Redesigned enzyme could help reverse damage from spinal cord injuries and strokes

A team of researchers from the University of Toronto Engineering and the University of Michigan have redesigned and improved a natural enzyme that shows promise for promoting the regrowth of nerve tissue after injury.

Their new version is more stable than the protein found in nature and could lead to new treatments to reverse nerve damage caused by traumatic injury or stroke.

“Stroke is the leading cause of disability in Canada and the third leading cause of death,” says Molly Shoichet, professor of engineering at the University of Toronto, lead author of a new study published in the journal Scientific advances.

“One of the main challenges in healing after this type of nerve injury is the formation of a glial scar.”

A glial scar is formed by cells and biochemicals that unite tightly around the damaged nerve. In the short term, this protective environment protects nerve cells from further injury, but in the long term, it can inhibit nerve repair.

About two decades ago, scientists discovered that a naturally occurring enzyme known as chondroitinase ABC – produced by a bacteria called Proteus vulgaris – could selectively break down 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 the recovery of a lost function.

But progress has been limited by the fact that chondroitinase ABC is not very stable where researchers want to use it.

“It is stable enough for the environment in which bacteria live, but inside the body it is very fragile,” says Shoichet. “It aggregates or agglutinates, causing it to lose its activity. It happens more quickly at body temperature than at room temperature. It is also difficult to deliver chondroitinase ABC because it is sensitive to chemical degradation. and the shear forces generally used in formulations. “

Different teams, including Shoichet’s, 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 to inject it slowly and gradually into damaged tissue, to ensure a constant concentration at the site of the injury.

But all of these approaches are just band-aid – they don’t solve the fundamental problem of instability.

In their latest article, Shoichet and colleagues tried a new approach: they altered the biochemical structure of the enzyme to create a more stable version.

“Like any protein, chondroitinase ABC is made up of building blocks called amino acids,” says Shoichet. “We have used computational chemistry to predict the effect of exchanging certain building blocks for others, with the aim of increasing overall stability while maintaining or enhancing the activity of the enzyme.”

“The idea was probably a bit crazy, because just like in nature, a single bad mutation can destroy structure,” says Mathew O’Meara, professor of computational medicine and bioinformatics at the University of Michigan and co-author main of the new paper.

“There are over 1,000 links in the chain that forms this enzyme, and for each link you have a choice of 20 amino acids,” he says. “There are too many choices to fake them all.”

To reduce search space, the team applied computer algorithms that mimicked the types of amino acid substitutions found in real organisms. This approach, known as consensus design, produces mutant forms of the enzyme that do not exist in nature, but which presumably resemble those that do.

In the end, the team ended up with three new candidate forms of the enzyme which were then produced and tested in the laboratory. All three were more stable than the wild type, but only one, which had 37 amino acid substitutions on over 1000 links in the chain, was both more stable and more active.

“Wild-type chondroitinase ABC loses most of its activity within 24 hours, while our redesigned enzyme is active for seven days,” says Marian Hettiaratchi, the article’s other co-lead author. A former postdoctoral fellow at Shoichet’s lab, Hettiaratchi is now a professor of bioengineering at the University of Oregon’s Phil and Penny Knight campus to accelerate scientific impact.

“It’s a huge difference. Our improved enzyme is expected to break down the glial scar even more effectively than the version commonly used by other research groups,” says Hettiaratchi.

The next step will be to deploy the enzyme in the same types of experiments where the wild type was previously used.

“When we started this project, we were advised not to try because it would be like looking for a needle in a haystack,” says Shoichet. “Having found this needle, we are studying this form of the enzyme in our models of stroke and spinal cord injury to better understand its potential as a therapy, alone or in combination with other strategies.”

Shoichet emphasizes the multidisciplinary nature of the project as the key to its success.

“We were able to take advantage of the authors’ complementary expertise to bring this project to fruition, and we were shocked and delighted to be so successful,” she says. “It far exceeded our expectations.”