Protein expression profiling of Orthosiphon aristatus (Blume) Miq and molecular dynamics simulation of transketolase with antidiabetic potential

Abstract

Orthosiphon aristatus (Blume) Miq is a well-known medicinal plant widely commercialized especially as tea product to promote health. However, to date, the proteome of O. aristatus has never been studied. Hence, this study used four approaches; one-dimensional (1D) and two-dimensional (2D) proteomics, antidiabetic assay and bioinformatics to analyze the leaves proteome of O. aristatus. Protein from fresh and dried leaves (microwave-drying, halogen oven-drying and freeze-drying) was extracted using phenol/sodium dodecyl sulphate (SDS) buffer with three preliminary washes and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to check the quality. Subsequently, antioxidant assays; ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were performed on the protein to identify a suitable drying method with minimum effect on plant’s beneficial content as it is highly consumed as tea product. Microwave-drying was the most suitable drying method for O. aristatus as it produced better protein quality (859.49 ± 5.98 µg/mL, 14 protein bands detected) with high antioxidant activity (71.5% free radical scavenging activity, 1426.13 Fe2+mM/g). Thus, only fresh and microwave-dried leaves protein were further used for subsequent study. Over 1000 proteins were identified from fresh and dried leaves using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) with 43 proteins found to be similar for both samples. The profiled proteins were obviously affected by heat stress, where proteins with known anticancer property such as thioredoxin were only present in fresh leaves whereas myrosinase and glutathione were only extracted from dried leaves. Protein expression profile of fresh and dried leaves using 2D approach was analyzed with PDQuest software. Fresh and dried leaves have 202 and 106 spots of different distribution pattern on 2D gel respectively. Via PDQuest analysis, 32 distinct spots were detected from fresh leaves but not dried leaves and 12 spots with 2-fold expression detected from both types of leaves. Fresh and dried leaves protein also showed potential antidiabetic activity from inhibitions of glucose diffusion, a-amylase and a-glucosidase. Subsequently, one of the identified proteins, Transketolase (TKT) which plays a crucial role in preventing diabetes complications, was studied further in terms of its thermostability using molecular dynamics (MD) simulation at different temperature (310 K, 368 K, 373 K, 423 K, 453 K and 473 K) and interaction with its ligand, thiamine diphosphate (TPP) via Autodock Vina. The MD results indicated that TKT is a thermostable protein but TKT and TKT-TPP complex may start to denature when exposed to temperature beyond boiling point. Gln237, Ser242, Cys246, Arg283 and Phe284 were identified as the possible TPP binding sites. Taken together, the findings provide a snapshot of fresh and dried leaves proteome of O. aristatus showing that the plant protein exhibited antioxidant and antidiabetic activities. The protein expression profile of O. aristatus could also be used for quality control purpose as a biomarker for O. aristatus. TKT bioinformatics information provided pharmacological insight that can accelerate drug design process.