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.

Centre for Reconstructive Neuroscience Institute of Experimental Medicine Czech Academy of Sciences Videnska 1083 14220 Prague 4 Czech Republic

Department of Clinical Neurosciences John van Geest Centre for Brain Repair University of Cambridge Cambridge CB2 0PY U K

Department of Physiology Development and Neuroscience University of Cambridge Cambridge CB2 0PY U K

School of Biomedical Sciences Faculty of Biological Sciences University of Leeds Leeds LS2 9JT U K

Wolfson Centre for Age Related Diseases Institute of Psychiatry Psychology and Neuroscience King's College London Guy's Campus London Bridge London SE1 1UL U K

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Properzi F.; Asher R. A.; Fawcett J. W. Chondroitin sulphate proteoglycans in the central nervous system: changes and synthesis after injury. Biochem. Soc. Trans. 2003, 31, 335–336. 10.1042/bst0310335. PubMed DOI

Sandvig A.; Berry M.; Barrett L. B.; Butt A.; Logan A. Myelin-, reactive glia-, and scar-derived CNS axon growth inhibitors: expression, receptor signaling, and correlation with axon regeneration. Glia 2004, 46, 225–251. 10.1002/glia.10315. PubMed DOI

Tran A. P.; Warren P. M.; Silver J. The Biology of Regeneration Failure and Success After Spinal Cord Injury. Physiol. Rev. 2018, 98, 881–917. 10.1152/physrev.00017.2017. PubMed DOI PMC

Yamagata T.; Saito H.; Habuchi O.; Suzuki S. Purification and properties of bacterial chondroitinases and chondrosulfatases. J. Biol. Chem. 1968, 243, 1523–1535. 10.1016/s0021-9258(18)93574-x. PubMed DOI

Huang W.; Matte A.; Suzuki S.; Sugiura N.; Miyazono H.; Cygler M. Crystallization and preliminary X-ray analysis of chondroitin sulfate ABC lyases I and II fromProteus vulgaris. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2000, 56, 904–906. 10.1107/s0907444900005102. PubMed DOI

Prabhakar V.; Raman R.; Capila I.; Bosques C. J.; Pojasek K.; Sasisekharan R. Biochemical characterization of the chondroitinase ABC I active site. Biochem 2005, 390, 395–405. 10.1042/bj20050532. PubMed DOI PMC

Huang W.-C.; Kuo W.-C.; Cherng J.-H.; Hsu S.-H.; Chen P.-R.; Huang S.-H.; Huang M.-C.; Liu J.-C.; Cheng H. Chondroitinase ABC promotes axonal re-growth and behavior recovery in spinal cord injury. Biochem. Biophys. Res. Commun. 2006, 349, 963–968. 10.1016/j.bbrc.2006.08.136. PubMed DOI

McKeon R. J.; Höke A.; Silver J. Injury-induced proteoglycans inhibit the potential for laminin-mediated axon growth on astrocytic scars. Exp. Neurol. 1995, 136, 32–43. 10.1006/exnr.1995.1081. PubMed DOI

Zuo J.; Neubauer D.; Dyess K.; Ferguson T. A.; Muir D. Degradation of chondroitin sulfate proteoglycan enhances the neurite-promoting potential of spinal cord tissue. Exp. Neurol. 1998, 154, 654–662. 10.1006/exnr.1998.6951. PubMed DOI

Moon L. D. F.; Asher R. A.; Rhodes K. E.; Fawcett J. W. Regeneration of CNS axons back to their target following treatment of adult rat brain with chondroitinase ABC. Nat. Neurosci. 2001, 4, 465–466. 10.1038/87415. PubMed DOI

Bradbury E. J.; Moon L. D. F.; Popat R. J.; King V. R.; Bennett G. S.; Patel P. N.; Fawcett J. W.; McMahon S. B. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 2002, 416, 636–640. 10.1038/416636a. PubMed DOI

