Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, ...associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys158-Ile159 (P1-P1′) peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (kcat/Km 4.8 × 105m–1 s–1) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 ± 1.9 nm uPA h–1, which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Stimulation of hepatocyte growth factor (HGF) signaling through the Met receptor is an attractive approach for promoting tissue repair and preventing fibrosis. Using structure-guided peptide phage ...display combined with an activity-based sorting strategy, we engineered allosteric activators of zymogen-like pro-HGF to bypass proteolytic activation and reversibly stimulate pro-HGF signaling through Met. Biochemical, structural and biological data showed that zymogen activator peptides (ZAPtides) potently and selectively bind the activation pocket within the serine protease-like β-chain of pro-HGF and display titratable activation of pro-HGF-dependent Met signaling, leading to cell survival and migration. To further demonstrate the versatility of our ZAPtide platform, we identified allosteric activators for pro-macrophage stimulating protein and a zymogen serine protease, Protein C, which also provides evidence for target selectivity. These studies reveal that ZAPtides use molecular mimicry of the trypsin-like N-terminal insertion mechanism and establish a new paradigm for selective pharmacological activation of plasminogen-related growth factors and zymogen serine proteases.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Apolipoprotein L1 (ApoL1) is a circulating innate immunity protein protecting against trypanosome infection. However, two ApoL1 coding variants are associated with a highly increased risk of chronic ...kidney disease. Here we present X-ray and NMR structures of the N-terminal domain (NTD) of ApoL1 and of its closest relative ApoL2. In both proteins, four of the five NTD helices form a four-helix core structure which is different from the classical four-helix bundle and from the pore-forming domain of colicin A. The reactivity with a conformation-specific antibody and structural models predict that this four-helix motif is also present in the NTDs of ApoL3 and ApoL4, suggesting related functions within the small ApoL family. The long helix 5 of ApoL1 is conformationally flexible and contains the BH3-like region. This BH3-like α-helix resembles true BH3 domains only in sequence and structure but not in function, since it does not bind to the pro-survival members of the Bcl-2 family, suggesting a Bcl-2-independent role in cytotoxicity. These findings should expedite a more comprehensive structural and functional understanding of the ApoL immune protein family.
The aspartic acid residues (Asp) present in the complementarity-determining regions (CDRs) of the light chains of two recombinant monoclonal antibodies (MAbs), MAb I and MAb II, are highly ...susceptible to isomerization due to the presence of glycine residues (Gly) on their C-terminal ends. Asp isomerization in these MAbs leads to formation of the isoaspartate (IsoAsp) and the cyclic imide (Asu) variants of these MAbs. Both MAb I and MAb II, employed in this study, elicit their pharmacological responses through binding human IgE. The formation of the MAb variants as a result of Asp isomerization significantly reduces the binding affinities of these antibodies to IgE, thereby reducing their potencies. Here we report on significant differences in the susceptibility of the MAb I and the MAb II to Asp isomerization. The molecular basis for these differences in rates of Asp isomerization was elucidated. The effect of primary sequence on Asp isomerization was evaluated using pentapeptide models of the MAbs, which included the labile Asp residues and their neighboring amino acid residues. The separation of the parent MAbs and pentapeptides from their isomerization products was achieved using hydrophobic interaction chromatography (HIC) and rp-HPLC, respectively. Structural characterization of the MAbs was performed using differential scanning calorimetry (DSC), circular dichroism (CD), and X-ray crystallography. Our investigations demonstrate that the differences in the Asp isomerization rates between MAb I and MAb II can be attributed to structural factors including the conformational flexibility and the extent of solvent exposure of the labile Asp residue.
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IJS, KILJ, NUK, PNG, UL, UM
IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases. Although neutralization of serum IgE with IgE-specific antibodies is in general an efficacious ...treatment for allergic asthma, one limitation of this approach is its lack of effect on IgE production. Here, we have developed a strategy to disrupt IgE production by generating monoclonal antibodies that target a segment of membrane IgE on human IgE-switched B cells that is not present in serum IgE. This segment is known as the M1' domain, and using genetically modified mice that contain the human M1' domain inserted into the mouse IgE locus, we demonstrated that M1'-specific antibodies reduced serum IgE and IgE-producing plasma cells in vivo, without affecting other immunoglobulin isotypes. M1'-specific antibodies were effective when delivered prophylactically and therapeutically in mouse models of immunization, allergic asthma, and Nippostrongylus brasiliensis infection, likely by inducing apoptosis of IgE-producing B cells. In addition, we generated a humanized M1'-specific antibody that was active on primary human cells in vivo, as determined by its reduction of serum IgE levels and IgE plasma cell numbers in a human PBMC-SCID mouse model. Thus, targeting of human IgE-producing B cells with apoptosis-inducing M1'-specific antibodies may be a novel treatment for asthma and allergy.
