Identification of endo-type chondroitin sulfate hydrolases applicable to neuronal regenerative therapy

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In this study, human hyaluronidase-4 (HYAL4) was demonstrated to be a chondroitin sulfate (CS)-specific endo-type hydrolase for the first time. Although mouse hyaluronidase-4 (mHyal4) was also demonstrated to be a CS-specific endo-type hydrolase, it has broader substrate specificity than HYAL4. A series of human/mouse hyaluronidase-4 chimeric proteins and point mutants were generated, and the amino acid residues responsible for the difference in substrate specificity were identified. To prepare an active enzyme applicable to neuronal regenerative therapy in a large scale, the expression system in yeast was constructed. Injury region was extracted from the rats with lateral hemisection of the spinal cord, and the composition of CS in the region as well as its sensitivity to mHyal4 was investigated.