Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor

Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9+, CD63+, and CD81+ EV subpopulations of <50 nm exomeres and 50-80 nm exosomes were isolated and fractionated using our recently developed on-line coupled immunoaffinity chromatography - asymmetric flow field-flow fractionation system. Adaptive Interaction Distribution Algorithm (AIDA), specifically designed for the analysis of complex biological interactions, was used with a four-step procedure for reliable estimation of the degree of heterogeneity in rate constant distributions. Interactions between exomere-sized EVs and anti-tetraspanin antibodies demonstrated two interaction sites with comparable binding kinetics and estimated dissociation constants Kd ranging from nM to fM. Exomeres exhibited slightly higher affinity compared to exosomes. The highest affinity with anti-tetraspanin antibodies was achieved with CD63+ EVs. The interaction of EV subpopulations with ICAM-1 involved in cell internalization of EVs was also investigated. EV - ICAM-1 interaction was also of high affinity (nM to pM range) with overall lower affinity compared to the interactions of anti-tetraspanin antibodies and EVs. Our findings proved that QCM is a valuable label-free tool for kinetic studies with limited sample concentration, and that advanced algorithms, such as AIDA, are crucial for proper determination of kinetic heterogeneity. To the best of our knowledge, this is the first kinetic study on the interaction between plasma-derived EV subpopulations and anti-tetraspanin antibodies and ICAM-1.Peer reviewe

Reliable Strategy for Analysis of Complex Biosensor Data

When using biosensors, analyte biomolecules of several different concentrations are percolated over a chip with immobilized ligand molecules that form complexes with analytes. However, in many cases of biological interest, e.g., in antibody interactions, complex formation steady-state is not reached. The data measured are so-called sensorgram, one for each analyte concentration, with total complex concentration vs time. Here we present a new four-step strategy for more reliable processing of this complex kinetic binding data and compare it with the standard global fitting procedure. In our strategy, we first calculate a dissociation graph to reveal if there are any heterogeneous interactions. Thereafter, a new numerical algorithm, AIDA, is used to get the number of different complex formation reactions for each analyte concentration level. This information is then used to estimate the corresponding complex formation rate constants by fitting to the measured sensorgram one by one. Finally, all estimated rate constants are plotted and clustered, where each cluster represents a complex formation. Synthetic and experimental data obtained from three different QCM biosensor experimental systems having fast (close to steady-state), moderate, and slow kinetics (far from steady-state) were evaluated using the four-step strategy and standard global fitting. The new strategy allowed us to more reliably estimate the number of different complex formations, especially for cases of complex and slow dissociation kinetics. Moreover, the new strategy proved to be more robust as it enables one to handle system drift, i.e., data from biosensor chips that deteriorate over time.Peer reviewe

Inactivation of Aconitase by Tetrahydrobiopterin in DArgic Cells: Relevance to PD

Oxidative damage is thought to be a major cause of the progression of dopamine (DA)rgic neurodegeneration as in Parkinson's disease. We have previously reported that tetrahydrobiopterin (BH4), an endogenous molecule required for DA synthesis, exerts oxidative stress to DA-producing cells and facilitates the production of DA quinone. It is known that aconitase, present in both mitochondrial and cytosolic forms, act as an reactive oxygen species (ROS) sensor, and that their inactivation leads to further generation of ROS. In the present study we investigated whether the BH4-associated vulnerability of DA cells might involve aconitase. In DArgic cell line CATH.a, BH4 treatment caused reduction of activity of both mitochondrial and cytosolic aconitases, and this appeared to be due to direct inactivation of the pre-existing enzyme molecules. Although most of the activity reduced by BH4 was increased upon reactivation reaction under a reducing condition, the restoration was not complete, suggesting that irreversible and covalent modification has occurred. The aconitase inactivation was exacerbated in the presence of DA and attenuated in the presence of tyrosine hydroxylase inhibitor a-methyl-p-tyrosine, suggesting the involvement of DA. The degree of inactivation increased when the cells were treated with the quinone reductase inhibitor dicoumarol and decreased in the presence of quinone reductase inducer sulforaphane. Taken together, BH4 appeared to lead to both reversible and irreversible inactivation of aconitase and that this is facilitated by the presence of DA and accumulation of DA quinone

Mouse LSECtin as a model for a human Ebola virus receptor

The biochemical properties of mouse LSECtin, a glycan-binding receptor that is a member of the C-type lectin family found on sinusoidal endothelial cells, have been investigated. The C-type carbohydrate-recognition domain of mouse LSECtin, expressed in bacteria, has been used in solid-phase binding assays, and a tetramerized form has been used to probe a glycan array. In spite of sequence differences near the glycan-binding sites, the mouse receptor closely mimics the properties of the human receptor, showing high affinity binding to glycans bearing terminal GlcNAcβ1-2Man motifs. Site-directed mutagenesis has been used to confirm that residues near the binding site that differ between the human and the mouse proteins do not affect this binding specificity. Mouse and human LSECtin have been shown to bind Ebola virus glycoprotein with equivalent affinities, and the GlcNAcβ1-2Man disaccharide has been demonstrated to be an effective inhibitor of this interaction. These studies provide a basis for using mouse LSECtin, and knockout mice lacking this receptor, to model the biological properties of the human receptor

Thermodynamic and kinetic approaches for evaluation of monoclonal antibody - Lipoprotein interactions

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool. (C) 2016 Elsevier Inc. All rights reserved.Peer reviewe

The pigmented life of a redhead.

As a redhead I have had a personal interest in red hair, freckles and sunburns since childhood. An observation of a formaldehyde-induced fluorescence in human epidermal melanocytes initiated my scientific interest in these cells. Prota and Nicolaus demonstrated that oxidation products of cysteinyldopas are the main components of pheomelanin. Our identification of 5-S-cysteinyldopa as the source of formaldehyde-induced fluorescence of normal and pathological melanocytes started a series of investigations into this amino acid, enzymatic and non-enzymatic oxidation of catecholic compounds and the metabolism of thiols. All melanocytes with functioning tyrosinase produce cysteinyldopas and the levels of 5-S-cysteinyldopa in serum and urine are related to the size and pigment forming activity of the melanocyte population. The determination of 5-S-cysteinyldopa in serum or urine is a sensitive diagnostic method in the detection of melanoma metastasis. Some non-specific formation of cysteinyldopa is present in the body, as demonstrated by 5-S-cysteinyldopa in individuals with tyrosinase-negative albinism

Galectin-3 alters the lateral mobility and clustering of beta 1-integrin receptors

Glycoprotein receptors are influenced by myriad intermolecular interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examined the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the alpha 5 beta 1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor reductions in lateral mobility. Treatment of cells with Gal-3 increased beta 1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics analysis to confirm that cells expressed endogenous Gal-3, and found that addition of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3-integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins