title: Intestinal cell targeting of a stable recombinant Cu-Zn SOD from Cucumis melo fused to a gliadin peptide.
abstract: The mRNA encoding full length chloroplastic Cu-Zn SOD (superoxide dismutase) of Cucumis melo (Cantaloupe melon) was cloned. This sequence was then used to generate a mature recombinant SOD by deleting the first 64 codons expected to encode a chloroplastic peptide signal. A second hybrid SOD was created by inserting ten codons to encode a gliadin peptide at the N-terminal end of the mature SOD. Taking account of codon bias, both recombinant proteins were successfully expressed and produced in Escherichia coli. Both recombinant SODs display an enzymatic activity of ~5000U mg(-1) and were shown to be stable for at least 4h at 37°C in biological fluids mimicking the conditions of intestinal transit. These recombinant proteins were capable in vitro, albeit at different levels, of reducing ROS-induced-apoptosis of human epithelial cells. They also stimulated production and release in a time-dependent manner of an autologous SOD activity from cells located into jejunum biopsies. Nevertheless, the fused gliadin peptide enable the recombinant Cu-Zn SOD to maintain a sufficiently sustained interaction with the intestinal cells membrane in vivo rather than being eliminated with the flow. According to these observations, the new hybrid Cu-Zn SOD should show promise in applications for managing inflammatory bowel diseases.
authors.affiliation: ISOCELL Pharma-53bd du General Martial Valin, Paris, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Intes
authors.forename: Muriel
authors.initials: M
authors.lastname: Bahut
authors.forename: Pascal
authors.initials: P
authors.lastname: Nicole
authors.forename: Alain
authors.initials: A
authors.lastname: Couvineau
authors.forename: Catherine
authors.initials: C
authors.lastname: Guette
authors.forename: Alphonse
authors.initials: A
authors.lastname: Calenda
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D018938:Caco-2 Cells; D049109:Cell Proliferation / Q000187:drug effects; D002736:Chloroplasts / Q000201:enzymology; D028463:Cucumis melo / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005772:Gastrointestinal Transit; D005903:Gliadin / Q000737:chemistry* / Q000235:genetics; D019073:HT29 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005903:Gliadin; D013482:Superoxide Dismutase