Surface Plasmon Resonance Measurements Research Articles

Abstract 3774: A high-throughput process for the development and characterization of chimeric antigen receptors (CARs)

Abstract INTRODUCTION: Chimeric antigen receptors (CARs) expressed by T cells bind to surface antigens via a single-chain variable fragment (scFv) and are able to elicit potent anti-tumor efficacy. To our knowledge no rapid CAR development platforms exist to facilitate the high-throughput generation and characterization of novel CARs to allow head-to-head comparisons and to determine the influence of scFv properties on CAR T cell activation, phenotype, and persistence. EXPERIMENTAL PROCEDURES: We have established a rapid process for the development of functional novel CARs using fully human antibody phage display, vector systems for bacterial and mammalian expression, as well as high-throughput functional and biochemical characterization assays. We validated individual parts of the workflow using different tumor antigens and completed the entire workflow for one model antigen expressed on the surface of multiple myeloma cells. An initial population of scFvs against the model antigen was selected from a large naïve fully-human antibody phage display library by panning. The reactivity of the polyclonal phage population was then confirmed by time-resolved fluorescence before reformatting into various monoclonal binder formats. We established a high-throughput flow cytometry assay simultaneously determining CAR surface expression and antigen binding. In addition, we adapted protocols for small-scale lentiviral transduction and expansion of primary CAR T cells followed by a sensitive, high-throughput luciferase-based cytotoxicity assay. Finally, in collaboration with Wasatch Microfluidics we performed high-throughput surface plasmon resonance measurements as well as epitope binning of candidate binding domains. RESULTS: We obtained 1,323 scFvs after two rounds of antibody phage selection. The subsequent bacterial screen of 163 clones identified 23 unique monoclonal binders. In a comparison of all 23 binders in different formats we determined that none of the traditional screening formats, including soluble scFv or scFv-Fc fusion constructs, correlated with CAR binding. CAR constructs with high expression and antigen binding were found to have affinities in the low nanomolar range (18-22nM) and evidenced strong killing of human cancer cell lines spontaneously expressing the antigen (53-79% killing at an effector:target ratio of 10:1), but not healthy cells expressing lower levels of the antigen. Importantly, in a murine xenograft model of multiple myeloma we observed specific killing of tumor cells and no overt toxicities. CONCLUSION: We show that our process allows the generation, screening, as well as functional and biochemical characterization of novel CARs within 2 months. Our approach enables researchers to easily carry out head-to-head comparisons allowing comprehensive lead identification and the determination of binding domain properties which may shape CAR T cell function and phenotype. Citation Format: Sabarinath Venniyil Radhakrishnan, Adam Miles, Djordje Atanackovic, Tim Luetkens. A high-throughput process for the development and characterization of chimeric antigen receptors (CARs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3774. doi:10.1158/1538-7445.AM2017-3774

Fabrication and characterization of calix[4]arene Langmuir–Blodgett thin film for gas sensing applications

In this work, the Langmuir–Blodgett (LB) technique was applied onto the different substrates such as quartz, quartz crystals and gold coated glass for fabricating LB thin films with 25,27-(Dipropylmorpholinoacetamido)-26,28-dihydroxycalix[4]arene. The characteristics of the calix[4]arene LB films were evaluated by UV–Vis, atomic force microscopy (AFM), quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) measurements. The mass deposited on the quartz crystal per bilayer was predicted as 2028.80 ng (7.65 ng mm−2) for the calix[4]arene LB film. The sensing abilities of this LB film towards the development of room temperature organic vapor sensing devices were also examined. QCM and SPR systems were fixed to investigate vapor sensing performance of macrocyclic LB films during exposure to volatile organic compounds (VOCs). The macrocyclic LB thin films were more sensitive to dichloromethane than other vapors. The sensitivity and detection limit values of the calix[4]arene QCM sensor to dichloromethane vapor were calculated as 2.023 Hz ppm−1 and 1.482 ppm, respectively. The sensitivities of the calix[4]arene LB films against organic vapors can be explained with the host–guest interaction between the cavity of molecule and the organic vapor molecules. It is determined that the calix[4]arene LB film is more sensitive to dichloromethane than other vapors at room temperature and the calix[4]arene is a promising material serving as a vapor sensing device.

New Approach for Thickness Determination of Solution-Deposited Graphene Thin Films.

