Alexander Wentzel

The potential of cystine-knot microproteins as novel pharmacophoric scaffolds in oral peptide drug delivery

Within this study, the potential of three clinically relevant microproteins (SE–AG–AZ, SE–EM and SE–EP) with cystine-knot architecture as pharmacophoric scaffolds for oral peptide delivery was investigated. Cystine-knot microproteins (CKM) were analysed regarding their stability towards the most important gastrointestinal secreted and membrane bound proteases in physiological concentrations. In addition, their permeation behaviour through freshly excised rat intestinal mucosa as well as important parameters such as aggregation behaviour, stability in rat plasma and isoelectric point were evaluated and compared to the properties of the model peptide drugs bacitracin and insulin. Aggregation studies indicate that under physiological conditions between 25 and 70% of the CKMs occur as monomers, whereas the rest forms di- and trimers. Pepsin and elastase cause no or only minor degradation to CKMs, whereas trypsin and chymotrypsin degrade CKMs extensively. Removing the theoretical chymotrypsin cleavage site from a CKM, however, led to stabilization towards this protease. Two of the three evaluated CKMs are stable against membrane bound proteases. Papp values were determined to be 5.96 ± 0.98 × 10− 6 and 6.63 ± 0.47 × 10− 6 cm/s. In conclusion, this study indicates that CKM are promising novel pharmacophoric scaffolds for oral peptide delivery.

A generic system for the Escherichia coli cell-surface display of lipolytic enzymes.

EstA is an outer membrane‐anchored esterase from Pseudomonas aeruginosa. An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell‐surface display of lipolytic enzymes. Flow cytometry analysis and measurement of lipase activity revealed that Bacillus subtilis lipase LipA, Fusarium solani pisi cutinase and one of the largest lipases presently known, namely Serratia marcescens lipase were all efficiently exported by the EstA autotransporter and also retained their lipolytic activities upon cell surface exposition. EstA provides a useful tool for surface display of lipases including variant libraries generated by directed evolution thereby enabling the identification of novel enzymes with interesting biological and biotechnological ramifications.

Inhibition of platelet aggregation by grafting RGD and KGD sequences on the structural scaffold of small disulfide-rich proteins

Disintegrins represent a group of disulfide-rich peptides ranging in size from 41 to over 80 residues and are antagonists of several integrin receptors. Disintegrins containing an RGD or KGD sequence are potent inhibitors of platelet aggregation as they block the binding of fibrinogen to αIIbβ3 integrin. The high affinity binding to αIIbβ3 in comparison to short linear peptides has been attributed to the localisation of the RGD or KGD sequence within a defined three-dimensional structure. Cystine knot microproteins are members of another family of small disulfide-rich peptides that consist of only 28–40 amino acid residues. They display numerous biological activities depending on the peptide sequence of loop regions that are fixed on a structural scaffold that is stabilised by three knot-forming disulfide bonds. In the present study we grafted RGD and KGD containing peptide sequences with seven and 11 amino acids, respectively, into two cystine knot microproteins, the trypsin inhibitor EETI-II and the melanocortin receptor binding domain of the human agouti-related protein AGRP, as well as into the small disintegrin obtustatin. The engineered proteins were much more potent to inhibit the fibrinogen binding, αIIbβ3 activation and platelet aggregation when compared to the grafted peptides. Differences that were observed between the engineered proteins indicate the importance of the structural scaffold and the amino acids neighbouring the grafted peptide sequences.

Sequence requirements of the GPNG beta-turn of the Ecballium elaterium trypsin inhibitor II explored by combinatorial library screening.

The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot. Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure. Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22–25 comprising a β-turn in native EETI-II is a possible candidate for a folding initiation site. We generated a molecular repertoire of EETI-II variants with variegated 22–25 tetrapeptide sequences and presented these proteins on the outer membrane ofEscherichia coli cells via fusion to the Igaβautotransporter. Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting. At least 1–5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides. Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in β-turns. The folding pathway of two selected variants, EETI-βNEDE and EETI-βTNNK, was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate. Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture.

Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA

Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.

Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries

We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed bacterial surface display vector. A carboxyterminally truncated intimin, an adhesin from enteropathogenic E. coli, serves as a carrier protein to present foreign peptides on the surface of E. coli K12 cells. Epitope-presenting cells were isolated by immunofluorescence staining of the bacterial cell population with monoclonal anti-E(rns) antibodies followed by fluorescence-activated cell sorting (FACS). Nucleotide sequence analysis of the coding sequence for the cloned target gene fragments of a few FACS-positive clones allowed the identification of the respective epitope sequence. A major linear antigenic determinant of the E(rns) protein could be identified by epitope mapping with a polyclonal anti-E(rns) serum. Furthermore, the high-density surface display of intimin-peptide fusions allowed us to use epitope-presenting bacteria directly as whole cell adsorbants for affinity purification of monospecific antibodies. Monospecific antibodies directed against the carboxyterminal fragment of E(rns) were isolated and used for immunostaining of transfected BHK-21 cells to validate the transient expression of E(rns). This demonstrates that gene-fragment libraries displayed on E. coli cells as fusion proteins with intimin are useful tools for rapid mapping of linear epitopes recognized by monoclonal antibodies (MAbs) and polyclonal sera and for the affinity purification of monospecific antibodies by adsorption to the E. coli surface exposed antigenic peptide.

