IIAEK Targets Intestinal Alkaline Phosphatase (IAP) to Improve Cholesterol Metabolism with a Specific Activation of IAP and Downregulation of ABCA1

Abstract

IIAEK (Ile-Ile-Ala-Glu-Lys, lactostatin) is a novel cholesterol-lowering pentapeptide derived from bovine milk β-lactoglobulin. However, the molecular mechanisms underlying the IIAEK-mediated suppression of intestinal cholesterol absorption are unknown. Therefore, we evaluated the effects of IIAEK on intestinal cholesterol metabolism in a human intestinal model using Caco-2 cells. We found that IIAEK significantly reduced the expression of intestinal cholesterol metabolism-associated genes, particularly that of the ATP-binding cassette transporter A1 (ABCA1). Subsequently, we chemically synthesized a novel molecular probe, IIXEK, which can visualize a complex of target proteins interacting with photoaffinity-labeled IIAEK by fluorescent substances. Through photoaffinity labeling and MS analysis with IIXEK for the rat small intestinal mucosa and intestinal lipid raft fractions of Caco-2 cells, we identified intestinal alkaline phosphatase (IAP) as a specific molecule interacting with IIAEK and discovered the common IIAEK-binding amino acid sequence, GFYLFVEGGR. IIAEK significantly increased IAP mRNA and protein levels while decreasing ABCA1 mRNA and protein levels in Caco-2 cells. In conclusion, we found that IIAEK targets IAP to improve cholesterol metabolism via a novel signaling pathway involving the specific activation of IAP and downregulation of intestinal ABCA1.

Keywords: ABCA1; Caco-2 cells; IIAEK; alkaline phosphatase; cholesterol; photoaffinity labeling.

Figure 1

Effect of IIAEK (Ile-Ile-Ala-Glu-Lys, lactostatin) containing cholesterol micelle on cholesterol metabolism in Caco-2 cells. (A) Effect of IIAEK containing cholesterol micelle on cholesterol absorption rate in Caco-2 cells. (B) Effect of IIAEK containing cholesterol micelle on cholesterol metabolism-associated gene expression in Caco-2 cells. Values are represented as means ± standard errors, represented by vertical bars (n = 6 per group). (C) Effect of IIAEK containing cholesterol micelle on ATP-binding cassette transporter A1 (ABCA1) protein level in Caco-2 cells by Western blot. (D) ABCA1 protein level was quantified with ImageJ and normalized to the level of β-actin. Values are represented as mean ± standard error, represented by vertical bars (n = 3 per group). (E) Effect of IIAEK containing cholesterol micelle on ABCA1 gene promoter activity in Caco-2 cells. Caco-2 cells were transfected with the human pGL3-ABCA1-Luc plasmid (WT: −928 to +107 bp) or the pGK3-ABCA1-Luc plasmid deletion mutants (ABC–536: −536 to +107 bp, ABC–126: −126 to +107 bp, ABC–36: −36 to +107 bp), using the pPGK β-galactosidase plasmid as an internal control. Data are presented as luciferase activity normalized to β-galactosidase activity. Values are represented as means ± standard error, represented by vertical bars (n = 5–9 per group). Asterisks indicate the difference from the control (* p < 0.05, ** p < 0.01, ***p < 0.001), as determined by Student’s t-test.

Figure 2

Chemical synthesis of the molecular probe, IIXEK. (A) Amide bond by 3-ethynyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl] benzoic acid and (S)-2,3-diaminopropionic acid. (B) Structure of IIXEK. IIXEK possesses chemically synthesized 3-ethynyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl] benzoic acid. This probe captures the target proteins interacting with IIAEK. (C) Click reaction, which occurs by introduction of rhodamine into IIXEK in the presence of copper as catalyst (Huisgen cycloaddition reaction). (D) Mass spectrometry of Cu-Catalyzed Azide (Azide-fluor 488) Alkyne (IIXEK) cycloaddition.

Figure 3

Extraction of intestinal lipid raft fractions from Caco-2 cells and photoaffinity labeling by IIXEK for the lipid raft fraction and rat intestinal mucosal protein. (A,B) Protein level of the lipid raft marker flotilin-1 in each fraction. (C) Alkaline phosphatase (AP) activity in each fraction. (D) Photoaffinity labeling of intestinal lipid raft proteins by IIXEK. The intestinal lipid raft fraction (Fr. 3) containing 250 μM IIXEK was irradiated with UV for 30 min for photoaffinity labeling. Subsequently, the fraction containing the fluorescent substance, rhodamine, which was added by a click reaction, was separated on SDS-PAGE. (E) Photoaffinity labeling of rat intestinal mucosal protein fractions by IIXEK. We collected the small intestinal mucosal proteins of 5-week-old male Wistar rats. The membrane protein fractions, which were derived from rat small intestinal mucosa, were collected with EB2B of TM-PEK and made into rat small intestinal mucosal fractions. Photoaffinity labeling was performed in the same way as described in Figure 3D.

Figure 4

IIXEK-binding amino acid sequences of proteins identified by nano LC-MS/MS analyses. (A) Human IAP (Uniprot accession No. P09923). (B) Human ATP synthase subunit beta, mitochondrial (Uniprot accession No. P06576). (C) Human Trifunctional enzyme subunit beta, mitochondrial (Uniprot accession No. P55084). (D) Rat IAP-2 (Uniprot accession No. P51740). (E) Rat ATP synthase subunit beta, mitochondrial (Uniprot accession No. P10719). Matched peptide sequences are bold-faced and underlined.

Figure 5

Effects of IIAEK on the expression of intestinal alkaline phosphatase (IAP) and the cholesterol metabolism-associated gene, ABCA1. Caco-2 cells were cultured in six-well Transwell plates. The cells were treated with serum-free medium containing 2 mM IIAEK or vehicle for 24 h. They were incubated with or without cholesterol micelle. (A,B) The total proteins were collected from the cells, and alkaline phosphatase (AP) activity was measured. Values are represented as means ± standard error represented by vertical bars (n = 6 per group). (C–F) Total RNA was collected from the cells. The IAP and ABCA1 mRNA levels were measured by real-time PCR and normalized to the mRNA expression level of 18S ribosomal RNA. Values are represented as means ± standard error represented by vertical bars (n = 6 per group). (G,I) The cell lysate was collected from the cell, separated on SDS-PAGE, and analyzed by Western blot analysis. (H,J) The IAP and ABCA1 protein levels were quantified with ImageJ and normalized to the level of β-actin. Values are represented as means ± standard error represented by vertical bars (n = 5 per group). Asterisks indicate the difference from the control (* p < 0.05, ** p < 0.01, *** p < 0.001) as determined by Student’s t-test.

Figure 6

IIAEK–IAP interaction hypothesis. IIAEK interacts with IAP and improves cholesterol metabolism by the specific activation of IAP and downregulation of intestinal ABCA1.