Solution processing-based fabrication techniques such as liquid phase exfoliation may enable economically feasible utilization of graphene and related nanomaterials in real-world devices in the near future. However, measurement of the thickness of the thin film structures fabricated by these approaches remains a significant challenge. By using surface plasmon resonance (SPR), a simple, accurate, and quick measurement of the deposited thickness for inkjet-printed graphene thin films is reported here. We show that the SPR technique is convenient and well-suited for the measurement of thin films formulated from nanomaterial inks, even at sub-10 nm thickness. We also demonstrate that the analysis required to obtain results from the SPR measurements is significantly reduced compared to that required for atomic force microscopy (AFM) or stylus profilometer, and much less open to interpretation. The gathered data implies that the film thickness increases linearly with increasing number of printing repetitions. In addition, SPR also reveals the complex refractive index of the printed thin films composed of exfoliated graphene flakes, providing a more rigorous explanation of the optical absorption than that provided by a combination of AFM/profilometer and the extinction coefficient of mechanically exfoliated graphene flakes. Our results suggest that the SPR method may provide a new pathway for the thickness measurement of thin films fabricated from any nanomaterial containing inks.

Carbon monoxide (CO) optical gas sensor based on ZnO thin films

Optical gas sensors provide an alternative over the conventional conductometric gas sensors. Carbon monoxide (CO) gas is generally regarded as one of the most dangerous air pollutants, hence, a room temperature operated CO gas sensor using ZnO sensing film deposited on Au coated prisms has been developed using an indigenously developed surface plasmon resonance (SPR) measurement setup. This system is found to be highly sensitive with very fast response towards a wide concentration range (0.5–100ppm) of CO gas at room temperature and hence pave way for commercial application of an efficient optical CO gas sensor.

A Sequence in the loop domain of hepatitis C virus E2 protein identified in silico as crucial for the selective binding to human CD81.

Hepatitis C virus (HCV) is a species-specific pathogenic virus that infects only humans and chimpanzees. Previous studies have indicated that interactions between the HCV E2 protein and CD81 on host cells are required for HCV infection. To determine the crucial factors for species-specific interactions at the molecular level, this study employed in silico molecular docking involving molecular dynamic simulations of the binding of HCV E2 onto human and rat CD81s. In vitro experiments including surface plasmon resonance measurements and cellular binding assays were applied for simple validations of the in silico results. The in silico studies identified two binding regions on the HCV E2 loop domain, namely E2-site1 and E2-site2, as being crucial for the interactions with CD81s, with the E2-site2 as the determinant factor for human-specific binding. Free energy calculations indicated that the E2/CD81 binding process might follow a two-step model involving (i) the electrostatic interaction-driven initial binding of human-specific E2-site2, followed by (ii) changes in the E2 orientation to facilitate the hydrophobic and van der Waals interaction-driven binding of E2-site1. The sequence of the human-specific, stronger-binding E2-site2 could serve as a candidate template for the future development of HCV-inhibiting peptide drugs.

Protein Trapping in Plasmonic Nanoslit and Nanoledge Cavities: The Behavior and Sensing.

A novel plasmonic nanoledge device was presented to explore the geometry-induced trapping of nanoscale biomolecules and examine a generation of surface plasmon resonance (SPR) for plasmonic sensing. To design an optimal plasmonic device, a semianalytical model was implemented for a quantitative analysis of SPR under plane-wave illumination and a finite-difference time-domain (FDTD) simulation was used to study the optical transmission and refractive index (RI) sensitivity. In addition, total internal reflection fluorescence (TIRF) imaging was used to visualize the migration of fluorescently labeled bovine serum albumin (BSA) into the nanoslits; and fluorescence correlation spectroscopy (FCS) was further used to investigate the diffusion of BSA in the nanoslits. Transmission SPR measurements of free prostate specific antigen (f-PSA), which is similar in size to BSA, were performed to validate the trapping of the molecules via specific binding reactions in the nanoledge cavities. The present study may facilitate further development of single nanomolecule detection and new nanomicrofluidic arrays for effective detection of multiple biomarkers in clinical biofluids.

