Purification and Functional Characterization of the Chloroform/Methanol-Soluble Protein 3 (CM3) From Triticum aestivum in Drosophila melanogaster

Abstract

Non-celiac wheat sensitivity (NCWS) has been proposed to be an independent disease entity that is characterized by intestinal (e.g., abdominal pain, flatulence) and extra-intestinal symptoms (e.g., headache, fatigue), which are propagated following the ingestion of wheat products. Increased activity of amylase trypsin inhibitors (ATIs) in modern wheat is suggested to be major trigger of NCWS, while underlying mechanisms still remain elusive. Here, we aimed to generate and functionally characterize the most abundant ATI in modern wheat, chloroform/methanol-soluble protein 3 (CM3), in vitro and in Drosophila melanogaster. We demonstrate that CM3 displays α-glucosidase but not α-amylase or trypsin inhibitory activity in vitro. Moreover, fruit flies fed a sucrose-containing diet together with CM3 displayed significant overgrowth of intestinal bacteria in a sucrose-dependent manner while the consumption of α-amylase and α-glucosidase inhibitors was sufficient to limit bacterial quantities in the intestine. Notably, both CM3 and acarbose-treated flies showed a reduced lifespan. However, this effect was absent in amylase inhibitor (AI) treated flies. Together, given α-glucosidase is a crucial requirement for disaccharide digestion, we suggest that inhibition of α-glucosidase by CM3 enhances disaccharide load in the distal gastrointestinal tract, thereby promoting intestinal bacteria overgrowth. However, it remains speculative if this here described former unknown function of CM3 might contribute to the development of gastrointestinal symptoms observed in NCWS patients which are very similar to symptoms of patients with small intestinal bacterial overgrowth.

Keywords: ATIs; CM3; Drosophila melanogaster; non-celiac wheat sensitivity; α-glucosidase.

Graphical Abstract

The effect of CM3 on carbohydrate digestive enzymes and intestinal bacteria in Drosophila melanogaster. Designed by Biorender.com.

Figure 1

Recombinant expression and purification of CM3. (A) A structural model of the CM3 protein was modeled from the respective protein sequence using the PHYRE2 protein fold recognition server. CM3 protein domains were predicted using the InterPro server CM3 protein domains were predicted using the InterPro server (https://www.ebi.ac.uk/interpro/download/) and the SMART server (http://smart.embl-heidelberg.de). Yellow: amino acids (aa) 1–25 = signal peptide, Purple: aa 29–52 = PS00426, conserved site of cereal trypsin/alpha-amylase inhibitors family. (B) Coomassie stained SDS-gel (left) and western blot (right) of fractions collected during Ni-NTA affinity cleaning of CM3. Recombinant protein was detected with anti-His antibody. (C) Experion automated gel electrophoresis was performed to measure the purity of CM3 under non-reducing (n. red.) or reducing conditions (red.) in duplicates. Arrows with SP indicate technically necessary system peaks. Arrow with CM3 indicates CM3 protein at predicted size. (D) LAL assay was performed to measure endotoxin level. CM3 was tested directly after dialysis and gliadin was used as positive control (pos. ctrl). One endotoxin unit (EU) equals ~0.1–0.2 ng endotoxin.

Figure 2

Effect of CM3 on digestive enzymes in vitro. (A) Effect of CM3 (2 μM), α-amylase inhibitor from Triticum aestivum (AI; 2 μM), or acarbose (2 μM) on trypsin activity was tested with bovine pancreatic trypsin. Positive control was trypsin inhibitor from chicken egg white (pos. ctrl) and PBS treated trypsin was used as a negative control (neg. ctrl). (B) Influence of CM3 (2 μM), AI (2 μM), or acarbose (1–100 μM) on amylase activity was measured utilizing salivary human α amylase. (C) Effect of CM3 (0.4 μM), AI (0.4 μM), or acarbose (500 μM) on α-glucosidase activity. Data are mean ± SD. All experiments were repeated at least three times. Significance was calculated with matched one-way ANOVA followed by Dunnett's comparing sample with control (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 3

Effect of CM3 on the life expectancy of Drosophila melanogaster. (A) Schematic representation of longevity experiments in which female and male W¹¹¹⁸ flies were treated with PBS, CM3, AI, or acarbose. Influence of CM3, acarbose or AI feeding on the survival rates of male and female flies combined (B), female flies (C), or male flies (D). Data were recorded until the last fly died. Significant differences between treatments were calculated by applying the Log-rank (Mantel-Cox) test (ns: not significant, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 4

Short term effect of CM3 on Drosophila melanogaster. (A) Gustatory assay of W¹¹¹⁸ flies treated with CM3, acarbose, AI, and PBS supplementation for 5 days. (B) Fruit flies were weighed after 10 days of CM3, acarbose, or AI treatment and compared to control (PBS) flies. (C) Relative fitness score of CM3, acarbose, or AI-treated flies was assessed by testing the climbing ability of these flies. Glucose (D), triglycerides (E), and protein (F) levels of treated flies were assessed using whole fly homogenates of five flies. (G) Relative amylase activity was measured in whole fly homogenates of 13 flies treated with PBS, CM3, acarbose, or AI. Data are mean ± SD (*p ≤ 0.05, **p ≤ 0.01).

Figure 5

Effect of CM3 on intestinal bacteria isolated from Drosophila melanogaster. (A) Schematic representation of the experimental setup to study the effect of CM3, acarbose, or AI on intestinal bacteria. (B) Gating strategy to identify and quantify living intestinal bacterial. Bacterial density of flies treated for 10 days with either sucrose-rich (C) or sucrose-free (D) diet supplemented with CM3, acarbose, AI, or PBS. Data are presented as box graph. Lines within the boxes indicate median values; whiskers—min and max. Significance was calculated with matched One-way ANOVA followed by Fisher's LSD comparing samples with PBS control (**p ≤ 0.01).