Proteolytic and N-Glycan Processing of Human α1-Antitrypsin Expressed in Nicotiana benthamiana

Author:

Castilho Alexandra1,Windwarder Markus2,Gattinger Pia1,Mach Lukas1,Strasser Richard1,Altmann Friedrich2,Steinkellner Herta1

Affiliation:

1. Departments of Applied Genetics and Cell Biology (A.C., P.G., L.M., R.S., H.S.) and

2. Chemistry (M.W., F.A.), University of Natural Resources and Life Sciences, 1190 Vienna, Austria

Abstract

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.

Publisher

Oxford University Press (OUP)

Subject

Plant Science,Genetics,Physiology

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