Redesigned enzymes may help repair spinal cord injury and stroke damage

A team of researchers at the University of Toronto and the University of Michigan have redesigned and enhanced natural enzymes that have the potential to promote neural tissue regrowth after injury.

Their newer version is more stable than naturally occurring proteins and could lead to new therapies for reversing nerve damage caused by trauma and stroke.

“Stroke is the leading cause of disability in Canada and the third leading cause of death,” Molly Shoiche, a professor of engineering at the University of Toronto, is a senior author of a new study published in this journal. Scientific progress..

“One of the major challenges to healing after this type of nerve injury is the formation of glial scars.”

Glial scars are formed by the tight binding of cells and biochemicals around damaged nerves. In the short term, this protective environment protects nerve cells from further damage, but in the long term, can interfere with nerve repair.

About 20 years ago, scientists discovered that a natural enzyme called chondroitinase ABC (produced by a bacterium called Proteus vulgaris) could selectively break down some of the biomolecules that make up glial scars. Did.

Chondroitinase ABC has been shown to promote nerve cell regrowth by altering the environment around damaged nerves. In animal models, it also helps restore lost functionality.

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

“It’s stable enough to the environment in which bacteria live, but in the body it’s very fragile,” says Scheche. “It clumps or clumps and loses activity. It occurs faster at body temperature than at room temperature. Also, chondroitinase ABC has the chemical degradation and shear forces commonly used in pharmaceuticals. It is also difficult to deliver because it is susceptible.”

Various teams, including the Shoichet team, have tried techniques to overcome this instability. Some have tried to wrap the enzyme in a biocompatible polymer or attach it to nanoparticles to prevent aggregation. Others have tried slowly and slowly injecting into the damaged tissue to ensure a consistent concentration at the site of injury.

But all these approaches are just band-aids-they do not address the underlying problem of instability.

In the latest paper, Shoichet and her co-workers have tried a new approach. We modified the biochemical structure of the enzyme to create a more stable version.

“Like other proteins, chondroitinase ABC is made up of building blocks called amino acids,” Shoichet says. “We used computational chemistry to predict the effects of exchanging some building blocks for others, with the goal of maintaining or improving enzyme activity while increasing overall stability. “

Mathew O’Meara, a professor of computational medicine and bioinformatics at the University of Michigan, and co-author, said: New paper.

“There are over 1,000 links in the chain that make up this enzyme, and you can choose from 20 amino acids for each link,” he says. “There are too many choices to simulate everything.”

To narrow the search space, the team applied computer algorithms that mimic the types of amino acid substitutions found in living organisms. This approach, called consensus design, produces variants of enzymes that do not occur in nature, but are similar to existing enzymes.

Eventually, the team created three new candidate forms of the enzyme, which were then produced and tested in the lab. All three were more stable than wild type, but only one with 37 amino acid substitutions from more than 1,000 links in the chain was more stable and more active.

“Wild-type chondroitinase ABC loses most of its activity within 24 hours, but the reengineered enzyme is active for 7 days,” says Marian Hettiaratchi, co-author of this paper. Former postdoctoral researcher at Schochette’s lab, Hetia Latch is now a professor of biotechnology at the University of Oregon’s Phil and Penny Knight campus to accelerate scientific impact.

“This is a big difference. Our improved enzymes are expected to break down glial scars more effectively than those commonly used in other research groups,” says Hettiaratchi. ..

The next step is to deploy the enzyme in the same type of experiment where the wild type was previously used.

“When we started this project, we were advised not to try, as it would be like looking for needles in a haystack,” said Shoichet. “Once we find that needle, we are investigating the form of this enzyme in models of stroke and spinal cord injury to better understand its therapeutic potential, either alone or in combination with other strategies.”

Shoichet points to the interdisciplinary nature of the project as the key to its success.

“We were able to leverage the complementary expertise of the author to deliver on this project, and we were overwhelmed with the shock of being so successful,” she says. “It far exceeded our expectations.”