variation. The results indicate that T.aestivum xylanase inhibitor interacts with several amino acid residues surrounding the active site of the enzyme. Three different amino acid substitutions in one particular residue (D11) completely abolished the interaction between T.aestivum xylanase inhibitor and B.subtilis xylanase A.     

Maintenance of the hydrophobic face of the diphtheria toxin amphipathic transmembrane helix 1 is essential for the efficient delivery of the catalytic domain to the cytosol of target cells

Abstract The transmembrane (T) domain of diphtheria toxin (DT) comprisesnine -helices and has been shown to play an essential role inthe efficent delivery of the catalytic (C) domain ofDT acrossthe eukaryotic cell membrane and into the cytosol. We have demonstratedrecently thatthe first three amphipathic helixes of the T domain,although not necessary for either channel formation or receptorbinding, are required for the efficient transmembrane deliveryof the Cdomain.In the present study,we have performed a detailedstructure-function analysis of T domainhelix 1 (TH1) of theDT-related fusion protein DAB₃₈₉lL-2. We performed exchangeandsite-directed mutagenesis of TH1 and the resulting mutantfusion toxins were analyzed by gel electrophoresis and testedfor their efficiencies in the delivery of the C domain to thecell cytosol. We demonstrate that the overall charge distributionand hydrophobicity of amino acids in the amphipathic helix TH1,rather than a specific amino acid sequence, are critical forthe function of this helix. The insertion of a charged residuein the hydrophobic face of TH1 abolishes cytotoxic activity,whereas replacement of a hydrophobic residue by a charged aminoacid in the hydrophilic face of the helix has little, if any,effect on cytotoxic activity. In addition,we have identifiedSer220 by site-directed mutagenesis as a residue that appearsto be criticalfor correct folding of the fusion toxin. Mutationsin this position result in fusion proteins that are extremelysensitive to proteolytic attack.     

Characterization of a ricin fusion toxin targeted to the interleukin-2 receptor

Fusion toxins are hybrid proteins consisting of peptide ligandslinked through amide bonds to polypeptide toxins. The liganddirects the molecule to the surface of target cells and thetoxin enters the cytosol and induces cell death. Ricin is anexcellent candidate for use in fusion toxins because of itsextreme potency, the extensive knowledge of its atomic structureand the lack of prior immunological exposure in patients. Wesynthesized a baculovirus transfer vector with the polyhedrinpromoter followed sequentially from the 5' end with DNA encodingthe gp67A leader sequence, the tripeptide ADP, IL-2 (interleukin-2),another ADP tripeptide and RTB (ricin toxin B chain) with lectinsitemutations W37S and Y248H. Recombinant baculovirus was generatedin Sf9 insect cells and used to infect Sf9 cells. RecombinantIL-2-RTB[W37S/Y248H] protein (fusion protein of IL-2 with modificationsW37S and Y248H) was recovered at high yields from day 6 insectcell supernatants, partially purified by affinity chromatographyand reassociated with RTA (ricin toxin A chain). The fusiontoxin was soluble, immunoreactive with antibodies to RTB, LL-2and RTA and had a molecular weight of 80 kDa by SDS-PAGE. Themolecule reacted poorly with asialofetuin, but bound stronglyto IL-2 receptor based on selective cytotoxicity to IL-2 receptorbearing cells. The specific cytotoxicity could be blocked withIL-2 but not lactose. Thus, we report a novel targeted fusiontoxin protein with full biological activity.     

X-ray structures of two single-residue mutants of DNase I: H134Q and Y76A

The structures of the single-residue mutants H134Q and Y76Aof bovine pancreatic DNase I have been determined and refinedincluding data to 2.3 and 2.4 Å resolution respectively,by X-ray crystallography. H134 is an essential catalytic residue,while Y76 contributes to the binding of DNA by providing a largevan der Waals contact area that stabilizes the wide minor grooveseen in DNase I-DNA complexes. The mutant proteins, which showstrongly reduced activities of 0.001% (H134Q) and 0.3% (Y76A),were expressed in E.coli and both crystallize in space-groupC2 with almost identical unit cells. The crystal packing schemeis different from that found in wild type crystals grown undervery similar conditions, presumably due to the absence of thecarbohydrate moiety. In both mutants the conformation of theprotein is nearly identical to that of the wild type enzymeand changes are confined to surface loops involved in packing.The disruption of the hydrogen bonds between H134, E78 and Y76in both mutants leads to an increased mobility and positionalshifts in the DNA-binding loop, mainly around residue Y76. Thisin turn may further reduce DNA-binding affinity and, thus, contributeto the low activity. In contrast, symmetry contacts involvingresidues 97–108 lead to a stabilization of the flexibleloop compared to wild type DNase I.     