Pizzorusso T.; Medini P.; Berardi N.; Chierzi S.; Fawcett J. W.; Maffei L. Reactivation of ocular dominance plasticity in the adult visual cortex. Science 2002, 298, 1248–1251. 10.1126/science.1072699. PubMed DOI

Warren P. M.; Steiger S. C.; Dick T. E.; MacFarlane P. M.; Alilain W. J.; Silver J. Rapid and robust restoration of breathing long after spinal cord injury. Nat. Commun. 2018, 9, 4843.10.1038/s41467-018-06937-0. PubMed DOI PMC

Tropea D.; Caleo M.; Maffei L. Synergistic effects of brain-derived neurotrophic factor and chondroitinase ABC on retinal fiber sprouting after denervation of the superior colliculus in adult rats. J. Neurosci. 2003, 23, 7034–7044. 10.1523/jneurosci.23-18-07034.2003. PubMed DOI PMC

Yick L. Axonal regeneration of Clarke’s neurons beyond the spinal cord injury scar after treatment with chondroitinase ABC. Exp. Neurol. 2003, 182, 160–168. 10.1016/s0014-4886(02)00052-3. PubMed DOI

Caggiano A. O.; Zimber M. P.; Ganguly A.; Blight A. R.; Gruskin E. A. Chondroitinase ABCI improves locomotion and bladder function following contusion injury of the rat spinal cord. J. Neurotrauma 2005, 22, 226–239. 10.1089/neu.2005.22.226. PubMed DOI

Cafferty W. B. J.; Bradbury E. J.; Lidierth M.; Jones M.; Duffy P. J.; Pezet S.; McMahon S. B. Chondroitinase ABC-Mediated Plasticity of Spinal Sensory Function. J. Neurosci. 2008, 28, 11998–12009. 10.1523/jneurosci.3877-08.2008. PubMed DOI PMC

Lin R.; Kwok J. C. F.; Crespo D.; Fawcett J. W. Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain. J. Neurochem. 2007, 104, 400–408. 10.1111/j.1471-4159.2007.05066.x. PubMed DOI

Tester N. J.; Plaas A. H.; Howland D. R. Effect of body temperature on chondroitinase ABC’s ability to cleave chondroitin sulfate glycosaminoglycans. J. Neurosci. Res. 2007, 85, 1110–1118. 10.1002/jnr.21199. PubMed DOI

Iseda T.; Okuda T.; Kane-Goldsmith N.; Mathew M.; Ahmed S.; Chang Y.-W.; Young W.; Grumet M. Single, High-Dose Intraspinal Injection of Chondroitinase Reduces Glycosaminoglycans in Injured Spinal Cord and Promotes Corticospinal Axonal Regrowth after Hemisection but Not Contusion. J. Neurotrauma 2008, 25, 334–349. 10.1089/neu.2007.0289. PubMed DOI

Tom V. J.; Kadakia R.; Santi L.; Houlé J. D. Administration of chondroitinase ABC rostral or caudal to a spinal cord injury site promotes anatomical but not functional plasticity. J. Neurotrauma 2009, 26, 2323–2333. 10.1089/neu.2009.1047. PubMed DOI PMC

Fuller D. D.; Lee K.-Z.; Tester N. J. The impact of spinal cord injury on breathing during sleep. Respir. Physiol. Neurobiol. 2013, 188, 344–354. 10.1016/j.resp.2013.06.009. PubMed DOI PMC

Tauchi R.; Imagama S.; Natori T.; Ohgomori T.; Muramoto A.; Shinjo R.; Matsuyama Y.; Ishiguro N.; Kadomatsu K. The endogenous proteoglycan-degrading enzyme ADAMTS-4 promotes functional recovery after spinal cord injury. J. Neuroinflammation 2012, 9, 53.10.1186/1742-2094-9-53. PubMed DOI PMC

