Surface Plasmon Resonance (SPR) Analysis of Binding Interactions of Proteins in Inner-Ear Sensory Epithelia

Drescher, Dennis G.; Ramakrishnan, Neeliyath A.; Drescher, Marian J.

doi:10.1007/978-1-59745-523-7_20

Cited by 85 publications

(55 citation statements)

References 7 publications

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“…6A shows the set of data for varying concentrations of C2F samples containing 61 M Ca 2ϩ . There was no mass transfer limitation (17) in the interaction, as determined by analyzing 40 nM C2F at several flow rates (5, 15, and 75 l/min). Using BIAevaluation software (GE Healthcare), the curves for the above concentrations of C2F (and C2D) were globally fit to a 1:1 Langmuir binding model to determine the rate constants and equilibrium constants.…”

Section: Speciesmentioning

confidence: 99%

“…Affinity-purified fusion proteins were immobilized on a CM5 chip (research grade; Biacore, Piscataway, NJ) by an amine-coupling reaction (16). Surface tests (17) were performed on each immobilized protein (ligand), with different concentrations of purified otoferlin C2 domain fusion protein (analyte). The reference surface was blocked with ethanolamine and thus contained no ligand.…”

mentioning

confidence: 99%

“…Otoferlin C2D and C2F domains (analytes) were diluted to a series of concentrations in HBS-P buffer, with added 61 M Ca 2ϩ for optimal binding. Kinetic values were determined using BIAevaluation software (Biacore), and the data were fitted with the model showing closest match (17). A 1:1 Langmuir binding model was generally selected.…”

mentioning

confidence: 99%

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Direct Interaction of Otoferlin with Syntaxin 1A, SNAP-25, and the L-type Voltage-gated Calcium Channel CaV1.3

Ramakrishnan

Drescher

2009

Journal of Biological Chemistry

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110

112

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The molecular mechanisms underlying synaptic exocytosis in the hair cell, the auditory and vestibular receptor cell, are not well understood. Otoferlin, a C2 domain-containing Ca 2؉ -binding protein, has been implicated as having a role in vesicular release. Mutations in the OTOF gene cause nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In the present study, we generated otoferlin fusion proteins containing two of the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and SNAP-25 in a Ca 2؉ -dependent manner (with optimal 61 M free Ca 2؉ required for binding). These interactions were greatly diminished for C2F with the P1825A mutation, possibly because of a reduction in tertiary structural change, induced by Ca 2؉ , for the mutated C2F compared with the native C2F. The otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity (K d ‫؍‬ 1.7 ؋ 10 ؊5 M) than for the C2F interaction (K d ‫؍‬ 2.6 ؋ 10 ؊9 M). In contrast, it was the otoferlin C2D domain that bound the Ca v 1.3 II-III loop, in a Ca 2؉ -dependent manner. The L1011P mutation in C2D rendered this binding insensitive to Ca 2؉ and considerably diminished. Overall, we demonstrated that otoferlin interacts with two main target-SNARE proteins of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the calcium channel, with the otoferlin C2F and C2D domains of central importance for binding. Because mutations in the otoferlin C2 domains that cause deafness in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the Ca v 1.3 calcium channel, it is these interactions that may mediate regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear hair cell function.Calcium is a key regulator of synaptic vesicle fusion (reviewed in Ref. 1). In mechanosensory hair cells, calcium microdomains (2) and possibly nanodomains (3) are formed when voltage-gated calcium channels open upon depolarization. Calcium at these sites is thought to activate protein interactions, leading to vesicle fusion. Some of the key players in this process are the target-SNARE 2 proteins, syntaxin 1A and SNAP-25, and the vesicle-SNARE, synaptobrevin (4). Vesicle-SNARE synaptotagmin 1 plays a crucial role as a calcium sensor at the neuronal synapse, modulating calcium channels and vesicle release by a Ca 2ϩ -dependent interaction with other SNARE proteins in the presence of lipid molecules (4 -6). However, in vertebrate mechanosensory hair cells, synaptotagmin 1 is not detected (7). Instead, fast neurotransmitter release in auditory and vestibular hair cells, facilitated largely by an L-type voltagegated calcium channel, Ca v 1.3 (8, 9), is thought to be modulated by a newly discovered protein, otoferlin, acting as the Ca 2ϩ sensor and vesicle-binding protein. When mutated, otoferlin causes DFNB9 nonsyndromic deafness (10). Gene sequences of different deaf families show that the OTOF gene can undergo mutation at multiple locatio...

