Abstract
The present study reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially sourced preparations of the small leucine-rich proteoglycans (SLRPs), decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans (PGs) using both mass spectrometry (MS) and Western blotting, with and without various enzymatic deglycosylations. Commercial ‘decorin’ and ‘biglycan’ were found to contain a mixture of PGs including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of ‘decorin’ and ‘biglycan’ on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulfate glycosaminoglycan (GAG) chains whereas fibromodulin only contains keratan sulfate and the large (>2500 kDa), highly glycosylated aggrecan contains both keratan and chondroitin sulfate. The different structure, molecular weight and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers’ funds and time.
Highlights
For several decades, it has been known that extracellular matrix (ECM) molecules can provide topographical cues and guidance for neuronal growth
Since maximum inhibition of DRG neurite growth was observed using ‘decorin’ at 500 μg/ml, experiments were repeated using this concentration to determine the effect of enzymatic removal of GAG chains from the ‘decorin’ on neurite growth
Molecules that contain GAG chains have previously been shown to influence neuronal growth with those containing chondroitin sulfate (CS) in particular being implicated in neurite inhibition [8]
Summary
It has been known that extracellular matrix (ECM) molecules can provide topographical cues and guidance for neuronal growth. Proteoglycans (PGs) are a major component of the ECM in several tissues in our body and have been shown to influence nerve growth in many systems from the developing central nervous system [1,2] to the intervertebral disc [3,4,5]. They are reported to inhibit outgrowth of sensory neurons [6], reduce astrogliosis or, indirectly, promote axon growth of adult sensory neurons [7], with chondroitin sulfate–PGs acting through receptor protein tyrosine phosphatase σ [8].
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