Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase

Abstract

Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.

Keywords: Click chemistry; Hedgehog acyltransferase; MBOAT; Protein palmitoylation.

Fig.1

Comparison of enzyme-linked immunosorbent assay (ELISA) (A) and click chemistry armed ELISA (click–ELISA) (B). (A) Analyte is captured on the plate and detected with primary antibody, followed by secondary antibody-HRP conjugate. Colorimetric readout is proportional to bound analyte. (B) Biotinylated substrate modified with YnC₁₅ alkyne tag is captured on a streptavidin-coated plate and functionalized with FLAG-tag via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). FLAG-tag is detected by α-FLAG-HRP conjugate. Colorimetric readout is proportional to alkyne-tagged substrate.

Fig.2

Solubilization of P100 membrane fractions. Total lysate (Tot. Lys.) was separated into S100 and P100 fractions at 100,000 rcf for 1 h. P100 membranes were solubilized in 1% (w/v) detergent for 1 h and then separated into P100(sol) and P100(n/sol) fractions at 100,000 rcf for 1 h. Samples were separated by SDS–PAGE and analyzed by immunoblotting with α-polyhistidine-HRP. n-octyl β-d-glucopyranoside (OTG), Triton X-100 (Triton), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and n-dodecyl β-d-maltopyranoside (DDM). Blot representative of three separate experiments.

Fig.3

Determination of click–ELISA parameters. All assays were performed at a total Shh(1–11) peptide concentration of 1 μM. Alkynylated Shh(1–11) peptides were click chemistry functionalized with 10 μM azido-FLAG, 1 mM CuSO₄, 1 mM TCEP, and 1 mM TBTA for 1 h unless otherwise stated. FLAG-labeled Shh(1–11) was detected with α-FLAG-HRP at 1:20,000 dilution for 1 h unless otherwise stated. TMB reactions were developed for 10 min unless otherwise stated. (A) TMB development reaction time course of YnC₁₅-Shh(1–11) (0.4 μM) in the presence of Shh(1–11) (0.6 μM). Assays were performed in duplicate, n = 3. (B) α-FLAG-HRP binding time course of YnC₁₅-Shh(1–11) (0.1 μM) in the presence of Shh(1–11) (0.9 μM) (n = 3). (C) Click reaction time course of YnC₁₅-Shh(1–11) (0.1 μM) in the presence of Shh(1–11) (0.9 μM) (n = 3). (D) P100(sol)-catalyzed acylation of Shh(1–11) (1 μM) in the presence of YnC₁₅-CoA (1 μM). Assays were performed in duplicate, n = 3.

Fig.4

Structures of RU-SKI inhibitors and activity as assessed by click–ELISA. Inhibitors were synthesized as described in the supplementary material and were assayed over a six-log unit serial dilution from 100 μM. Samples were background corrected against heat-inactivated P100(sol) low control and normalized to vehicle-only high control. Assays were performed in duplicate, n = 3.

Fig.5

Validation of SDS stop conditions for click–ELISA kinetic analysis. The click–ELISA protocol was performed as described in Fig. 3. (A) Plate binding time course for YnC₁₅-Shh(1–11) (0.1 μM) and Shh(1–11) (0.9 μM) in the presence of 0.9% SDS. Assays were performed in duplicate, n = 3. (B) Click–ELISA signal in the presence of varying total peptide concentrations of 40% YnC₁₅-Shh(1–11)/Shh(1–11) mixture in the presence of 0.9% SDS. Assays were performed in duplicate, n = 3. (C) P100(sol)-catalyzed acylation of Shh(1–11) (1 μM) in the presence of YnC₁₅-CoA (1 μM) stopped by the addition of SDS to 0.9% (10%, w/v, 10 μl). Assays were performed in duplicate, n = 3.