Human pancreatic RNase1-human epidermal growth factor fusion: an entirely human 'immunotoxin analog' with cytotoxic properties against squamous cell carcinomas

The gene encoding human pancreatic ribonuclease 1 (hpRNasel) was fused with a gene encoding human epidermal growth factor (hEGF). The hybrid human protein was isolated from Escherichia coli inclusion bodies, refolded and purified to homogeneity. The fusion protein competed with 125I-hEGF for binding to hEGF receptors (EGFR) and had ribonucleolytic activities approaching those of hpRNase1. Several conformations having different enzymatic activities could be detected after reversed-phase high-performance liquid chromatographic analysis, the less hydrophobic molecules being the most active. The hybrid protein was specifically cytotoxic to A431, an EGFR overexpressing squamous carcinoma cell line, with an IC50 of approximately 10(-7) M. In contrast, recombinant hpRNase1 had an IC50 higher than 10(-4) M. A mixture of free hEGF and free hpRNasel was not more cytotoxic than hpRNasel alone and no cytotoxicity was detected in EGFR-deficient control cells. Taken together, these data suggest that this construct might be useful for targeted therapy of esophageal, lung and other squamous cell carcinomas and also breast cancers overexpressing EGFR, which correlate with a poor prognosis and cannot be cured by surgery alone. Engineering hybrid molecules with endogenous human proteins for targeted therapy may alleviate the dose-limiting immunogenicity and toxicity of conventional immunotoxins.      

Chimeric protein for selective cell attachment onto cellulosic substrates

We have developed a fusion protein (CBD-LG) incorporating a cellulose-binding domain and an antibody binding domain, protein LG, to provide an adaptor molecule for cell separation with regenerated cellulose hollow fiber arrays. A single hollow fiber cell adhesion assay utilizing a CD34+ cell line, KG1a, was used to investigate whether ligand affinity interactions were strong enough for cell attachment and separation. CBD-LG efficiently captured CD34+ cells labeled with the mouse IgG2a monoclonal antibody MHCD3400. However, it was not possible to bind CD34+ cells labeled with an IgG1 antibody (HPCA-2). The low affinity of HPCA-2 for LG was overcome by secondary antibodies: KG1a cells that were dual labeled with HPCA-2 followed by rat anti-mouse IgG1 adhered inside hollow fibers coated with CBD-LG. Alternatively, immobilized rabbit polyclonal anti-mouse IgG1 captured cells labeled with HPCA-2. The development of an adaptor molecule to display recombinant domains at the surface of hollow fibers will be an effective tool to investigate cellular ligand-receptor interactions, a necessary step in the development of hollow fiber bioreactors for manufacture of human cellular products.     

Monovalent antibody scFv fragments selected to modulate T-cell activation by inhibition of CD86-CD28 interaction

Beside the interaction of the antigen-presenting major histocompatibility complex with the T-cell receptor, a co-stimulatory signal is required for T-cell activation in an immune response. To reduce immune-mediated graft rejection in corneal transplantation, where topical application of drugs in ointments or eye-drops may be possible, we selected single-chain antibody fragments (scFv) with binding affinity to rat CD86 (B7.2) that inhibit the co-stimulatory signal. We produced the IgV-like domain of rat CD86 as a fusion protein in Escherichia coli by refolding from inclusion bodies. This protein was used as a target for phage display selection of scFv from HuCAL-1, a fully artificial human antibody library. Selected binding molecules were shown to specifically bind to rat CD86 and inhibit the interaction of CD86 with CD28 and CTLA4 (CD152) in flow cytometry experiments. In an assay for CD86-dependent co-stimulation, the selected scFv fragment successfully inhibited the proliferation of T-cells induced by CD86-expressing P815 cells.