Surface Plasmon Resonance Studies of the Hybridization Behavior of DNA-Modified Gold Nanoparticles with Surface-Attached DNA Probes

Colloidal gold nanoparticles (AuNPs) have been extensively investigated as amplification tags to improve the sensitivity of surface plasmon resonance (SPR) biosensors. When using the so-called AuNP-enhanced SPR technique for DNA detection, the density of single-stranded DNA (ssDNA) on both the AuNPs and planar gold substrates is of crucial importance. Thus, in this work, we carried out a systematical study about the influence of surface ssDNA density onto the hybridization behavior of various DNA-modified AuNPs (DNA-AuNPs) with surface-attached DNA probes by using surface plasmon resonance spectroscopy. The lateral densities of the ssDNA on both the AuNPs and planar gold substrates were controlled by using different lengths of oligo-adenine sequence (OAS) as anchoring group. Besides SPR measurements, the amount of the captured DNA-AuNPs after the hybridization was further identified via atomic force microscope (AFM). SPR and AFM results clearly indicated that a higher ssDNA density on either the AuNPs or the gold substrates would give rise to better hybridization efficiency. Moreover, SPR data showed that the captured DNA-AuNPs could not be removed from SPR sensor surfaces using various dehybridization solutions regardless of surface ssDNA density. Consequently, it is apparent that the hybridization behavior of DNA-AuNPs was different from that of solution-phase ssDNA. Based on these data, we hypothesized that both multiple recognitions and limited accessibility might account for the hybridization of DNA-AuNPs with surface-attached ssDNA probes.

Alkylation of phosphorothioated thrombin binding aptamers improves the selectivity of inhibition of tumor cell proliferation upon anticoagulation

BackgroundRecently, aptamers have been extensively researched for therapy and diagnostic applications. Thrombin-binding aptamer is a 15nt deoxyribonucleic acid screened by SELEX, it can specifically bind to thrombin and inhibit blood coagulation. Since it is also endowed with excellent antitumor activity, the intrinsic anticoagulation advantage converted to a main potential side effect for its further application in antiproliferative therapy. MethodsSite-specific alkylation was conducted through nucleophilic reaction of phosphorothioated TBAs using bromide reagents. Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements were used to evaluate anticoagulation activity, and a CCK-8 assay was used to determine cell proliferation activity. ResultsThe CD spectra of the modified TBAs were weakened, and their affinity for thrombin was dramatically reduced, as reflected by the KD values. On the other hand, their inhibition of A549 cells was retained. ConclusionsIncorporation of different alkyls apparently disrupted the binding of TBA to thrombin while maintaining the antitumor activity. General significanceA new modification strategy was established for the use of TBA as a more selective antitumor agent.

Reversible assembly of protein-DNA nanostructures triggered by mediated electron transfer

Stable protein-DNA nanostructures have been assembled by reconstitution of the multi-ligand binding flavoprotein dodecin on top of flavin-terminated dsDNA monolayers on gold electrodes. These structures could be disassembled by electrochemical flavin reduction via mediated electron transfer. For this purpose a negative potential was applied at the Au working electrode in the presence of the redox mediator bis-(ammoniumethyl)-4,4′-bipyridinium tetrabromide. The stepwise formation of the flavin-terminated dsDNA monolayers as well as the binding and electrochemically triggered release of apododecin were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) measurements. The assembly and disassembly of the protein-DNA nanostructures were fully reversible processes, which could be carried out multiple times at the same flavin-dsDNA modified surface. When a negative potential was applied in the absence of a redox mediator apododecin could not be released, i.e. direct electron transfer was not possible. As alternative redox mediators also methylene blue and phenosafranine were studied, but in the presence of these molecules apododecin was released without applying a potential, probably because the tricyclic aromatic compounds are able to replace the flavins at the binding sites.

Sequence-Controlled Glycopolymers via Step-Growth Polymerization of Precision Glycomacromolecules for Lectin Receptor Clustering.

A versatile approach for the synthesis of sequence-controlled multiblock copolymers, using a combination of solid phase synthesis and step-growth polymerization by photoinduced thiol-ene coupling (TEC) is presented. Following this strategy, a series of sequence-controlled glycopolymers is derived from the polymerization of a hydrophilic spacer macromonomer and different glycomacromonomers bearing between one to five α-d-Mannose (Man) ligands. Through the solid phase assembly of the macromonomers, the number and positioning of spacer and sugar moieties is controlled and translates into the sequence-control of the final polymer. A maximum M̅n of 16 kDa, corresponding to a X̅n of 10, for the applied macromonomers is accessible with optimized polymerization conditions. The binding behavior of the resulting multiblock glycopolymers toward the model lectin Concanavalin A (ConA) is studied via turbidity assays and surface plasmon resonance (SPR) measurements, comparing the ability of precision glycomacromolecules and glycopolymers to bind to and cross-link ConA in dependence of the number of sugar moieties and overall molecular weight. The results show that there is a clear correlation between number of Man ligands and Con A binding and clustering, whereas the length of the glycooligomer- or polymer backbone seems to have no effect.