Pakulska M. M.; Tator C. H.; Shoichet M. S. Local delivery of chondroitinase ABC with or without stromal cell-derived factor 1α promotes functional repair in the injured rat spinal cord. Biomaterials 2017, 134, 13–21. 10.1016/j.biomaterials.2017.04.016. PubMed DOI

Hyatt A. J. T.; Wang D.; Kwok J. C.; Fawcett J. W.; Martin K. R. Controlled release of chondroitinase ABC from fibrin gel reduces the level of inhibitory glycosaminoglycan chains in lesioned spinal cord. J. Controlled Release 2010, 147, 24–29. 10.1016/j.jconrel.2010.06.026. PubMed DOI

Azizi M.; Farahmandghavi F.; Joghataei M. T.; Zandi M.; Imani M.; Bakhtiari M.; Omidian H. ChABC-loaded PLGA nanoparticles: A comprehensive study on biocompatibility, functional recovery, and axonal regeneration in animal model of spinal cord injury. Int. J. Pharm. 2020, 577, 119037.10.1016/j.ijpharm.2020.119037. PubMed DOI

Cafferty W. B. J.; Yang S.-H.; Duffy P. J.; Li S.; Strittmatter S. M. Functional Axonal Regeneration through Astrocytic Scar Genetically Modified to Digest Chondroitin Sulfate Proteoglycans. J. Neurosci. 2007, 27, 2176–2185. 10.1523/jneurosci.5176-06.2007. PubMed DOI PMC

Muir E. M.; Fyfe I.; Gardiner S.; Li L.; Warren P.; Fawcett J. W.; Keynes R. J.; Rogers J. H. Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells. J. Biotechnol. 2010, 145, 103–110. 10.1016/j.jbiotec.2009.11.002. PubMed DOI PMC

Warren P. M.; Andrews M. R.; Smith M.; Bartus K.; Bradbury E. J.; Verhaagen J.; Fawcett J. W.; Kwok J. C. F. Secretion of a mammalian chondroitinase ABC aids glial integration at PNS/CNS boundaries. Sci. Rep. 2020, 10, 11262.10.1038/s41598-020-67526-0. PubMed DOI PMC

Day P.; Alves N.; Daniell E.; Dasgupta D.; Ogborne R.; Steeper A.; Raza M.; Ellis C.; Fawcett J.; Keynes R.; Muir E. Targeting chondroitinase ABC to axons enhances the ability of chondroitinase to promote neurite outgrowth and sprouting. PLoS One 2020, 15, e022185110.1371/journal.pone.0221851. PubMed DOI PMC

Bartus K.; James N. D.; Didangelos A.; Bosch K. D.; Verhaagen J.; Yáñez-Muñoz R. J.; Rogers J. H.; Schneider B. L.; Muir E. M.; Bradbury E. J. Large-scale chondroitin sulfate proteoglycan digestion with chondroitinase gene therapy leads to reduced pathology and modulates macrophage phenotype following spinal cord contusion injury. J. Neurosci. 2014, 34, 4822–4836. 10.1523/jneurosci.4369-13.2014. PubMed DOI PMC

Burnside E. R.; De Winter F.; Didangelos A.; James N. D.; Andreica E.-C.; Layard-Horsfall H.; Muir E. M.; Verhaagen J.; Bradbury E. J. Immune-evasive gene switch enables regulated delivery of chondroitinase after spinal cord injury. Brain 2018, 141, 2362.10.1093/brain/awy158. PubMed DOI PMC

Prabhakar V.; Capila I.; Bosques C. J.; Pojasek K.; Sasisekharan R. Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification. Biochem 2005, 386, 103–112. 10.1042/bj20041222. PubMed DOI PMC

Prabhakar V.; Capila I.; Raman R.; Srinivasan A.; Bosques C. J.; Pojasek K.; Wrick M. A.; Sasisekharan R. The Catalytic Machinery of Chondroitinase ABC I Utilizes a Calcium Coordination Strategy to Optimally Process Dermatan Sulfate†. Biochem 2006, 45, 11130–11139. 10.1021/bi0605484. PubMed DOI

