Novel ubiquitin-derived high affinity binding proteins with tumor targeting properties.

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.

Dihydrolipoamide dehydrogenase of Pseudomonas aeruginosa is a surface-exposed immune evasion protein that binds three members of the factor H family and plasminogen.

The opportunistic human pathogen Pseudomonas aeruginosa causes a wide range of diseases. To cross host innate immune barriers, P. aeruginosa has developed efficient strategies to escape host complement attack. In this study, we identify the 57-kDa dihydrolipoamide dehydrogenase (Lpd) as a surface-exposed protein of P. aeruginosa that binds the four human plasma proteins, Factor H, Factor H-like protein-1 (FHL-1), complement Factor H-related protein 1 (CFHR1), and plasminogen. Factor H contacts Lpd via short consensus repeats 7 and 18-20. Factor H, FHL-1, and plasminogen when bound to Lpd were functionally active. Factor H and FHL-1 displayed complement-regulatory activity, and bound plasminogen, when converted to the active protease plasmin, cleaved the chromogenic substrate S-2251 and the natural substrate fibrinogen. The lpd of P. aeruginosa is a rather conserved gene; a total of 22 synonymous and 3 nonsynonymous mutations was identified in the lpd gene of the 5 laboratory strains and 13 clinical isolates. Lpd is surface exposed and contributes to survival of P. aeruginosa in human serum. Bacterial survival was reduced when Lpd was blocked on the surface prior to challenge with human serum. Similarly, bacterial survival was reduced up to 84% when the bacteria was challenged with complement active serum depleted of Factor H, FHL-1, and CFHR1, demonstrating a protective role of the attached human regulators from complement attack. In summary, Lpd is a novel surface-exposed virulence factor of P. aeruginosa that binds Factor H, FHL-1, CFHR1, and plasminogen, and the Lpd-attached regulators are relevant for innate immune escape and most likely contribute to tissue invasion.

Regulation of the Ca(2+) channel TRPV6 by the kinases SGK1, PKB/Akt, and PIKfyve.

The serum- and glucocorticoid-inducible kinase SGK1 and the protein kinase PKB/Akt presumably phosphorylate and, by this means, activate the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3), which has in turn been shown to regulate transporters and channels. SGK1-regulated channels include the Ca(2+) channel TRPV6, which is expressed in a variety of epithelial and nonepithelial cells including tumor cells. SGK1 and protein kinase B PKB/Akt foster tumor growth. The present study thus explored whether TRPV6 is regulated by PIKfyve. TRPV6 was expressed in Xenopus laevis oocytes with or without additional coexpression of constitutively active (S422D)SGK1, constitutively active (T308D,S473D)PKB, wild-type PIKfyve, and (S318A)PIKfyve lacking the SGK1 phosphorylation site. TRPV6 activity was determined from the current (I(Ca)) resulting from TRPV6-induced Ca(2+) entry and subsequent activation of Ca(2+)-sensitive endogenous Cl(-) channels. TRPV6 protein abundance in the cell membrane was determined utilizing immunohistochemistry and Western blotting. In TRPV6-expressing oocytes I(H) was increased by coexpression of (S422D)SGK1 and by (T308D,S473D)PKB. Coexpression of wild-type PIKfyve further increased I(H) in TRPV6 + (S422D)SGK1-expressing oocytes but did not significantly modify I(Ca) in oocytes expressing TRPV6 alone. (S318A)PIKfyve failed to significantly modify I(Ca) in the presence and absence of (S422D)SGK1. (S422D)SGK1 increased the TRPV6 protein abundance in the cell membrane, an effect augmented by additional expression of wild-type PIKfyve. We conclude that PIKfyve participates in the regulation of TRPV6.

Repression by Fur is not the main mechanism controlling the iron-inducible isiAB operon in the cyanobacterium Synechocystis sp. PCC 6803.

The iron deficiency-dependent regulation of isiAB transcription in Synechocystis sp. PCC 6803 was analyzed by fusion of modified isiAB promoter fragments to gfp and in vivo quantification of Gfp fluorescence. For the putative Fur box only a slight repressing impact on promoter activity could be shown. In a heteroallelic fur mutant a corresponding incomplete repression of isiAB transcription under iron-replete conditions confirmed the role of Fur in isiAB regulation. However, a 90 bp region upstream of the putative -35 box of the isiAB promoter was essential for full promoter activity under iron-deplete conditions. This pattern indicates a dual promoter regulation by both a repressing mechanism exhibited via the Fur system and an unknown activating mechanism.

Indication for a new lipolytic enzyme family: isolation and characterization of two esterases from a metagenomic library.