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Section: Speciesmentioning

confidence: 99%

mentioning

confidence: 99%

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Direct Interaction of Otoferlin with Syntaxin 1A, SNAP-25, and the L-type Voltage-gated Calcium Channel CaV1.3

Ramakrishnan

Drescher

2009

Journal of Biological Chemistry

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110

112

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“…This can be achieved by optimizing electrostatic interactions between the negatively charged carboxylated surface and the ligand. Ligands are commonly dissolved in 10 mM sodium acetate to maintain low ionic strength (Drescher et al, 2009;Johnsson et al, 1991). The pH of this solution should be at least one pH unit below the ligand's pI (isoelectric point) to ensure that the ligand is positively charged and thus present at high concentrations at the surface.…”

Section: Assay Development Using the LI Spr Systemmentioning

confidence: 99%

Monitoring human serum albumin cell cultures using surface plasmon resonance (SPR) spectroscopy

Henseleit

Pohl

Bley

et al. 2015

J. Sens. Sens. Syst.

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Abstract. Continuously monitoring cell cultures is essential for both controlling critical parameters and improving understanding of key processes. An ideal technique in this context is surface plasmon resonance (SPR) spectroscopy, which essentially exploits changes in the angle of incident light that occur when molecules bind to a surface. It provides the ability to monitor real-time changes in small concentrations of various molecules, with no need for additional labels or sample preparation. Here we present an SPR-based immunoassay for monitoring concentrations of human serum albumin (HSA), and compare its sensitivity when used in conjunction with a Biacore platform and the cheaper, smaller li SPR system. In conjunction with either system, the immunoassay can detect HSA (a hepatocyte viability marker) at concentrations typically present in three-dimensional hepatocyte cultures mimicking the liver used to evaluate effects of drug candidates before exposure to humans or animals. Furthermore, in conjunction with the li SPR system, it is sufficiently sensitive to measure the much lower HSA levels present in skin-hepatocyte co-cultures.

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“…In this study, a surface performance test was performed using mannan (10 µg/ml), a known MR binding ligand, to check the receptor activity following MR immobilisation [296]. Regeneration is an important step in which the receptor is revived from previous binding to be prepared for a new binding cycle [297]. It is essential to have a baseline at the same level of the initial baseline following each regeneration, otherwise, the next measurement cycle is inconsistent and the results cannot be trusted [294].…”

Section: Methods Developmentmentioning

confidence: 99%

Mannosylated lipopetides as model vaccines for targeting the mannose receptor

Sedaghat¹

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The mannose receptor (MR) is an important component of the immune system and understanding the structural and conformational characteristics of this receptor is a key aspect of targeted vaccine design. Improved understanding of the role of carbohydrate recognition domains (CRDs) 4-7 in recognising glycosylated ligands present on the surface of pathogens such as C. albicans, P. carinii, L. donovani, and M. tuberculosis has given new insight into MR vaccine development. Initial studies identified mannan and its derivatives to be important ligands in MR targeting, providing essential knowledge about structural properties of the MR. During the last decade many attempts have been made to target this receptor for applications including vaccine and drug development.In the present study, a library of vaccine constructs comprising fluorescently-labelled mannosylated lipid dendrimers that contained the ovalbumin CD4 + epitope, OVA323-339, as the model peptide antigen were synthesised using fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). The vaccine constructs were designed with an alanine spacer between the O-linked mannose moieties to investigate the impact of distance between the mannose units on receptor-mediated uptake and/or binding in antigen presenting cells.Mannosylated building blocks were prepared with a Lewis acid reaction and the reaction was successfully modified and monitored for a better yield. This was followed by solid phase synthesis of mannosylated peptides with an azide functional group and a fluorescent tag. Considering the presence of multi-components on the designed mannosylated peptide, different methods of preparation was used and a high yield of the final mannosylated peptides with an azide functional group and a fluorescent tag were prepared. Further, OVA323-339 lipopeptides with an alkyne functional group were prepared using microwave assisted peptide synthesis. Final vaccine structures were prepared by attaching azide and alkyne functional groups using click chemistry. The same methods were applied for the preparation of a library of control vaccine structures.In vitro uptake studies performed on macrophage (F4/80 + ) and dendritic (CD11c + ) cells showed significant uptake and/or binding for vaccine constructs containing mannose and lipid, and also for the lipopeptide vaccine construct without mannose when compared to the controls (containing no lipid, no mannose and no mannose or lipid). Further, in vitro mannan competition assays demonstrated that uptake of the mannosylated and lipidated vaccine constructs was MR mediated. To address the specificity of receptor uptake, surface plasmon resonance (SPR) was performed usingBiacore technology which confirmed a high affinity of the mannosylated and lipidated vaccine constructs towards the human recombinant MR in vitro when compared with vaccine constructs without mannose. These studies also confirmed that both mannose and lipid moieties play significant II roles in receptor-mediated uptake on APCs, potentially faci...

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Surface Plasmon Resonance (SPR) Analysis of Binding Interactions of Proteins in Inner-Ear Sensory Epithelia

Cited by 85 publications

References 7 publications

Direct Interaction of Otoferlin with Syntaxin 1A, SNAP-25, and the L-type Voltage-gated Calcium Channel CaV1.3

Direct Interaction of Otoferlin with Syntaxin 1A, SNAP-25, and the L-type Voltage-gated Calcium Channel CaV1.3

Monitoring human serum albumin cell cultures using surface plasmon resonance (SPR) spectroscopy

Mannosylated lipopetides as model vaccines for targeting the mannose receptor

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