Hiyama K.; Okada S. Action of Chondroitinases. J. Biochem. 1976, 80, 1201–1207. 10.1093/oxfordjournals.jbchem.a131390. PubMed DOI

Klüppel M. Efficient secretion of biologically active Chondroitinase ABC from mammalian cells in the absence of an N-terminal signal peptide. Mol. Cell. Biochem. 2011, 351, 1–11. 10.1007/s11010-010-0705-1. PubMed DOI

Guo Y.; Klüppel M.; Tang H.; Tan S.; Zhang P.; Chen Z. Lentivirus-mediated transfection of chondroitinase ABC gene without the bacterial leader sequence enables long-term secretion of functional chondroitinase ABC in human bone marrow stromal cells. Biotechnol. Lett. 2016, 38, 893–900. 10.1007/s10529-016-2046-y. PubMed DOI

Coulson-Thomas Y. M.; Coulson-Thomas V. J.; Filippo T. R.; Mortara R. A.; da Silveira R. B.; Nader H. B.; Porcionatto M. A. Adult bone marrow-derived mononuclear cells expressing chondroitinase AC transplanted into CNS injury sites promote local brain chondroitin sulphate degradation. J. Neurosci. Methods 2008, 171, 19–29. 10.1016/j.jneumeth.2008.01.030. PubMed DOI

Jin Y.; Ketschek A.; Jiang Z.; Smith G.; Fischer I. Chondroitinase activity can be transduced by a lentiviral vector in vitro and in vivo. J. Neurosci. Methods 2011, 199, 208–213. 10.1016/j.jneumeth.2011.05.007. PubMed DOI PMC

Curinga G. M.; Snow D. M.; Mashburn C.; Kohler K.; Thobaben R.; Caggiano A. O.; Smith G. M. Mammalian-produced chondroitinase AC mitigates axon inhibition by chondroitin sulfate proteoglycans. J. Neurochem. 2007, 102, 275–288. 10.1111/j.1471-4159.2007.04530.x. PubMed DOI

Kwok J. C. F.; Afshari F.; García-Alías G.; Fawcett J. W. Proteoglycans in the central nervous system: plasticity, regeneration and their stimulation with chondroitinase ABC. Restor. Neurol. Neurosci. 2008, 26, 131–145. PubMed

Properzi F.; Carulli D.; Asher R. A.; Muir E.; Camargo L. M.; van Kuppevelt T. H.; ten Dam G. B.; Furukawa Y.; Mikami T.; Sugahara K.; Toida T.; Geller H. M.; Fawcett J. W. Chondroitin 6-sulphate synthesis is up-regulated in injured CNS, induced by injury-related cytokines and enhanced in axon-growth inhibitory glia. Eur. J. Neurosci. 2005, 21, 378–390. 10.1111/j.1460-9568.2005.03876.x. PubMed DOI

Shaya D.; Hahn B.-S.; Park N. Y.; Sim J.-S.; Kim Y. S.; Cygler M. Characterization of Chondroitin Sulfate Lyase ABC fromBacteroides thetaiotaomicronWAL2926†. Biochem 2008, 47, 6650–6661. 10.1021/bi800353g. PubMed DOI

Lee H.; McKeon R. J.; Bellamkonda R. V. Sustained delivery of thermostabilized chABC enhances axonal sprouting and functional recovery after spinal cord injury. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 3340–3345. 10.1073/pnas.0905437106. PubMed DOI PMC

Alves J. N.; Muir E. M.; Andrews M. R.; Ward A.; Michelmore N.; Dasgupta D.; Verhaagen J.; Moloney E. B.; Keynes R. J.; Fawcett J. W.; Rogers J. H. AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations. J. Neurosci. Methods 2014, 227, 107–120. 10.1016/j.jneumeth.2014.02.010. PubMed DOI