We have isolated several novel esterase genes from a sheep rumen metagenomic library using the activity-based cluster screening approach as a highly efficient screening technology. The two most remarkable esterase genes, designated estGK1 and estZ3, were further examined. Sequence analysis of estGK1 and estZ3 revealed that they encoded proteins covering 322 and 317 amino acids, respectively. Both proteins were biochemically characterized. EstGK1 and EstZ3 have only minor overall sequence similarity to known esterases. We propose that, together with other hypothetical enzymes, they constitute a new family of lipolytic enzymes. EstGK1 harbors the catalytic serine in the conserved pentapeptide GHSQG, which is typical for lipases, whereas EstZ3 and several other hypothetical proteins contain the pentapeptide SHSQG, a new variation of the conserved motif in lipolytic enzyme families.

Immune evasion of the human pathogenic yeast Candida albicans: Pra1 is a Factor H, FHL-1 and plasminogen binding surface protein.

The pathogenic yeast Candida albicans utilizes human complement regulators, like Factor H and Factor H like protein-1 (FHL-1) for immune evasion. By screening a C. albicans cDNA expression library, we identified the pH-regulated antigen 1 (Pra1) as a novel Factor H and FHL-1 binding protein. Consequently Pra1 was recombinantly expressed in Pichia pastoris and purified from culture supernatant. Recombinant Pra1 binds Factor H, FHL-1 and also plasminogen. Attached to Pra1, the three human proteins are functionally active. Factor H and FHL-1 inactivate complement and plasminogen can be activated to plasmin which then degrades the extra-cellular matrix component fibrinogen. Polyclonal Pra1 anti-serum was generated and used to localize Pra1 on the surface and also in the culture supernatant of both yeast cells and the hyphal form of C. albicans. Furthermore Pra1 expression was up-regulated upon induction of hyphal growth. Pra1, released by Candida cells binds back to the surface of Candida hyphae and in addition enhances the complement regulatory activity of Factor H in the fluid phase. A Pra1 overexpression strain, with about twofold higher levels of Pra1 on the surface binds more Factor H, and plasminogen. In summary, C. albicans Pra1 is a yeast immune evasion protein that binds host immune regulators and acts at different sites. As a surface protein, Pra1 acquires the two human complement regulators Factor H, FHL-1 and plasminogen, mediates complement evasion, as well as extra-cellular matrix interaction and/or degradation. As a released protein, Pra1 enhances complement control in direct vicinity of the yeast and thus generates an additional protective layer which controls host complement attack.

The host immune regulator factor H interacts via two contact sites with the PspC protein of Streptococcus pneumoniae and mediates adhesion to host epithelial cells.

Pneumococcal surface protein C (PspC) of Streptococcus pneumoniae is a key virulence factor that mediates adhesion to host cells and immune evasion of the host complement. PspC binds the host immune and complement regulator factor H, which is composed of 20 short consensus repeats (SCR). This interaction contributes to pneumococcal virulence. In this study, we identified within the factor H protein two separate PspC binding regions, which were localized to SCR8-11 and SCR19-20, by using recombinant factor H deletion constructs for Western blotting assays and surface plasmon resonance studies. A detailed analysis of binding epitopes in these SCR by peptide spot arrays identified several linear binding regions within the sequences of SCR8-11 and SCR19-20. In addition, the factor H binding site was mapped within the pneumococcal PspC protein to a 121-aa-long stretch positioned in the N terminus (residues 38-158). Factor H attached to the surface of pneumococci via PspC significantly enhanced pneumococcal adherence to host epithelial and endothelial cells. This adhesion was specific and was blocked with a truncated N-terminal factor H-binding fragment of PspC. In conclusion, the acquisition of factor H by pneumococci via PspC occurs via two contact sites located in SCR8-11 and SCR19-20, and factor H attached to the surface of the pneumococcus promotes adhesion to both host epithelial and endothelial cells.

Gpm1p is a factor H-, FHL-1-, and plasminogen-binding surface protein of Candida albicans.

The human pathogenic yeast Candida albicans utilizes host complement regulators for immune evasion. Here we identify the first fungal protein that binds Factor H and FHL-1. By screening a protein array of 4088 proteins of Saccharomyces cerevisiae, phosphoglycerate mutase (ScGpm1p) was identified as a Factor H- and FHL-1-binding protein. The homologous C. albicans Gpm1p (CaGpm1p) was cloned and recombinantly expressed as a 36-kDa His-tagged protein. Purified CaGpm1p binds the host complement regulators Factor H and FHL-1, but not C4BP. The CaGpm1p binding regions in the host proteins were localized; FHL-1 binds via short consensus repeats (SCRs) 6 and 7, and Factor H utilizes two contact regions that are located in SCRs 6 and 7 and in SCRs 19 and 20. In addition, recombinant CaGpm1p binds plasminogen via lysine residues. CaGpm1p is a surface protein as demonstrated by immunostaining and flow cytometry. A C. albicans gpm1(-/-) mutant strain was generated that did not grow on glucose-supplemented but on ethanol- and glycerol-supplemented medium. Reduced binding of Factor H and plasminogen to the null mutant strain is in agreement with the presence of additional binding proteins. Attached to CaGpm1p, each of the three host plasma proteins is functionally active. Factor H and FHL-1 show cofactor activity for cleavage of C3b, and bound plasminogen is converted by urokinase-type plasminogen activator to proteolytically active plasmin. Thus, the surface-expressed CaGpm1p is a virulence factor that utilizes the host Factor H, FHL-1, and plasminogen for immune evasion and degradation of extracellular matrices.

