Substrate Specificity and Biochemical Characteristics of an Engineered Mammalian Chondroitinase ABC.

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Fawcett, James W 
Kwok, Jessica CF 

Chondroitin sulfate proteoglycans inhibit regeneration, neuroprotection, and plasticity following spinal cord injury. The development of a second-generation chondroitinase ABC enzyme, capable of being secreted from mammalian cells (mChABC), has facilitated the functional recovery of animals following severe spinal trauma. The genetically modified enzyme has been shown to efficiently break down the inhibitory extracellular matrix surrounding cells at the site of injury, while facilitating cellular integration and axonal growth. However, the activity profile of the enzyme in relation to the original bacterial chondroitinase (bChABC) has not been determined. Here, we characterize the activity profile of mChABC and compare it to bChABC, both enzymes having been maintained under physiologically relevant conditions for the duration of the experiment. We show that this genetically modified enzyme can be secreted reliably and robustly in high yields from a mammalian cell line. The modifications made to the cDNA of the enzyme have not altered the functional activity of mChABC compared to bChABC, ensuring that it has optimal activity on chondroitin sulfate-A, with an optimal pH at 8.0 and temperature at 37 °C. However, mChABC shows superior thermostability compared to bChABC, ensuring that the recombinant enzyme operates with enhanced activity over a variety of physiologically relevant substrates and temperatures compared to the widely used bacterial alternative without substantially altering its kinetic output. The determination that mChABC can function with greater robustness under physiological conditions than bChABC is an important step in the further development of this auspicious treatment strategy toward a clinical application.

3403 Macromolecular and Materials Chemistry, 34 Chemical Sciences, 3406 Physical Chemistry, 40 Engineering, 4004 Chemical Engineering, Physical Injury - Accidents and Adverse Effects, Traumatic Head and Spine Injury, Rehabilitation, Regenerative Medicine, Neurodegenerative, Spinal Cord Injury, 5.2 Cellular and gene therapies, 5 Development of treatments and therapeutic interventions
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ACS Omega
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American Chemical Society (ACS)
MRC (MR/V002694/1)
Medical Research Council (MR/R004463/1)