Researchers here report on their successful redesign of the chondroitinase ABC enzyme, capable of degrading a form of scarring that forms following nervous system injury. This scarring inhibits regrowth of nerves, and thus suppressing or removing it may be beneficial. Chondroitinase ABC achieves this goal to some degree, but is impractical to use because of its instability in the body. This redesign may have improved stability to a large enough degree to make the enzyme a practical basis for therapies that improve nerve regrowth.
One of the major challenges to healing after the kind of nerve injury resulting from stroke or spinal cord damage is the formation of a glial scar. A glial scar is formed by cells and biochemicals that knit together tightly around the damaged nerve. In the short term, this protective environment shields the nerve cells from further injury, but in the long term it can inhibit nerve repair.
About two decades ago, scientists discovered that a natural enzyme known as chondroitinase ABC – produced by a bacterium called Proteus vulgaris – can selectively degrade some of the biomolecules that make up the glial scar. By changing the environment around the damaged nerve, chondroitinase ABC has been shown to promote regrowth of nerve cells. In animal models, it can even lead to regaining some lost function. But progress has been limited by the fact that chondroitinase ABC is not very stable in the places where researchers want to use it. “It aggregates, or clumps together, which causes it to lose activity. This happens faster at body temperature than at room temperature. It is also difficult to deliver chondroitinase ABC because it is susceptible to chemical degradation and shear forces typically used in formulations.”
Various teams have experimented with techniques to overcome this instability. Some have tried wrapping the enzyme in biocompatible polymers or attaching it to nanoparticles to prevent it from aggregating. Others have tried infusing it into damaged tissue slowly and gradually, in order to ensure a consistent concentration at the injury site. But all of these approaches fail to address the fundamental problem of instability. Now researchers tried a new approach: they altered the biochemical structure of the enzyme in order to create a more stable version. In the end, the team ended up with three new candidate forms of the enzyme that were then produced and tested in the lab. All three were more stable than the wild type, but only one, which had 37 amino acid substitutions out of more than 1,000 possible substitution locations, was both more stable and more active.
“The wild type chondroitinase ABC loses most of its activity within 24 hours, whereas our re-engineered enzyme is active for seven days. This is a huge difference. Our improved enzyme is expected to even more effectively degrade the glial scar than the version commonly used by other research groups.” The next step will be to deploy the enzyme in the same kinds of experiments where the wild type was previously used.