Engineered antibody paratopes with limited sequence diversity permit assessment of the roles played by different amino acid side chains in creating the high-affinity, high-specificity interactions ...characteristic of antibodies. We describe a paratope raised against the human ErbB family member HER2, using a binary diversity tryptophan/serine library displayed on phage. Fab37 binds to the extracellular domain of HER2 with sub-nanomolar affinity. An X-ray structure at 3.2 Å resolution reveals a contact paratope composed almost entirely of tryptophan and serine residues. Mutagenesis experiments reveal which of these side chains are more important for direct antigen interactions and which are more important for conformational flexibility. The crystal lattice contains an unprecedented trimeric arrangement of HER2 closely related to previously observed homodimers of the related epidermal growth factor receptor.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Non-immune (naïve) antibody phage libraries have become an important source of human antibodies. The synthetic phage antibody library described here utilizes a single human framework with a template ...containing human consensus complementarity-determining regions (CDRs). Diversity of the libraries was introduced at select CDR positions using tailored degenerate and trinucleotide codons that mimic natural human antibodies. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 semaphorins, is expressed on endothelial cells and neurons. NRP1 is required for vascular development and is expressed widely in the developing vasculature. To investigate the possibility of function blocking antibodies to NRP1 as potential therapeutics, and study the consequence of targeting NRP1 in murine tumor models, panels of antibodies that cross-react with human and murine NRP1 were generated from a designed antibody phage library. Antibody (YW64.3) binds to the CUB domains (a1a2) of NRP1 and completely blocks Sema3A induced neuron collapse; antibody (YW107.4.87) binds to the coagulation factor V/VIII domains (b1b2) of NRP1 and blocks VEGF binding and VEGF induced cell migration. YW107.4.87 inhibits tumor growth in animal xenograft models. These antibodies have provided valuable tools to study the roles of NRP1 in vascular and tumor biology.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
To better understand how the relatively flat antigen-combining sites of antibodies interact with the concave shaped substrate-binding clefts of proteases, we determined the structures of two ...antibodies in complex with the trypsin-like hepatocyte growth-factor activator (HGFA). The two inhibitory antibodies, Ab58 and Ab75, were generated from a human Fab phage display library with synthetic diversity in the three complementarity determining regions (H1, H2, and H3) of the heavy chain, mimicking the natural diversity of the human Ig repertoire. Biochemical studies and the structures of the Fab58:HGFA (3.5-Å resolution) and the Fab75:HGFA (2.2-Å resolution) complexes revealed that Ab58 obstructed substrate access to the active site, whereas Ab75 allosterically inhibited substrate hydrolysis. In both cases, the antibodies interacted with the same protruding element (99-loop), which forms part of the substrate-binding cleft. Ab58 inserted its H1 and H2 loops in the cleft to occupy important substrate interaction sites (S3 and S2). In contrast, Ab75 bound at the backside of the cleft to a region corresponding to thrombin exosite II, which is known to interact with allosteric effector molecules. In agreement with the structural analysis, binding assays with active site inhibitors and enzymatic assays showed that Ab58 is a competitive inhibitor, and Ab75 is a partial competitive inhibitor. These results provide structural insight into antibody-mediated protease inhibition. They suggest that unlike canonical inhibitors, antibodies may preferentially target protruding loops at the rim of the substrate-binding cleft to interfere with the catalytic machinery of proteases without requiring long insertion loops.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Two-in-One antibodies with dual action Fabs Eigenbrot, Charles; Fuh, Germaine
Current opinion in chemical biology,
June 2013, 2013-Jun, 2013-06-00, 20130601, Volume:
17, Issue:
3
Journal Article
Peer reviewed
•Multi-specific antibodies can arise spontaneously.•Dual action Fabs can be engineered in vitro.•Dual action Fabs are non-promiscuous and specific for two distinct antigens.•Two-in-One antibodies may ...have effects not possible with two-antibody combinations.
Mechanisms and biological roles of antibody multi-specificity are topics of high interest. Evidence of conformational flexibility in antigen-combining sites and their utility in the recognition of different antigens appeared two decades ago. In the last three years an appreciation has emerged that recognition of very much more diverse protein antigens is within the scope of multi-specificity and also that this is sometimes, but not always, associated with structural plasticity. The ability to invent non-promiscuous, high affinity antibodies which are ‘specific’ for more than one antigen such as the recently described dual-specific Two-in-One DAF antibodies, provides not only insights into the evolution of antibody specificity but also a promise of powerful medicines fully within the paradigm of targeted therapies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Summary Activation of intracellular mitogenic signal transduction pathways driven by the ErbB family of receptor tyrosine kinases has been implicated in the development and/or progression of a ...variety of cancers. Studies on ErbB receptors in osteosarcoma have focused on HER-2 and have produced conflicting results with few studies evaluating the expression of the epidermal growth factor receptor (EGFR). In this study, we determined the level of expression of EGFR and the mutational status of the EGFR receptor in a subset of osteosarcoma tumor samples as well as in a series of established bone tumor–derived cell lines. EGFR protein expression was detected in the form of strong membranous staining by immunohistochemistry in 21 (57%) of 37 cases analyzed. Six of 12 (50%) osteosarcoma cell lines revealed moderate to high expression levels of EGFR. Two somatic alterations (E829E and R831C) were identified in the cytoplasmic domain of the EGFR gene in 1 of 10 tumor samples. The significance of these findings for the pathobiology of osteosarcomas will be investigated further.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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