We may not be buying what the label says on the tin: a cautionary tale from a study of the influence of proteoglycans on nerve growth

Owen, S.J.; Fuller, H.R.; Jones, P.; Caterson, B.; Shirran, S.L.; Botting, C.H.; Roberts, S.

Abstract

Introduction

Commercially sourced preparations of reagents are an essential tool for all our laboratory studies and are assumed to have a certain degree of purity. During a study to determine how different proteoglycan substrates might control nerve growth in the intervertebral disc, we found confounding results when comparing the influence of aggrecan [containing chondroitin (CS) and keratan sulphate (KS)] with decorin and biglycan [both containing CS and dermatan sulphate (DS)]. These prompted us to carry out detailed chemical analyses – the results of which surprised us greatly.

Materials and Methods

Dorsal root ganglia (DRG) excised from embryonic chickens (Day 10) were cultured on substrates containing type I collagen and either commercially sourced ‘decorin’ or ‘biglycan’ (both intact and deglycosylated with chondroitinase-AC (removes CS) or –ABC (removes CS and DS)). Neurite outgrowth from ≥20 explants were assessed and scored for each condition. Deglycosylated ‘decorin’ and ‘biglycan’ were probed for decorin, biglycan, aggrecan, fibromodulin, lumican, KS and CS stubs via Western blotting. Additionally, deglycosylated ‘decorin’ and ‘biglycan’ were separated by SDS-PAGE and the resulting Coomassie-stained bands excised and trypsin digested prior to mass spectrometry analysis for protein identification. Bovine nasal cartilage extracts and aggrecan isolated from human intervertebral disc were used as positive controls.

Results

Both commercial ‘decorin’ and ‘biglycan’ inhibited DRG neurite outgrowth in a dose-dependent manner. Deglycosylation with chondroitinase ABC significantly reduced the inhibitory effect of ‘decorin’ but, surprisingly, not of ‘biglycan’. Western blot and mass spectrometry analysis confirmed that the commercial preparations of ‘decorin’ and ‘biglycan’ BOTH contained decorin and biglycan, as well as fibromodulin and aggrecan. The presence of these impurities with their KS glycosaminoglycan chains provided an explanation to the unexpected findings in our nerve growth work.

Discussion

If impurities in the commercially sourced biomolecules had not produced unexpected data, no compositional analysis would have been done and the wrong conclusions deduced about how individual proteoglycans controlled nerve growth. These erroneous findings could have been published in good faith. What should we in the scientific community do in the light of these results? Can we put pressure on companies to provide a more comprehensive analysis of the reagents that we purchase?

• Journal Article Type: Meeting Abstract
• Conference Name: British Society for Matrix Biology Spring 2017 Meeting: Matrix Proteoglycans: active participants in cell-extracellular matrix communication
• Online Publication Date: Aug 28, 2017
• Publication Date: Aug 28, 2017
• Deposit Date: Jun 13, 2023
• Journal: International Journal of Experimental Pathology
• Print ISSN: 0959-9673
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 98
• Issue: 3
• Pages: A4
• Publisher URL: https://onlinelibrary.wiley.com/doi/10.1111/iep.12235