title: Proteolytic and N-glycan processing of human α1-antitrypsin expressed in Nicotiana benthamiana.
abstract: Plants are increasingly being used as an expression system for complex recombinant proteins. However, our limited knowledge of the intrinsic factors that act along the secretory pathway, which may compromise product integrity, renders process design difficult in some cases. Here, we pursued the recombinant expression of the human protease inhibitor α1-antitrypsin (A1AT) in Nicotiana benthamiana. This serum protein undergoes intensive posttranslational modifications. Unusually high levels of recombinant A1AT were expressed in leaves (up to 6 mg g(-1) of leaf material) in two forms: full-length A1AT located in the endoplasmic reticulum displaying inhibitory activity, and secreted A1AT processed in the reactive center loop, thus rendering it unable to interact with target proteinases. We found that the terminal protein processing is most likely a consequence of the intrinsic function of A1AT (i.e. its interaction with proteases [most likely serine proteases] along the secretory pathway). Secreted A1AT carried vacuolar-type paucimannosidic N-glycans generated by the activity of hexosaminidases located in the apoplast/plasma membrane. Notwithstanding, an intensive glycoengineering approach led to secreted A1AT carrying sialylated N-glycan structures largely resembling its serum-derived counterpart. In summary, we elucidate unique insights in plant glycosylation processes and show important aspects of postendoplasmic reticulum protein processing in plants.
authors.affiliation: Departments of Applied Genetics and Cell Biology (A.C., P.G., L.M., R.S., H.S.) andChemistry (M.W., F.A.), University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Castilho
authors.affiliation: Departments of Applied Genetics and Cell Biology (A.C., P.G., L.M., R.S., H.S.) andChemistry (M.W., F.A.), University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Windwarder
authors.affiliation: Departments of Applied Genetics and Cell Biology (A.C., P.G., L.M., R.S., H.S.) andChemistry (M.W., F.A.), University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
authors.forename: Pia
authors.initials: P
authors.lastname: Gattinger
authors.affiliation: Departments of Applied Genetics and Cell Biology (A.C., P.G., L.M., R.S., H.S.) andChemistry (M.W., F.A.), University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
authors.forename: Lukas
authors.initials: L
authors.lastname: Mach
authors.affiliation: Departments of Applied Genetics and Cell Biology (A.C., P.G., L.M., R.S., H.S.) andChemistry (M.W., F.A.), University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
authors.forename: Richard
authors.initials: R
authors.lastname: Strasser
authors.affiliation: Departments of Applied Genetics and Cell Biology (A.C., P.G., L.M., R.S., H.S.) andChemistry (M.W., F.A.), University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Departments of Applied Genetics and Cell Biology (A.C., P.G., L.M., R.S., H.S.) andChemistry (M.W., F.A.), University of Natural Resources and Life Sciences, 1190 Vienna, Austria herta.steinkellner@boku.ac.at.
authors.forename: Herta
authors.initials: H
authors.lastname: Steinkellner
mesh_terms: D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D011994:Recombinant Proteins; D055571:Secretory Pathway; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin; D057057:Serine Proteases