Immune evasion of the human pathogen Pseudomonas aeruginosa: elongation factor Tuf is a factor H and plasminogen binding protein.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause a wide range of clinical symptoms and infections that are frequent in immunocompromised patients. In this study, we show that P. aeruginosa evades human complement attack by binding the human plasma regulators Factor H and Factor H-related protein-1 (FHR-1) to its surface. Factor H binds to intact bacteria via two sites that are located within short consensus repeat (SCR) domains 6-7 and 19-20, and FHR-1 binds within SCR domain 3-5. A P. aeruginosa Factor H binding protein was isolated using a Factor H affinity matrix, and was identified by mass spectrometry as the elongation factor Tuf. Factor H uses the same domains for binding to recombinant Tuf and to intact bacteria. Factor H bound to recombinant Tuf displayed cofactor activity for degradation of C3b. Similarly Factor H bound to intact P. aeruginosa showed complement regulatory activity and mediated C3b degradation. This acquired complement control was rather effective and acted in concert with endogenous proteases. Immunolocalization identified Tuf as a surface protein of P. aeruginosa. Tuf also bound plasminogen, and Tuf-bound plasminogen was converted by urokinase plasminogen activator to active plasmin. Thus, at the bacterial surface Tuf acts as a virulence factor and binds the human complement regulator Factor H and plasminogen. Acquisition of host effector proteins to the surface of the pathogen allows complement control and may facilitate tissue invasion.

Factor H and atypical hemolytic uremic syndrome: mutations in the C-terminus cause structural changes and defective recognition functions.

Atypical hemolytic uremic syndrome is a disease that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Mutations in the complement regulator factor H are associated with the inherited form of the disease, and >60% of the mutations are located within the C terminus of factor H. The C-terminus of factor H, represented by short consensus repeat 19 (SCR19) and SCR20, harbors multiple functions; consequently, this study aimed to examine the functional effects of clinically reported mutations in these SCR. Mutant factor H proteins (W1157R, W1183L, V1197A, R1210C, R1215G, and P1226S) were recombinantly expressed and functionally characterized. All six mutant proteins showed severely reduced heparin, C3b, C3d, and endothelial cell binding. By peptide spot analyses, four linear regions that are involved in heparin, C3b, and C3d binding were localized in SCR19 and SCR20. A three-dimensional homology model of the two domains suggests that these four regions form a common binding site across both domains. In addition, this structural model identifies two types of residues: Type A residues are positioned on the SCR surface and are represented by mutants W1157R, W1183L, R1210C, and R1215G; and type B residues are buried within the SCR structure and affect mutations V1197A and P1226S. Mutations of both types of residue result in the same functional defects, namely the reduced binding of factor H to surface-attached C3b molecules and reduced complement regulatory activity at the cell surfaces. The buried type B mutations seem to affect ligand interaction of factor H more severely than the surface-exposed mutations.

Two factor H-related proteins from the mouse: expression analysis and functional characterization.

Complement factor H-related (FHR) proteins display structural and functional similarities to each other and to the complement regulator factor H (FH). FHRs have been identified in various species, including human, rat, and the fish barred sand bass. As mice provide a useful model system to study the physiological role of FHRs in vivo, we aimed at characterizing murine FHR proteins. Two putative FHRs of approximately 100 and 38 kDa were detected in mouse plasma using FH-specific antiserum. In a liver cDNA library, three murine FHR-encoding transcripts were identified. Two clones code for related FHR proteins termed FHR-C and FHR-C_v1, which in secreted form are composed of 14 and 13 short consensus repeat (SCR) domains, homologous to SCRs 6-17 and 19-20 of FH. The third transcript, FHR-B, is derived from a separate gene and codes for a secreted protein composed of five SCR domains. FHR-B displays homology to SCRs 5-7 and 19-20 of FH. Expression of FHR-B in various tissues was analyzed by real-time polymerase chain reaction and was identified at high levels in liver, kidney and heart. In liver, FHR-B transcript level was even higher than that of FH. In addition, FHR-B was expressed as a recombinant 37-kDa protein, and this recombinant FHR-B interacted with the ligands heparin and human C3b. Using mouse plasma, the native presumptive FHR proteins were also analyzed in binding assays. In summary, we identify two FHR proteins in mice and for the first time characterize a murine FHR as a heparin- and C3b-binding protein.