Integration of a miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism

Yi Sun,Yuen-Kee Tsui,Minmin Lyu,Victor Leung,Kenneth Cheung,Mengqi Yu,Richard Kao

doi:10.1038/s41598-022-04805-y

Abstract

Defective biosynthesis or function of proteoglycans causes pathological conditions in a variety of tissue systems. Osteoarthritis (OA) is a prevalent degenerative joint disorder characterized by progressive cartilage destruction caused by imbalanced proteoglycan synthesis and degradation. Identifying agents that regulate proteoglycan metabolism may benefit the development of OA-modifying therapeutics. High-throughput screening (HTS) of chemical libraries has paved the way for achieving this goal. However, the implementation and adaptation of HTS assays based on proteoglycan measurement remain underexploited. Using primary porcine chondrocytes as a model, we report a miniaturized dimethyl-methylene blue (DMMB) assay, which is commonly used to quantitatively evaluate sulfated glycosaminoglycan (GAG) content, with an optimized detection range and reproducibility and its integration with HTS. Treatment with TGF-β1 and IL1-α, known as positive and negative proteoglycan regulators, respectively, supported the assay specificity. A pre-test of chemical screening of 960 compounds identified both stimulators (4.48%) and inhibitors (6.04%) of GAG production. Fluorophore-assisted carbohydrate electrophoresis validated the activity of selected hits on chondroitin sulfate expression in an alginate culture system. Our findings support the implementation of this simple colorimetric assay in HTS to discover modifiers of OA or other diseases related to dysregulated proteoglycan metabolism.

Highlights

Proteoglycans are macromolecules containing a core protein backbone to which one or more sulfated glycosaminoglycans (GAGs) are covalently attached, mostly in the form of chondroitin sulfate (CS), keratin sulfate and heparin sulfate chains
We set out to investigate dimethylmethylene blue (DMMB) assay conditions in a miniaturized, 96-well format that enables maximal detection of CS, the major GAGs expressed in chondrocytes
Shi et al demonstrated a modulatory effect of BNTA on extracellular matrices in chondrocytes via SOD3 induction[22]

Summary

Introduction

Proteoglycans are macromolecules containing a core protein backbone to which one or more sulfated glycosaminoglycans (GAGs) are covalently attached, mostly in the form of chondroitin sulfate (CS), keratin sulfate and heparin sulfate chains. Proteoglycans, in particular the large chondroitin sulfate-rich proteoglycan aggrecan, contribute to structural and mechanical integrity[15]. Strategies for probing regulators of proteoglycan metabolism in chondrocytes could be valuable to the understanding and treatment of OA. Previous studies explored regulators of chondrogenesis in mesenchymal stromal cells[21] and ATDC5 c ells[22] by assessing proteoglycan content by Alcian blue staining in high-throughput screening (HTS). Because of its simplicity and sensitivity, the dimethylmethylene blue (DMMB) assay has been commonly used for GAG measurement to determine proteoglycan c ontent[23]. Using porcine chondrocytes as a model, we report an optimized, miniaturized DMMB assay-based GAG detection system and its integration with a robotic HTS platform to enable chemical library screening of potential proteoglycan metabolism modifiers

Methods

Results

Conclusion