Proteolytic and N-glycan processing of human α1-antitrypsin expressed in Nicotiana benthamiana.

Alexandra Castilho,Lukas Mach,Friedrich Altmann,Markus Windwarder,Herta Steinkellner,Pia Gattinger,Richard Strasser

doi:10.1104/pp.114.250720

Alexandra Castilho, Lukas Mach + Show 5 more

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https://doi.org/10.1104/pp.114.250720

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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.

Highlights

Plants are increasingly being used as an expression system for complex recombinant proteins
Two signals of 40 and 52 kD were detected in total soluble protein (TSP) extracts, and the 52-kD band corresponded to the size of the full-length protein
A band corresponding to the full-length size was present in StrepA1AT purified from TSP (Fig. 2A)

Summary

Introduction

Plants are increasingly being used as an expression system for complex recombinant proteins. We pursued the recombinant expression of the human protease inhibitor a1-antitrypsin (A1AT) in Nicotiana benthamiana This serum protein undergoes intensive posttranslational modifications. The biosynthesis and physiological significance of this N-glycan formation has yet to be completely explained (Strasser et al, 2007; Liebminger et al, 2011) Another process not fully understood in plants is subcellular localization of proteins. Recombinant proteins designed for secretion are frequently located in the endoplasmic reticulum (ER) and as a consequence, carry oligomannosidic carbohydrates instead of the desired complex-type glycans (Loos et al, 2011; Schneider et al, 2014a). One human protein that is pharmaceutically interesting, and needed in large amounts at high quality, is a1-antitrypsin (A1AT) This highly glycosylated protease inhibitor from the serpin superfamily interacts with a wide variety of proteases (Gettins, 2002). Sialylated N-glycans are a well-known requisite for the plasma half-life of A1AT (Mast et al, 1991; Lindhout et al, 2011; Lusch et al, 2013); the difficulties associated with obtaining them hamper the generation of biologically active A1AT in many expression systems

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