title: Efficient secretion of biologically active Chondroitinase ABC from mammalian cells in the absence of an N-terminal signal peptide.
abstract: Proteoglycans carrying chondroitin sulfate side chains have been shown to fulfill important biological functions in development, disease, and signaling. One area of considerable interest is the functional importance of chondroitin sulfates as inhibitors of the regeneration of axonal projections in the mammalian central nervous system. In animal models of spinal cord injury, injections of the enzyme Chondroitinase ABC from the bacterium Proteus vulgaris into the lesion site leads to degradation of chondroitin sulfates, and promotes axonal regeneration and significant functional recovery. Here, a mammalian expression system of an epitope-tagged Chondroitinase ABC protein is described. It is demonstrated that the addition of a eukaryotic secretion signal sequence to the N-terminus of the bacterial Chondroitinase ABC sequence allowed secretion, but interfered with function of the secreted enzyme. In contrast, expression of the Chondroitinase ABC gene without N-terminal eukaryotic secretion sequence or bacterial hydrophobic leader sequence led to efficient secretion of a biologically active Chondroitinase ABC protein from both immortalized and primary cells. Moreover, the C-terminal epitope tag could be utilized to follow expression of this protein. This novel Chondroitinase ABC gene is a valuable tool for a better understanding of the in vivo roles of chondroitin sulfates in mammalian development and disease, as well as in gene therapy approaches, including the treatment of spinal chord injuries.
authors.affiliation: Department of Pediatrics, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60614-4314, USA. m-kluppel@northwestern.edu
authors.forename: Michael
authors.initials: M
authors.lastname: Klüppel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D019765:Chondroitin ABC Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D021382:Protein Sorting Signals; D017386:Sequence Homology, Amino Acid
chemical_list: D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D021382:Protein Sorting Signals; D019765:Chondroitin ABC Lyase