Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet

Abdiche, Yasmina; Malashock, Dan; Pinkerton, Alanna; Pons, Jaume

doi:10.1016/j.ab.2008.03.035

Cited by 347 publications

(281 citation statements)

References 9 publications

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“…Soluble LRP6 ECD Directly Binds to Wnt3a and Inhibits Wnt3a-mediated ␤-Catenin Stabilization-To study the interaction between LRP6 and Wnt3a, we developed a binding assay based on biolayer interferometry (32). Site-specifically biotinylated LRP6 E1E4 was obtained by co-expressing a C-terminal His 6 -Avi-tagged (33) version of LRP6 E1E4 with biotin ligase in insect cells.…”

Section: Expression Of Lrp6 Extracellular Domain and Fz8 Crd In Insectmentioning

confidence: 99%

Reconstitution of a Frizzled8·Wnt3a·LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Bourhis

Tam²,

Franke³

et al. 2010

Journal of Biological Chemistry

196

236

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Wnt/␤-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/␤-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD⅐Wnt3a⅐LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth ␤-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/␤-catenin signaling by Dkk1.Since its discovery more than 30 years ago, the Wnt signaling pathway has captured the interest of researchers due to its role in development, progression of cancer, and self-renewal and differentiation of stem cells (1, 2). Wnt signaling is initiated at the cell surface where secreted Wnt glycoprotein forms a complex with the receptors Frizzled (Fz) 2 (3) and low density lipoprotein receptor-related proteins 5 or 6 (LRP5/6) (4, 5). This results in stabilization of intracellular ␤-catenin ("Wnt/␤-catenin pathway") and its translocation to the nucleus. Nuclear ␤-catenin then initiates T-cell factor-dependent transcription of downstream Wnt target genes (6, 7). The interactions of LRP5/6 and Fz with Wnt are critical for mediating Wnt/␤-catenin signaling (5,8,9). Underscoring the central role that these receptors play in Wnt signaling, suppression of either Fz or LRPs has been shown to generate phenotypes associated with Wnt mutations (10). Furthermore, mechanisms have evolved to regulate the Wnt⅐Fz⅐LRP complex through secreted proteins that interfere with these extracellular interactions. One such example is Dickkopf-1 (Dkk1), which inhibits Wnt signaling by binding to LRP6 (9,11,12). Dkk1 has been shown to play a crucial role in bone formation, vertebrate embryogenesis, and suppression of cancer cell proliferation (13-15). Therefore, Wnt ligands, receptors Fz and LRP, and their natural inhibitors are all of great therapeutic interest. Indeed, recent findings show that the soluble extracellular domain of Fz8 interferes with Wnt-driven tumor growth in vivo (16).The detailed molecular arrangement of the Wnt⅐Fz⅐LRP ternary complex at the cell surface is ...

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Section: Expression Of Lrp6 Extracellular Domain and Fz8 Crd In Insectmentioning

confidence: 99%

Reconstitution of a Frizzled8·Wnt3a·LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Bourhis

Tam²,

Franke³

et al. 2010

Journal of Biological Chemistry

196

236

View full text Add to dashboard Cite

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“…Octet assay harnesses biolayer interferometry to detect and quantify molecular interactions. 44 In brief, the sample plate was agitated at 600 rpm and the prewet SA sensors were dipped into 10 mg/ml biotinylated HER2-ECD for 15 min. After equilibration in phosphate buffered saline (PBS) for 3 min, the HER2-ECD coated sensors were dipped into 50 mg/ml hertuzumab, hertuzumab-vcMMAE, and trastuzumab containing wells of the microplate for 15 min.…”

Section: Conjugation Of Hertuzumab-vcmmaementioning

confidence: 99%

An anti-HER2 antibody conjugated with monomethyl auristatin E is highly effective in HER2-positive human gastric cancer

Yu²,

Jiang

et al. 2016

Cancer Biology & Therapy

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Antibody-drug conjugate (ADC) is a novel class of therapeutics for cancer target therapy. This study assessed antitumor activity of ADC with an antimitotic agent, monomethyl auristatin E (MMAE) and a humanized monoclonal anti-HER2 antibody, hertuzumab, in gastric cancer. The efficacy of hertuzumab-MC-Val-Cit-PAB-MMAE (hertuzumab-vcMMAE) on human epidermal growth factor receptor 2 (HER2) positive human gastric cancer cells, NCI-N87, was evaluated in vitro and in vivo. The cytotoxicity of hertuzumab was significantly enhanced after conjugation with MMAE. Compared to trastuzumab, hertuzumab had a higher affinity to HER2 and had more potent antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. After conjugation with MMAE, the binding specificity for HER2 was not affected. Furthermore, the internalization of hertuzumab-vcMMAE in HER2 positive gastric cancer cells was verified. Although the conjugation of hertuzumab and MMAE decreased the ADCC effect, the overall cytotoxicity was dramatically increased in HER2 positive gastric cancer cells. In vitro data on this hertuzumab-vcMMAE has exerted much stronger antitumor activity compared to trastuzumab-DM1 in HER2 positive gastric cancer cells. A single administration of hertuzumab-vcMMAE at 5 or 10 mg/kg showed high potency and a sustained tumor inhibitory effect on NCI-N87 xenografts in mice. In conclusion, hertuzumab-vcMMAE conjugate is a highly effective anti-HER2 targeted therapy for HER2-positive gastric cancer.

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“…The experimental binding kinetic studies of biomolecules are carried out mainly by optical label-free biosensors [6,7,8,9] and label-techniques [2,3,4,5]. The mechanistic assay system model, based on antibody-analyte binding reaction kinetics, has proven to be applicable to the prediction of a rapid generic three-component sandwich-type (immunometric) immunoassay [4,5,10].…”

Section: Introductionmentioning

confidence: 99%

Modelling of multi-component immunoassay kinetics — A new node-based method for simulation of complex assays

Ylander

Hänninen

2010

Biophysical Chemistry

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To cite this version:Pilvi J. Ylander, Pekka Hänninen. Modelling of multi-component immunoassay kinetics -a new node-based method for simulation of complex assays. Biophysical Chemistry, Elsevier, 2010, 151 (3) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. This method provides an easy and quick way to study complex binding reactions since simulation networks are simple to construct directly from the reaction scheme. This presented new "NODE"-method is compared with the well known mechanistic assay model. A C C E P T E D

show abstract

Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet

Cited by 347 publications

References 9 publications

Reconstitution of a Frizzled8·Wnt3a·LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Reconstitution of a Frizzled8·Wnt3a·LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

An anti-HER2 antibody conjugated with monomethyl auristatin E is highly effective in HER2-positive human gastric cancer

Modelling of multi-component immunoassay kinetics — A new node-based method for simulation of complex assays

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