Cell-Based Sensors for the Detection of EGF and EGF-Stimulated Ca2+ Signaling.

Epidermal growth factor (EGF)-mediated activation of EGF receptors (EGFRs) has become an important target in drug development due to the implication of EGFR-mediated cellular signaling in cancer development. While various in vitro approaches are developed for monitoring EGF-EGFR interactions, they have several limitations. Herein, we describe a live cell-based sensor system that can be used to monitor the interaction of EGF and EGFR as well as the subsequent signaling events. The design of the EGF-detecting sensor cells is based on the split-intein-mediated conditional protein trans-cleavage reaction (CPC). CPC is triggered by the presence of the target (EGF) to activate a signal peptide that translocates the fluorescent cargo to the target cellular location (mitochondria). The developed sensor cell demonstrated excellent sensitivity with a fast response time. It was also successfully used to detect an agonist and antagonist of EGFR (transforming growth factor-α and Cetuximab, respectively), demonstrating excellent specificity and capability of screening the analytes based on their function. The usage of sensor cells was then expanded from merely detecting the presence of target to monitoring the target-mediated signaling cascade, by exploiting previously developed Ca2+-detecting sensor cells. These sensor cells provide a useful platform for monitoring EGF-EGFR interaction, for screening EGFR effectors, and for studying downstream cellular signaling cascades.

Intein-Mediated Protein Engineering for Cell-Based Biosensors.

Cell-based sensors provide a flexible platform for screening biologically active targets and for monitoring their interactions in live cells. Their applicability extends across a vast array of biological research and clinical applications. Particularly, cell-based sensors are becoming a potent tool in drug discovery and cell-signaling studies by allowing function-based screening of targets in biologically relevant environments and enabling the in vivo visualization of cellular signals in real-time with an outstanding spatiotemporal resolution. In this review, we aim to provide a clear view of current cell-based sensor technologies, their limitations, and how the recent improvements were using intein-mediated protein engineering. We first discuss the characteristics of cell-based sensors and present several representative examples with a focus on their design strategies, which differentiate cell-based sensors from in vitro analytical biosensors. We then describe the application of intein-mediated protein engineering technology for cell-based sensor fabrication. Finally, we explain the characteristics of intein-mediated reactions and present examples of how the intein-mediated reactions are used to improve existing methods and develop new approaches in sensor cell fabrication to address the limitations of current technologies.

Characterization and identification of essential residues of the glycoside hydrolase family 64 laminaripentaose-producing-ß-1, 3-glucanase.

Laminaripentaose-producing ß-1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of ß-1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to >90% homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residues--Asp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.

Crystal structures of Bacillus cereus NCTU2 chitinase complexes with chitooligomers reveal novel substrate binding for catalysis: a chitinase without chitin binding and insertion domains.

Chitinases hydrolyze chitin, an insoluble linear polymer of N-acetyl-d-glucosamine (NAG)(n), into nutrient sources. Bacillus cereus NCTU2 chitinase (ChiNCTU2) predominantly produces chitobioses and belongs to glycoside hydrolase family 18. The crystal structure of wild-type ChiNCTU2 comprises only a catalytic domain, unlike other chitinases that are equipped with additional chitin binding and insertion domains to bind substrates into the active site. Lacking chitin binding and chitin insertion domains, ChiNCTU2 utilizes two dynamic loops (Gly-67-Thr-69 and Ile-106-Val-112) to interact with (NAG)(n), generating novel substrate binding and distortion for catalysis. Gln-109 is crucial for direct binding with substrates, leading to conformational changes of two loops with a maximum shift of ∼4.6 Šalong the binding cleft. The structures of E145Q, E145Q/Y227F, and E145G/Y227F mutants complexed with (NAG)(n) reveal (NAG)(2), (NAG)(2), and (NAG)(4) in the active site, respectively, implying various stages of reaction: before hydrolysis, E145G/Y227F with (NAG)(4); in an intermediate state, E145Q/Y227F with a boat-form NAG at the -1 subsite, -1-(NAG); after hydrolysis, E145Q with a chair form -1-(NAG). Several residues were confirmed to play catalytic roles: Glu-145 in cleavage of the glycosidic bond between -1-(NAG) and +1-(NAG); Tyr-227 in the conformational change of -1-(NAG); Asp-143 and Gln-225 in stabilizing the conformation of -1-(NAG). Additionally, Glu-190 acts in the process of product release, and Tyr-193 coordinates with water for catalysis. Residues Asp-143, E145Q, Glu-190, and Tyr-193 exhibit multiple conformations for functions. The inhibitors zinc ions and cyclo-(l-His-l-Pro) are located at various positions and confirm the catalytic-site topology. Together with kinetics analyses of related mutants, the structures of ChiNCTU2 and its mutant complexes with (NAG)(n) provide new insights into its substrate binding and the mechanistic action.

Structural simulation and protein engineering to convert an endo-chitosanase to an exo-chitosanase.

To obtain an enzyme for the production of chito-disaccharides (GlcN(2)) by converting endo-chitosanase to exo-chitosanase, we chose an endo-chitosanase from Bacillus circulans MH-K1 (Csn) as the candidate for protein engineering. Using molecular modeling, two peptides with five amino acids (PCLGG) and six amino acids (SRTCKP) were designed and inserted after the positions of D(115) and T(222) of Csn, respectively. The inserted fragments are expected to form loops that might protrude from opposite walls of the substrate-binding cleft, thus forming a 'roof' over the catalytic site that might alter the product specificity. The chimeric chitosanase (Chim-Csn) and wild-type chitosanase (WT-Csn) were both over-expressed in Escherichia coli and purified nearly to homogeneity. The products formed from chitosan were analyzed by ESI-MS (electrospray ionization-mass spectrometry). A mixture of GlcN(2), GlcN(3) and GlcN(4) was obtained with WT-Csn, whereas Chim-Csn formed, with a smaller catalytic rate (3% of WT-Csn activity), GlcN(2) as the dominant product. Measurements of viscosity showed that, with similar amounts of enzyme activity, Chim-Csn catalyzed the hydrolysis of chitosan with a smaller rate of viscosity decrease than WT-Csn. The results indicate that, on inserting two surface loops, the endo-type chitosanase was converted into an exo-type chitosanase, which to our knowledge is the first chitosanase that releases GlcN(2) from chitosan as the dominant product.