Effect of Probe-Probe Distance on the Stability of DNA Hybrids on Surfaces.

We have used temperature gradient surface plasmon resonance (SPR) measurements to quantitatively evaluate how the stability of different types of hybrids formed with DNA probes on surfaces is affected by probe spacing. SPR sensors with different average surface densities of probes were prepared by coadsorbing probes with lateral spacers strands comprised of phosphorothioated adenine nucleotides (A15*). Increasing the fraction of A15* spacers in the immobilization solution results in larger distances between probes on the sensor, determined here using a combination of SPR and X-ray photoelectron spectroscopy (XPS) measurements. The hybridization activities of probes were simultaneously measured over a temperature range that spanned the denaturation temperature (Tm) of hybrids by applying a spatial temperature gradient across the sensor surface. The resulting temperature profiles of hybridization activity show how the stability of hybrids increases as either the distance between probes or the ionic strength of the hybridization buffer increase. Additionally, hybridization activity profiles sharpen as the spacing between probes increases, indicating more homogeneous hybridization behavior of probes. The results provide quantitative experimental data for testing theoretical models of stability, supporting models that account for both repulsive interactions between DNA strands and local variability in probe surface density.

Use of surface plasmon resonance in the binding study of vitamin D, metabolites and analogues with vitamin D binding protein.

Vitamin D3 and its metabolites are lipophilic molecules with low aqueous solubility and must be transported bound to plasma carrier proteins, primarily to vitamin D binding protein (DBP). The biological functions of vitamin D3 metabolites are intimately dependent on the protein, hence the importance of determining their affinity for DBP. In this study, we developed a novel procedure for measuring the kinetic and equilibrium constants of human-DBP with vitamin D3 and three metabolites: 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], 25-hydroxyvitamin D3 (25OHD3) and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] by surface plasmon resonance (SPR). At the same time, five different analogues, synthetized in our laboratory, were evaluated in order to compare the affinity values with the metabolites. Real-time SPR measurements showed that 25OHD3 and 24,25(OH)2D3 had higher affinity (0.3μM) than 1,25(OH)2D3 (5μM), with the higher affinity of 25OHD3 and 24,25(OH)2D3 due to dissociation constants 1 order of magnitude slower. In the case of the analogues, the affinity values were lower, with 1-hydroxy-25-nitro-vitamin D3 (NO2-446), structurally closer to 1,25(OH)2D3, showing the highest value with a K D of 50μM. (24R)-1,25-dihydroxyvitamin-24-buthyl-28-norvitamin D2 (Bu-471) and (24R)-1,25-dihydroxyvitamin-24-phenyl-28-norvitamin D2 (Ph-491), structurally similar, had affinities of 180 and 170μM, respectively. (22R,23E)-1-hydroxy-22-ethenyl-25-methoxy-23-dehydrovitamin D3 (MeO-455) and 1-hydroxy-20(R)-[5(S)-(2,2-dimethyltetrahydropyran-5-yl)]-22,23-dinor vitamin D3 (Oxan-429) had affinities of 310 and 100μM, respectively. The binding of the metabolites and analogues was reversible allowing the rapid capture of data for replicates. The kinetic and equilibrium data for both the metabolites and the analogues fitted to the Langmuir model describing a 1:1 interaction. Graphical Abstract Label-free, real time binding study between vitamin D binding protein immmobilized on the surface of a SPR sensor chip and the vitamin D, metabolites and analogues passed over it as analytes.

Chip-based digital surface plasmon resonance sensing platform for ultrasensitive biomolecular detection

A chip-based ultrasensitive surface plasmon resonance (SPR) sensor in a checkerboard nanostructure on plastic substrates is presented for digital detection. The sensing elements on the checkerboard are composed of silver-capped nanoslit arrays, which were fabricated using the thermal-embossing nanoimprint method, to meet the demand for low-cost and rapid fabrication. Sharp Fano resonances in the optimized nanoslit arrays provide high-intensity sensitivities (20,000% per refractive index unit), with an element size of 12.5µm. The polarization-dependent transmission in the checkerboard pattern produces optical isolation between sensing elements and results in a crosstalk lower than 1%. Protein–antibody experiments demonstrated that the digital detection limit was up to 1pg/mL, which is approximately 1000 times lower than that of conventional analog detection. For a 140µm×140µm checkerboard pattern, the dynamic range was approximately 100 times higher than that of conventional surface plasmon resonance measurements. This new digital detection method is very useful for detecting ultralow concentrations of analytes with a nonuniform distribution on the sensor surface.

