Combinatorial use of chondroitin sulphate lyase subtypes in the treatment of nerve injury

Abstract

Chondroitin sulphate (CS) proteoglycans are up-regulated in the extracellular matrix of injured nerves and constitute barriers to axonal re-growth. Chondroitin sulphate ABC lyases (ChABC) from Proteus vulgaris have been exploited to cleave CS moieties in the injured environment and thus to improve prospects of nerve regeneration. Albeit the occurrence of two subtypes (ChABC I, an endolyase, and ChABC II, an exolyase), only ChABC I is commercially available and tested for medical use. Reports indicated activity decay of ChABC I in the injured environment and the need for repeated doses to overcome barrier effects of CS. To address this, we prepared recombinant ChABC I & II and assessed effects of limit digestion products of CS on the enzyme activities. ChABC II was found more sensitive to competitive inhibition by CS disaccharides than ChABC I by CS tetrasaccharides. Use of the two ChABC subtypes in combination resolved the product inhibition and improved enzymatic efficacy. We then used TGF-beta1 to induce CS production by astrocytes in culture, mimicking reactive glia in vivo. In co-cultures of such astrocytes with cortical neurons under treatment with combinations of the two subtypes, neurites were found longer than in co-cultures treated with one of the subtypes. Co-cultures treated with the combination of subtypes revealed strong chondroitin 4-sulphate (C-4-S) stub signal. The limit digestion products of C-4-S were then tested for their effects on neurite extension. Cortical neuron cultures treated with the CS disaccharide indicated increases in neurite extension, but not the tetrasaccharide. Taken together, the combination of ChABC subtypes not only relieves product inhibition on enzyme activity, but the increases in CS disaccharides can also contribute to axonal growth.