Biomedical subjects

Harald Kolmar

Publications and source records attributed to Harald Kolmar.

13 recordsLinked to original sources

PubMed2007

Functional cell-surface display of a lipase-specific chaperone.

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.

Biochemistry↗

PubMed2006

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 EC(50) 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.

Animals↗

PubMed2006

The crystal structure of SdsA1, an alkylsulfatase from Pseudomonas aeruginosa, defines a third class of sulfatases.

Pseudomonas aeruginosa is both a ubiquitous environmental bacterium and an opportunistic human pathogen. A remarkable metabolic versatility allows it to occupy a multitude of ecological niches, including wastewater treatment plants and such hostile environments as the human respiratory tract. P. aeruginosa is able to degrade and metabolize biocidic SDS, the detergent of most commercial personal hygiene products. We identify SdsA1 of P. aeruginosa as a secreted SDS hydrolase that allows the bacterium to use primary sulfates such as SDS as a sole carbon or sulfur source. Homologues of SdsA1 are found in many pathogenic and some nonpathogenic bacteria. The crystal structure of SdsA1 reveals three distinct domains. The N-terminal catalytic domain with a binuclear Zn2+ cluster is a distinct member of the metallo-beta-lactamase fold family, the central dimerization domain ensures resistance to high concentrations of SDS, whereas the C-terminal domain provides a hydrophobic groove, presumably to recruit long aliphatic substrates. Crystal structures of apo-SdsA1 and complexes with substrate analog and products indicate an enzymatic mechanism involving a water molecule indirectly activated by the Zn2+ cluster. The enzyme SdsA1 thus represents a previously undescribed class of sulfatases that allows P. aeruginosa to survive and thrive under otherwise bacteriocidal conditions.

Amino Acid Sequence↗

PubMed2006

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 alpha(IIb)beta(3) integrin. The high affinity binding to alpha(IIb)beta(3) 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, alpha(IIb)beta(3) 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.

Amino Acid Sequence↗

PubMed2006

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. P(app) values were determined to be 5.96 +/- 0.98 x 10(-6) and 6.63 +/- 0.47 x 10(-6) cm/s. In conclusion, this study indicates that CKM are promising novel pharmacophoric scaffolds for oral peptide delivery.

Administration, Oral↗

PubMed2005

Barnase fusion as a tool to determine the crystal structure of the small disulfide-rich protein McoEeTI.

We present a fusion system suited to determine the crystal structure of small disulfide-rich proteins. McoEeTI, a hybrid inhibitor cystine knot microprotein, was produced as a soluble fusion to a catalytically inactive variant of the RNAse barnase in Escherichia coli. Functioning as a versatile tag, barnase facilitated purification, crystallization and high-resolution structure determination. Flexibility of the linker region allows for different relative orientations of barnase and the fusion partner in two crystallographically independent molecules and may thereby facilitate crystal packing. Nevertheless, the linker region is well ordered in both molecules. This system may prove more generally useful to determine the crystal structure of peptides and small proteins.

Amino Acid Sequence↗

PubMed2005

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.

Crystallography, X-Ray↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

A fusion protein system for the recombinant production of short disulfide bond rich cystine knot peptides using barnase as a purification handle.

The inhibitor cystine knot (ICK) structural motif has been found in several small proteins and peptides from plants, insects, marine molluscs, and also in human. It is defined by a triple beta-sheet that is held together by three intramolecular disulfide bonds built by six conserved cysteine residues that generate a highly rigid and stable fold. We describe a procedure for the production of ICK peptides with correct disulfide bond connectivities via expression in Escherichia coli as fusion proteins with an enzymatically inactive variant of the Bacillus amyloliquefaciens RNAse barnase. Barnase directs the fused peptide to the culture medium and the fusion protein can be isolated by combined cation exchange/reverse-phase chromatography. The ICK peptides are released from the barnase expression and purification handle either by cyanogen bromide or by protease cleavage to give pure and correctly folded cystine knot peptides.

Amino Acid Sequence↗

PubMed2005

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.

Adhesins, Bacterial↗

PubMed2005

Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II.

MCoTI-I and MCoTI-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTI-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue #10 within the open-chain variant of MCoTI-II by the non-natural isosteric nucleo amino acid AlaG [beta-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTI-I and MCoTI-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity.

Alanine↗

PubMed2004

Bacteria displaying interleukin-4 mutants stimulate mammalian cells and reflect the biological activities of variant soluble cytokines.

We describe a novel procedure that allows the rapid determination of cytokine activity on cells that express their cognate receptor. The four-helix bundle cytokine interleukin-4 (IL-4) was inducibly expressed as a fusion with the E. coli outer-membrane protein intimin, such that IL-4 was presented on the surfaces of the bacteria. Expression and accessibility of the cytokine on the cell exteriors were monitored by Western blotting and fluorescence microscopy, making use of two epitopes flanking the IL-4 component of the fusion protein. To demonstrate the biological activity of the immobilized cytokine, a Ba/F3-derived cell line stably transfected with both the bipartite human IL-4 receptor and an IL-4-specific luciferase reporter gene construct was employed. Bacterial cells displaying interleukin-4 elicited a specific, dose-dependent response in the reporter cells. Two variants of IL-4 with previously characterized (partial) antagonistic properties were also expressed as membrane-bound fusion proteins and were tested for their activity in the immobilized state. In comparison with bacteria displaying wild-type IL-4, E. coli clones presenting variants IL-4 Y124G and Y124D showed diminished or abolished activity, respectively, on murine reporter cells. The relative signaling potencies of the immobilized IL-4 variants thus closely mirror the agonistic properties of the corresponding soluble cytokines. This approach should be generally applicable for the mutational analysis of numerous signal mediators that trigger cellular responses through dimerization of transmembrane receptors.

Animals↗

PubMed2004

Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts.

Enzyme libraries displayed on the surface of microbial cells or microbeads can be screened with fluorogenic substrates that provide a physical linkage of the reaction product to the corresponding enzyme. Libraries exceeding 10(9) different variants can be quantitatively analysed and screened by flow cytometry at a rate of 30 000 cells/second. The promise of screening methods based on fluorescence-activated cell sorting for directed enzyme evolution is being realized and significantly improved enzymes have been reported recently.

Bacteria↗