Binding of human plasminogen and high-molecular-mass kininogen by cell surface-exposed proteins of Candida parapsilosis.

Pathogenic microbes can recruit to their cell surface human proteins that are components of important proteolytic cascades involved in coagulation, fibrinolysis and innate immune response. Once located at the bacterial or fungal surface, such deployed proteins might be utilized by pathogens to facilitate invasion and dissemination within the host organism by interfering with functionality of these systems or by exploiting specific activity of the bound enzymes. Aim of the study presented here was to characterize this phenomenon in Candida parapsilosis (Ashford) Langeron et Talice - an important causative agent of systemic fungal infections (candidiases and candidemias) in humans. We have investigated the interactions of fungal surface-exposed proteins with plasminogen (HPG) and high-molecular-mass kininogen (HK) - the crucial components of human fibrinolytic system and proinflammatory/procoagulant contact-activated kinin-forming system, respectively. After confirming ability of the fungal surface-exposed proteins to bind HPG and HK, four of them - two agglutinin-like sequence (Als) proteins CPAR2_404780 and CPAR2_404800, a heat shock protein Ssa2 and a moonlighting protein 6-phosphogluconate dehydrogenase 1 - were purified using ion-exchange chromatography, gel filtration and chromatofocusing. Then, their affinities to HPG and HK were characterized with surface plasmon resonance measurements. The determined dissociation constants for the investigated protein-protein complexes were within a 10-7 M order for the HPG binding and in a range of 10-8-10-9 M for the HK binding. Detailed characterization of adsorption of these two important plasma proteins on the fungal cell surface may help to increase our understanding of molecular mechanisms of C. parapsilosis-dependent candidiasis.

Taste-Masking Effect of Chlorogenic Acid (CGA) on Bitter Drugs Evaluated by Taste Sensor and Surface Plasmon Resonance on the Basis of CGA-Drug Interactions.

The purpose of this study was to evaluate the taste-masking effects of chlorogenic acid (CGA) on bitter drugs using taste sensor measurements and surface plasmon resonance (SPR) analysis of CGA-drug interactions. Six different bitter drugs were used: amlodipine besylate (AMD), diphenhydramine hydrochloride (DPH), donepezil hydrochloride (DNP), rebamipide (RBM), diclofenac sodium (DCF) and etodolac (ETD). Taste sensor outputs were significantly inhibited by the addition of CGA to all drugs. The inhibition ratio of the taste sensor output decreased in the following order DPH>DNP>AMD≈DCF≈RBM≈ETD. The association rate constant (ka) for the interaction between drugs and CGA as evaluated by SPR measurement also decreased in the following order DPH>DNP>AMD>DCF≈ETD≈RBM. It was suggested that basic drugs (AMD, DNP, DPH) associate more easily with CGA than acidic drugs (DCF, RBM, ETD). The inhibition ratios (%) of the taste sensor output of bitter drugs caused by CGA and the association rate constants (ka) between the drugs and CGA were significantly correlated (rs=0.886, p<0.05, Spearman's correlation test). Our findings suggest that the taste-masking effects of CGA are due to its direct association with the drugs. CGA may therefore be a useful taste-masking agent for basic drugs.

Investigating how vesicle size influences vesicle adsorption on titanium oxide: a competition between steric packing and shape deformation.

Understanding the adsorption behavior of lipid vesicles at solid-liquid interfaces is important for obtaining fundamental insights into soft matter adsorbates as well as for practical applications such as supported lipid bilayer (SLB) fabrication. While the process of SLB formation has been highly scrutinized, less understood are the details of vesicle adsorption without rupture, especially at high surface coverages. Herein, we tackle this problem by employing simultaneous quartz crystal microbalance-dissipation (QCM-D) and localized surface plasmon resonance (LSPR) measurements in order to investigate the effect of vesicle size (84-211 nm diameter) on vesicle adsorption onto a titanium oxide surface. Owing to fundamental differences in the measurement principles of the two techniques as well as a mismatch in probing volumes, it was possible to determine both the lipid mass adsorbed near the sensor surface as well as the total mass of adsorbed lipid and hydrodynamically coupled solvent in the adsorbed vesicle layer as a whole. With increasing vesicle size, the QCM-D frequency signal exhibited monotonic behavior reaching an asymptotic value, whereas the QCM-D energy dissipation signal continued to increase according to the vesicle size. In marked contrast, the LSPR-tracked lipid mass near the sensor surface followed a parabolic trend, with the greatest corresponding measurement response occurring for intermediate-size vesicles. The findings reveal that the maximum extent of adsorbed vesicles contacting a solid surface occurs at an intermediate vesicle size due to the competing influences of vesicle deformation and steric packing. Looking forward, such information can be applied to control the molecular self-assembly of phospholipid assemblies as well as provide the basis for investigating deformable, soft matter adsorbates.

