Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC

PLOS ONE, Nov 2019

Background There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. Methodology/Principal findings To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3’UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Conclusion/Significance Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.

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Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC

November Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC Elizabeth Muir 0 1 Mansoor Raza 0 1 Clare Ellis 0 1 Emily Burnside 0 1 Fiona Love 0 1 Simon Heller 0 1 Matthew Elliot 0 1 Esther Daniell 0 1 Debayan Dasgupta 0 1 Nuno Alves 0 1 Priscilla Day 0 1 James Fawcett 1 Roger Keynes 0 1 0 Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom, 2 John Van Geest Centre for Brain Repair, University of Cambridge , Forvie Site, Cambridge , United Kingdom 1 Editor: Catherine Faivre-Sarrailh, Aix Marseille University , FRANCE - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing interests: The authors have declared that no competing interests exist. Background There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. Methodology/Principal findings To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Conclusion/Significance Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins. Introduction While much is known about expressing mammalian proteins in bacterial cells, little is known about the requirements for passage of a bacterial protein through the secretory pathway of mammalian cells. We have previously shown that strategic removal of at least three N-glycosylation sites is required to achieve secretion of chondroitinase ABC (ChABC), a bacterial enzyme from Proteus Vulgaris by mammalian cells [ 1 ]. Here we have addressed whether it is possible to increase the efficiency of enzyme secretion by introducing further modifications to the bacterial gene. We removed additional N-glycosylation sites from regions where glycosylation could potentially adversely affect substrate binding. We also assessed the use of alternative leader sequences to direct enzyme secretion from the cells. Further, we evaluated the effect of directing secretion of the enzyme to the neuronal growth cone on neurite outgrowth. There is currently no effective treatment for promoting regeneration of injured nerves in patients following brain trauma or spinal cord injury. The principal cause of disability is the regenerative failure of mammalian CNS axons, which is due in part to up-regulation of axon growth-inhibitory chondroitin sulphate proteoglycans (CSPGs) in the region of injury [ 2 ]. ChABC promotes axon regeneration following CNS injury by removing axon growthinhibitory CSPGs in the lesion site, and by promoting neural plasticity [ 3,4 ]. This latter action, involving formation of new synaptic connections by intact undamaged neurons, has the beneficial consequence of promoting functional recovery. Additionally, we have shown recently that application of the enzyme also promotes the accumulation of anti-inflammatory (M2-like) macrophages at the lesion site [5]. These promote wound resolution and markedly reduce the secondary cavity formation and glial scarring that typically follows injury. ChABC treatment has further bee (...truncated)


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Elizabeth Muir, Mansoor Raza, Clare Ellis, Emily Burnside, Fiona Love, Simon Heller, Matthew Elliot, Esther Daniell, Debayan Dasgupta, Nuno Alves, Priscilla Day, James Fawcett, Roger Keynes. Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC, PLOS ONE, 2017, Volume 12, Issue 11, DOI: 10.1371/journal.pone.0186759