A collaborative team of researchers from the University of Toronto Engineering and the University of Michigan have redesigned a naturally occurring enzyme that has demonstrated the ability to promote nerve regrowth in nerve damage models, increasing its activity and stability.
Glial scar formation: A barrier to nerve regeneration
The central nervous system (CNS), the "processing hub" of the body, comprises the brain and spinal cord. It is responsible for integrating incoming sensory information and directing the majority of bodily functions.
Damage to the nerves of the CNS can result from a variety of medical conditions, such as a stroke, or through physical injury – spinal cord injury (SCI) being a common example, affecting approximately 12,000 people each year in the U.S alone.
The symptoms of nerve damage can prove extremely debilitating for a patient depending on the extent of the damage. In SCI patients specifically, advances in modern medicine, such as the development of novel antibiotics to treat potential infections, mean that patients are living for longer. Nonetheless, over 27 million SCI patients across the globe remain disabled.1 As such, exploring approaches to repairing nerve damage and encouraging nerve regeneration is a major topic in neuroscience that carries great clinical significance.
A major barrier in this research space is the formation of a glial scar post-nerve injury.
What is a glial scar?
CNS injury triggers a cascade of stress responses that ultimately induce the activation of astrocytes, a "star" shaped glial cell, and accompanied production of cytokines, chemokines, neurotrophic factors and growth factors. The astrocytes create a web of plasma membrane extensions that aim to fill the gap where damage has occurred. This process causes a change in the extracellular matrix environment of the injury, and there is secretion of molecules including chondroitin sulfate (CS) and dermatin sulfate (DS) proteoglycans.2 The glial scar has good intentions: it aims to protect the nerve site immediately after damage has occurred and create a wall between injured and healthy tissue. But ultimately, the formation of the scar inhibits nerve regeneration and hinders a patient's recovery.
Chondroitinase ABC promotes nerve recovery in animal models
Consequently, the glial scar has been a major focus of nerve regeneration research over the past few decades. An enzyme known as chondroitinase ABC, or ChABC, derived from the bacterium Proteus vulgaris has been explored as a potential treatment option for CNS nerve damage.3 The enzyme acts by degrading the side chains of chondroitin sulfate proteoglycans (CSPGs), which removes the regeneration inhibition characteristic of the glial scar in a variety of nerve injury models, including mice, cats, dogs and rhesus monkey models of nerve injury. A review published earlier this year by Griffin and Bradke noted: "Ultimately, removal of CSPGs has proven to be consistently advantageous for nearly two decades and we anticipate the next steps taken towards clinical translation of CSPG‐targeting therapies." 4
Unfortunately, a challenge in the application of ChABC for nerve injury treatment is that it isn't very stable. “It’s stable enough for the environment that the bacteria live in, but inside the body it is very fragile,” said Professor Molly Shoichet of the Shoichet Lab at the University of Toronto in a press release. “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.”
Redesigning nature's work
Shoichet is the senior author of a study published in Science Advances which explores the impact of computationally reengineering the enzyme by inducing amino acid changes to create a more stable version.5
“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.”
Starting with an X-ray crystal structure for the enzyme ChABC, the team used the Protein Repair One-Stop Shop (PROSS) to predict stabilizing mutations.
This produced a large amount of data that needed to be narrowed down. “There are more than 1,000 links in the chain that forms this enzyme, and for each link you have 20 amino acids to choose from,” said Professor Mathew O'Meara, a professor in computational medicine and bioinformatics at the University of Michigan. O'Meara, who is the co-lead author of the paper, added: "There are too many choices to simulate them all.”
The team took inspiration from nature, exploring the amino acid substitutions that exist in real organisms. This process is known as consensus design; it relies on the idea that these mutant forms of ChABC don't actually exist naturally, but it's extremely likely that they are similar to mutant forms that do exist.
Greater activity and stability
Ultimately, the researchers discovered three new candidate forms of ChABC that they expressed in E.coli and assessed their enzymatic activity against CS and DS substrates. Only one form, ChABC-37-SH3, demonstrated higher initial enzymatic activity against both CS and DS compared to the wild type enzyme, whereas ChABC-55-SH3 and ChABC-92-SH3 demonstrated significantly lower activity. All three mutant forms were more stable than the wild type.
“The wild type chondroitinase ABC loses most of its activity within 24 hours, whereas our re-engineered enzyme is active for seven days,” Marian Hettiaratchi, the other co-lead author of the paper, said. In the paper, the authors note that this highlights a limitation of the PROSS method: "This indicates that, while PROSS successfully predicted increased protein structural stability, one limitation of this approach is that it cannot reliably predict enzymatic activity."
The researchers indicate in the press release that their next steps will be to explore the use of this enzyme in similar experiments to where the wild type protein has previously been adopted. In the discussion, they emphasize the utility of PROSS in optimizing a therapeutic agent: "More broadly, with this first demonstration of the use of PROSS to optimize a therapeutic agent, such as ChABC, we highlight the versatility of this method and open up this approach for others to use in optimizing the stability and activity of other particularly sensitive proteins."
1. Yang T, Dai Y, Chen G and Cui S. Dissecting the dual role of the glial Scar and scar-forming astrocytes in spinal cord injury. Front. Cell. Neurosci. 2020;14(1662-5102):10.3389/fncel.2020.00078.
2. He Y, Liu X, Chen Z. Glial Scar—a promising target for improving outcomes after CNS injury. Journal of Molecular Neuroscience. 2020;70(3):340-352. doi:10.1007/s12031-019-01417-6.
3. Mahajan R. Chondroitinase ABC Enzyme: A potential treatment option for spinal cord injury. Int J Appl Basic Med Res. 2018;8(4):203. doi:10.4103/ijabmr.IJABMR_336_18.
4. Griffin JM, Bradke F. Therapeutic repair for spinal cord injury: combinatory approaches to address a multifaceted problem. EMBO Mol Med. 2020;12(3):e11505. doi:10.15252/emmm.201911505.
5. Hettiaratchi MH, O’Meara MJ, O’Meara TR, Pickering AJ, Letko-Khait N, Shoichet MS. Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability. Sci Adv. 2020;6(34):eabc6378. doi:10.1126/sciadv.abc6378.