title: Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells.
abstract: Although many eukaryotic proteins have been secreted by transfected bacterial cells, little is known about how a bacterial protein is treated as it passes through the secretory pathway when expressed in a eukaryotic cell. The eukaryotic N-glycosylation system could interfere with folding and secretion of prokaryotic proteins whose sequence has not been adapted for glycosylation in structurally appropriate locations. Here we show that such interference does indeed occur for chondroitinase ABC from the bacterium Proteus vulgaris, and can be overcome by eliminating potential N-glycosylation sites. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. As these proteoglycans are known to inhibit regeneration of axons in the mammalian central nervous system, the modified chondroitinase gene is a potential tool for gene therapy to promote neural regeneration, ultimately in human spinal cord injury.
authors.affiliation: Department of Physiology Development and Neuroscience, University of Cambridge, Downing St., Cambridge CB2 3EG, UK.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Muir
authors.forename: Ian
authors.initials: I
authors.lastname: Fyfe
authors.forename: Sonya
authors.initials: S
authors.lastname: Gardiner
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Philippa
authors.initials: P
authors.lastname: Warren
authors.forename: James W
authors.initials: JW
authors.lastname: Fawcett
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Keynes
authors.forename: John H
authors.initials: JH
authors.lastname: Rogers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D019765:Chondroitin ABC Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011514:Proteus vulgaris / Q000201:enzymology* / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000502:physiology*
chemical_list: D011994:Recombinant Proteins; D019765:Chondroitin ABC Lyase