Grafting of thrombopoietin‐mimetic peptides into cystine knot miniproteins yields high‐affinity thrombopoietin antagonists and agonists

Thrombopoietin is the primary regulator of platelet production. We exploited two naturally occurring miniproteins of the inhibitor cystine knot family as stable and rigid scaffolds for the incorporation of peptide sequences that have been shown to act as high‐affinity thrombopoietin antagonists. Several miniproteins that antagonistically block thrombopoietin‐mediated receptor activation were identified using a microscale reporter assay. Covalent miniprotein dimerization yielded potent bivalent c‐Mpl receptor agonists with EC50 values in the low nanomolar or picomolar range. One selected miniprotein‐derived thrombopoietin agonist was almost as active as natural thrombopoietin with regard to stimulation of megakaryocyte colony formation from human bone marrow mononuclear cells, and elicited doubling of platelet counts in mice. Our data suggest that dimeric cystine knot miniproteins have considerable potential for the future development of small and stable receptor agonists. This approach may provide a promising strategy for pharmaceutical interference with other receptors activated by ligand‐induced dimerization.

Structure of Ecballium Elaterium Trypsin Inhibitor II (Eeti-II): A Rigid Molecular Scaffold

The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold.

Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation

Intimins from pathogenic bacteria promote intimate bacterial adhesion to epithelial cells. Several structurally similar domains form on the bacterial cell surface an extended rigid rod that exposes the carboxy-terminal domain, which interacts with the translocated intimin receptor. We constructed a series of intimin-derived fusion proteins consisting of carboxy-terminally truncated intimin and the immunoglobulin light-chain variable domain REIv, ubiquitin, calmodulin, beta-lactamase inhibitor protein, or beta-lactamase. By systematically investigating the intimin-mediated cell surface exposure of these passenger domains in the presence or absence of compounds that interfere with outer membrane stability or passenger domain folding, we acquired experimental evidence that intimin-mediated protein export across the outer membrane requires, prior to export, the maintenance of a translocation-competent conformation that may be distinct from the final protein structure. We propose that, during export, competition exists between productive translocation and folding of the passenger domain in the periplasm into a stable conformation that is not compatible with translocation through the bacterial outer membrane. These results may expand understanding of the mechanism by which intimins are inserted into the outer membrane and expose extracellular domains on the cell surface.

Head-to-Tail Cyclized Cystine-Knot Peptides by a Combined Recombinant and Chemical Route of Synthesis

Cyclic peptides form an important class of naturally occurring or synthetic compounds with a large variety of biological activities as, for example, hormones, ion carriers, cancerostatics, antibiotics, antimycotics, or toxins. Biological studies with cyclopeptides have often indicate increased metabolic stability, improved receptor selectivity, and improved activity profiles in comparison with their linear counterparts. Among the group of natural circular peptides and proteins isolated in the last few years from microorganisms, plants, and even from humans, cyclotides provide an especially interesting topology. This family of circular plant proteins displays a head-totail cyclized peptide backbone together with a cystine knot (CK) motif based on disulfide bonds formed by six conserved Cys residues (Figure 1). Two disulfide bonds and their connecting backbone segments form a ring that is penetrated by the third disulfide bond to give a pseudo-knot structure that is ACHTUNGTRENNUNGinvariably associated with the nearby b sheet structure. The cystine knot in combination with the cyclic backbone appears to be a highly efficient motif for structure stabilization, resulting in exceptional conformational rigidity, together with stability against denaturing conditions, as well as against proteolytic degradation. CK-containing peptides are found in almost 20 different protein families with activities such as ion channel blocking (conotoxins and spider toxins), protease inhibition (squash inhibitors), and antiinsecticidal activity (plant cyclotides). Head-to-tail macrocyclic cystine knot peptides have been isolated from plants in the Rubiaceae, Violaceae, and Cucurbitaceae families. Several members of these family have been introduced as versatile scaffolds in drug design and biomolecular engineering. Because of their sizes, in the range of 30–40 amino acids, ACHTUNGTRENNUNGcyclotides are amenable both to recombinant production through bacterial expression and to chemical synthesis. In both routes, two steps of post-synthetic processing—oxidation of six cysteines to form three disulfide bonds and head-to-tail cyclization—are required to obtain the final cyclic product. Although the processes by which cyclotide backbone cyclization occurs naturally are largely unknown, two major strategies have been applied to generate synthetic macrocyclic CK peptides. The first approach relies on recombinant synthesis and makes use of modified protein splicing elements known as inteins to form a C-terminal thioester that reacts with the N terminus to result in macrocyclization. The second strategy is based on a solid-phase synthesis of the target peptide, followed by oxidation and cyclization. Fully deprotected peptides have successfully been “zipped” into macrocycles, followed by oxidation and cystine knot formation. Here we present a strategy for the backbone cyclization of already folded miniproteins based on the formation of a stable hydrazone. This method takes advantage of the combination of cheap and high-yielding recombinant production of linear peptide precursors that are already folded and oxidized. Chemical synthesis efficiently provides the artificial linkage of the termini, not interfering with the fold of the knotted motif stabilized by disulfide bonds. In a comparison of linear and cyclized derivatives, an increased efficiency in tryptase inhibition is reported for a representative iminocyclotide; this also indicates Figure 1. Structure of the cystine knot peptide McoEeTI. b Strand secondary structure elements are indicated as arrows. The disulfide bonds forming the cystine knot architecture are indicated as sticks; cysteine residues are numbered from I to VI beginning from the N terminus. The macrocycleforming loop was tentatively added and is shown as a dashed line.