Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC
| Autor: | Debayan Dasgupta, Esther Daniell, Matthew Elliot, Nuno Alves, James W. Fawcett, Clare Ellis, Fiona Love, Elizabeth M. Muir, Roger J. Keynes, Priscilla Day, Simon Heller, Mansoor Raza, Emily Burnside |
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| Přispěvatelé: | Muir, Elizabeth [0000-0002-8821-5051], Apollo - University of Cambridge Repository |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Macroglial Cells Glycosylation Physiology Glycobiology lcsh:Medicine Chondroitin ABC Lyase Biochemistry Substrate Specificity 0302 clinical medicine Nerve Fibers Animal Cells Medicine and Health Sciences Post-Translational Modification lcsh:Science 3' Untranslated Regions chemistry.chemical_classification Neurons Mammals Multidisciplinary Chemistry Transfection Cell biology Protein Transport Cell Processes Female Cellular Types Research Article Signal peptide Neurite Growth Cones Chondroitin ABC lyase Glial Cells Protein Sorting Signals Research and Analysis Methods Fluorescence Cell Line 03 medical and health sciences Dogs Bacterial Proteins Neurites Animals Humans Secretion Growth cone Molecular Biology Techniques Molecular Biology lcsh:R Biology and Life Sciences Proteins Protein Secretion Cell Biology Neuronal Dendrites Axons Actins Rats 030104 developmental biology Enzyme Cell culture Cellular Neuroscience lcsh:Q Schwann Cells Physiological Processes Protein Processing Post-Translational 030217 neurology & neurosurgery Neuroscience |
| Zdroj: | PLoS ONE, Vol 12, Iss 11, p e0186759 (2017) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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