The Mechanisms of Chondroitin Sulphate Lyases Treatment in Promotion of Axonal Growth

Abstract

In Injured nerves, chondroitin sulphate (CS) is upregulated forming barriers with astrocytes/fibroblasts and other extracellular matrix molecules, and thereby hampering nerve regeneration. Cleavage of CS using chondroitin sulphate lyases (Proteus vulgaris) promises axon regrowth through the barrier but the enzymatic efficacy remains to be improved. Two subtypes, endolyase and exolyase, have been found coexisting in the original host but only the former has been exploited for treatment of injured nerve tracts. We hypothesise that the two subtypes are necessary for enzymatic efficacy on CSs. We therefore prepared recombinant enzymes of both subtypes. Enzyme kinetics study revealed feedback inhibition by limit digestion products: that of the endolyase by tetrasaccharides and that of the exolyase by the disaccharides. When the two subtypes were used in combination, the digestion efficiency increased. We then used TGF beta-1 to induce CS production by astrocytes in culture, mimicking reactive glia in injured nerves. In co-cultures of such astrocytes with cortical neurons, treatment with combinations of the two subtypes resulted in increased neurite lengths as compared to co-cultures treated with one of the subtypes. The limit digestion products of CS were further tested for their effects on neurite extension on astrocytes that had been treated with TGF beta-1. The CS disaccharides, both 4- and 6-sulphated but not the tetrasaccharides, promoted neurite extension significantly. Taken together, the combinatorial use of the ChABC subtypes not only improved efficacy of enzyme activity on the axon-restrictive CS moiety, but also increased the yield of CS disaccharides which contributed to axonal growth.