Use of chondroitin sulphate exolyase and endolyase to decrease axon-restrictive chondroitin sulphates in neural environments

Abstract

Chondroitin sulphate proteoglycans (CSPGs) upregulated at sites of nerve injury restrict axonal regrowth and hamper nerve regeneration. Digestion of the CS moieties in vivo with chondroitin sulphate ABC endolyase (ChABC I from Proteus vulgaris) applied to the injured site decreases restrictiveness of the injured environment. Less is known about in vivo efficacy of the exolyase, ChABC II, which is used by the original host as a working partner of ChABC I. To address this, we cloned both the Type I and Type II enzymes and over-expressed them as recombinant enzymes in E. coli. Exhaustive digestion of CS with rChABC I yielded oligosaccharides as limit digestion products while that with rChABC II only yielded limited amounts of disaccharides. Treatment of CS with combinations of rChABC I and II resulted in complete digestion to disaccharides. To test if the oligosaccharide products exerted inhibition on rChABC I, tetrasaccharides were purified from the reaction mixture; these were found to inhibit ChABC I in a competitive manner. Use of rChABC I & II in combination therefore relieves the inhibition and allows CS digestion to proceed to completion. Further tests will elucidate if the enzyme combination improves digestion of CS in the neural environment and consequently axonal growth.