stigmasterol and 25-hydroxycholesterol

Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (β-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.

Topics: Aspergillus oryzae; Biological Transport; Carrier Proteins; Ergosterol; Gene Expression; Hydroxycholesterols; Ketocholesterols; Protein Binding; Protein Domains; Receptors, Steroid; Spores, Fungal; Stigmasterol

Intake of plant sterols has long been shown to reduce cholesterol absorption and subsequently plasma cholesterol concentrations. Despite competition between plant sterols and cholesterol for incorporation into mixed micelles as a suggested major mechanism for the inhibition of cholesterol absorption by plant sterols, studies exist to support an alternative mechanism. For example, another mechanism may be the action of plant sterols to reduce cholesterol absorption at the cellular level. This study was undertaken to test the hypothesis that plant sterols can modulate the expression of transporters such as Niemann-Pick C1-like 1 (NPC1L1) and scavenger receptor class B, type I (SR-BI) to lower intestinal cholesterol absorption. FHs 74 Int cells, a human small intestine epithelial cell line, were used as a model of enterocytes. The cells were treated with 25alpha-hydroxycholesterol (25 micromol/L) or 250 micromol/L of sitosterol, stigmasterol, and cholesterol for 24 hours to measure genes involved in cholesterol absorption and metabolism by quantitative real-time polymerase chain reaction. 25Alpha-hydroxycholesterol, cholesterol, and sitosterol significantly reduced the messenger RNA (mRNA) expression of NPC1L1 and hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, whereas SR-BI mRNA was not altered by the sterols. Western blot analysis confirmed the reduction in NPC1L1 by sterols. Depletion of cellular cholesterol by mevinolin, a cholesterol synthesis inhibitor, increased NPC1L1 and HMG-CoA reductase mRNA; and repletion of cholesterol abolished the increase. Sitosterol, but not stigmasterol, reduced the mRNA levels of NPC1L1 and HMG-CoA reductase to a similar extent of cholesterol. In conclusion, sitosterol can inhibit the expression of NPC1L1 in the enterocytes, which could be an alternate mechanism for plant sterols to reduce intestinal cholesterol uptake.

Topics: Anticholesteremic Agents; Cells, Cultured; Cholesterol; Dose-Response Relationship, Drug; Down-Regulation; Enterocytes; Humans; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intestinal Absorption; Intestine, Small; Membrane Proteins; Membrane Transport Proteins; Phytosterols; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scavenger Receptors, Class B; Sitosterols; Sterol Regulatory Element Binding Protein 2; Stigmasterol