A novel recombinant soluble splice variant of Met is a potent antagonist of the hepatocyte growth factor/scatter factor-Met pathway.

PURPOSE: The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), are involved in a wide range of biological activities, including cell proliferation, motility, invasion, and angiogenesis. The HGF/SF-Met signaling pathway is frequently activated in a variety of cancers, and uncontrolled Met activation correlates with highly invasive tumors and poor prognosis. In this study, we investigated the inhibitory effect of a novel soluble splice variant of Met on the HGF/SF-Met pathway. EXPERIMENTAL DESIGN: Using our alternative splicing modeling platform LEADS, we have identified a novel splice variant of the Met receptor, which encodes a truncated soluble form of the receptor. This variant was produced as a recombinant Fc-fused protein named Cgen-241A and was tested in various cell-based assays representing different outcomes of the HGF/SF-Met pathway. RESULTS: Cgen-241A significantly inhibited HGF/SF-induced Met phosphorylation as well as cell proliferation and survival. In addition, Cgen-241A showed a profound inhibitory effect on cell scattering, invasion, and urokinase up-regulation. The inhibitory effects of Cgen-241A were shown in multiple human and nonhuman cell types, representing different modes of Met activation. Furthermore, Cgen-241A showed direct binding to HGF/SF. CONCLUSIONS: Taken together, our results indicate that Cgen-241A is a potent antagonist of the HGF/SF-Met pathway, underlining its potential as a therapeutic agent for the treatment of a wide variety of human malignancies that are dependent on this pathway.

Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction.

Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high-affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. In order to characterize the interaction between different cohesins and dockerins, we have developed matching fusion-protein systems, which harbor either the cohesin or the dockerin component. For this purpose, corresponding plasmid cassettes were designed, which encoded for the following carrier proteins: (i) a thermostable xylanase with an appended His-tag; and (ii) a highly stable cellulose-binding module (CBM). The resultant xylanase-dockerin and CBM-cohesin fusion products exhibited high expression levels of soluble protein. The expressed, affinity-purified proteins were extremely stable, and the functionality of the cohesin or dockerin component was retained. The fusion protein system was used to establish a sensitive and reliable, semi-quantitative enzyme-linked affinity assay for determining multiple samples of cohesin-dockerin interactions in microtiter plates. A variety of cohesin-dockerin systems, which had been examined previously using other methodologies, were revisited applying the affinity-based enzyme assay, the results of which served to verify the validity of the approach.

Cohesin-dockerin interaction in cellulosome assembly: a single Asp-to-Asn mutation disrupts high-affinity cohesin-dockerin binding.

The cohesive cellulosome complex is sustained by the high-affinity cohesin-dockerin interaction. In previous work, we demonstrated that a single Thr-to-Leu replacement in the Clostridium thermocellum dockerin component differentiates between non-recognition and high-affinity recognition by the interspecies rival cohesin from C. cellulolyticum. In this report, we show that a single Asp-to-Asn substitution on the cohesin counterpart also disrupts normal recognition of the dockerin. The Asp34 carboxyl group of the cohesin appears to play a central role in the resultant hydrogen-bonding network as an acceptor of two crucial hydrogen bonds from Ser45 of the dockerin domain. The results underscore the fragile nature of the intermolecular contact interactions that maintain this very high-affinity protein--protein interaction.

Pinpoint mapping of recognition residues on the cohesin surface by progressive homologue swapping.

The high affinity cohesin-dockerin interaction dictates the suprastructural assembly of the multienzyme cellulosome complex. The connection between affinity and species specificity was studied by exploring the recognition properties of two structurally related cohesin species of divergent specificity. The cohesins were examined by progressive rounds of swapping, in which corresponding homologous stretches were interchanged. The specificity of binding of the resultant chimeric cohesins was determined by enzyme-linked affinity assay and complementary protein microarray. In succeeding rounds, swapped segments were systematically contracted, according to the binding behavior of previously generated chimeras. In the fourth and final round we discerned three residues, reputedly involved in interspecies binding specificity. By replacing only these three residues, we were able to convert the specificity of the resultant mutated cohesin, which bound preferentially to the rival dockerin with approximately 20% capacity of the wild-type interaction. These residues represent but 3 of the 16 contact residues that participate in the cohesin-dockerin interaction. This approach allowed us to differentiate, in a structure-independent fashion, between residues critical for interspecies recognition and binding residues per se.