Altered enzyme offers hope for spinal injury and stroke

An enzyme proven to help regrow damaged nerve tissue in animals but too unstable for use in humans has been redesigned for stability in research co-led by Marian Hettiaratchi of the Phil and Penny Knight Campus for Accelerating Scientific Impact at the University of Oregon.

With stability added, the enzyme found in many types of bacteria, chondroitinase ABC, could potentially be repurposed to help reverse nerve damage caused by strokes and as a treatment for spinal cord injuries.

A major challenge for healing in such cases is the formation of glial scars that are rapidly formed by cells and biochemicals that knit together around damaged nerves. Initially the scarring offers a protective shield but over time it inhibits nerve repair.

“A glial scar is similar to other scars that form when you injure other parts of the body in that it protects the tissue from further damage, but glial scars don’t heal and remodel into healthy tissue like other scars typically do,” Hettiaratchi said. “Chondroitinase ABC is a promising therapeutic protein, because it can degrade components of the glial scar, allowing healthy neurons to regrow.”

Natural chondroitinase ABC, she noted, loses most of its activity within 24 hours. The redesigned version, detailed recentlyin Science Advances, is active for seven days. The enzyme was discovered two decades ago in the bacterium Proteus vulgaris. In animals, it has helped return lost function after severe spinal cord injury.

The research began while Hettiaratchi was a postdoctoral scientist at the University of Toronto before she joined the UO in January. The research team included scientists from the University of Toronto and the University of Michigan.

The natural enzyme is stable in its bacterial environment but once inside a body it is fragile, said Molly Shoichet, a professor of chemical engineering at Toronto and the study’s senior author.

“It aggregates, or clumps together, which causes it to lose activity,” Shoichet said. “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, including Shoichet’s, 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.

None of those approaches, however, addressed the instability.

In the new project, which was funded by the Natural Sciences and Engineering Council of Canada and the Canada First Research Excellence Fund, the research team altered the enzyme’s biochemical structure.

“Like any protein, chondroitinase ABC is made up of building blocks called amino acids,” Shoichet said. “We used computational chemistry to predict the effect of swapping out some building blocks for others, with a goal of increasing the overall stability while maintaining or improving the enzyme’s activity.”

The team had a lot of possible directions, said Mathew O’Meara, a professor of computational medicine and bioinformatics at the University of Michigan and co-lead author.

To narrow down the search space, he said, 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 don’t exist in nature but are plausibly like those that do.

In the end, the team ended up with three new candidate forms of the enzyme, but only one was both more stable and more active.

The next step will be to test the enzyme in the same kinds of experiments where the natural enzyme was previously used.

“This approach has not yet been used by tissue engineers interested in delivering therapeutic proteins for regenerative medicine,” Hettiaratchi said. “Since we’ve overcome one of the key challenges of using chondroitinase ABC, we hope that this will open new opportunities for the use of this enzyme as a therapeutic.”

Hettiaratchi’s work at the Knight Campus focuses on developing new strategies to deliver proteins to heal injured human tissues.

“This project is a great example of an interdisciplinary approach to science where we leveraged expertise in computational biology to improve the activity of a therapeutic protein,” Hettiaratchi said. “Since many proteins demonstrate similar sensitivity and instability at body temperature, we are also interested in using this computational approach to improve the activity of other therapeutic proteins in the future.”

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