Automated Glycan Assembly of Oligo-N-Acetyllactosamine and Keratan Sulfate Probes to Study Virus-Glycan Interactions

Summary Oligo- N -acetyllactosamine (LacNAc) and keratan sulfate (KS) glycans exert crucial functions in disease-relevant processes, including cancer formation, inflammation, and viral infection. To facilitate structure-activity studies with these glycans, we established a universal strategy to synthesize linear and branched LacNAc as well as differentially sulfated KS oligosaccharides by automated glycan assembly. We synthesized oligosaccharides as long as hexamers by combining four monosaccharide building blocks. Key to the strategy was installing three orthogonal protection groups, 9-fluorenylmethoxycarbonyl (Fmoc), levulinoyl (Lev) ester, and 2-naphthylmethyl (Nap) ether, which were selectively removed from a common oligosaccharide precursor for differential sulfation. Microarrays presenting the synthetic oligosaccharides revealed a specific interaction between a disulfated KS tetrasaccharide and the adeno-associated virus AAVrh10 gene-therapy vector, which was further corroborated by surface plasmon resonance studies. Thus, KS represents a novel receptor candidate for AAVrh10. These insights could have implications for cell-type-specific gene-delivery approaches.

Influence of calcium blockers on the SPR of erythrocytes

One of the promising areas of research is the impact of calcium channel blockers (CB) of biological fluids. This paper shows that the CB impact on a biological fluid can be efficiently combine with the surface plasmon resonance (SPR). It is shown that the addition of CB at the SPR measurements affect the stability of membranes and acts differently on the kinetics of erythrocytes ligament in the different groups of people.

Interactions of in vitro selected fluorogenic peptide aptamers with calmodulin.

We examined the importance of aptamer usage under the same condition as the selection process by employing the previously selected aptamers for calmodulin (CaM) which includes a non-natural fluorogenic amino acid, 7-nitro-2,1,3-benzoxadiazole. We added five amino acids at the N-terminus which was employed for the selection and then we tested the affinity and selectivity for CaM binding. Surface plasmon resonance and fluorescence measurements showed that the additional amino acids for one of the aptamers drastically improved binding affinity to CaM, indicating the importance of aptamer use under the same conditions as the selection process. Such drastic improvement in affinity was not observed for the sequence which had been reported previously. Nuclear magnetic resonance data identified that the primary binding site is located in a C-terminal of CaM and the additional residues enhance interactions with CaM. We found that the addition of the common sequence, which was employed for ribosome display, makes the affinity of a selected peptide as strong as the previously reported peptide.

Identification of DNAJA1 as a novel interacting partner and a substrate of human transglutaminase 2.

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional member of the transglutaminase enzyme family. It has been implicated to have roles in many physiological and pathological processes such as differentiation, apoptosis, signal transduction, adhesion and migration, wound healing and inflammation. Previous studies revealed that TG2 has various intra- and extra-cellular interacting partners, which contribute to these processes. In the present study, we identified a molecular co-chaperone, DNAJA1, as a novel interacting partner of human TG2 using a GST pull-down assay and subsequent mass spectrometry analysis, and further confirmed this interaction via ELISA and surface plasmon resonance measurements. Interaction studies were also performed with domain variants of TG2 and results suggest that the catalytic core domain of TG2 is essential for the TG2-DNAJA1 interaction. Cross-linking activity was not essential for the interaction since DNAJA1 was also found to interact with the catalytically inactive form of TG2. Furthermore, we have showed that DNAJA1 interacts with the open form of TG2 and regulates its transamidation activity under both in vitro and in situ conditions. We also found that DNAJA1 is a glutamine donor substrate of TG2. Since DNAJA1 and TG2 are reported to regulate common pathological conditions such as neurodegenerative disorders and cancer, the findings in the present paper open up possibilities to explore molecular mechanisms behind TG2-regulated functions.