Targeting and post‐translational processing of human α₁‐antichymotrypsin in BY‐2 tobacco cultured cells†

Abstract

Summary The post‐translational processing of human α1‐antichymotrypsin (AACT) in Bright Yellow‐2 (BY‐2) tobacco cells was assessed in relation to the cellular compartment targeted for accumulation. As determined by pulse‐chase labelling experiments and immunofluorescence microscopy, AACT sent to the vacuole or the endoplasmic reticulum (ER) was found mainly in the culture medium, similar to a secreted form targeted to the apoplast. Unexpectedly, AACT expressed in the cytosol was found in the nucleus under a stable, non‐glycosylated form, in contrast with secreted variants undergoing multiple post‐translational modifications during their transit through the secretory pathway. All secreted forms of AACT were N‐glycosylated, with the presence of complex glycans as observed naturally on human AACT. Proteolytic trimming was also observed for all secreted variants, both during their intracellular transit and after their secretion in the culture medium. Overall, the targeting of human AACT to different compartments of BY‐2 tobacco cells led to the production of two protein products: (i) a stable, non‐glycosylated protein accumulated in the nucleus; and (ii) a heterogeneous mixture of secreted variants resulting from post‐translational N‐glycosylation and proteolytic processing. Overall, these data suggest that AACT is sensitive to resident proteases in the ER, the Golgi and/or the apoplast, and that the production of intact AACT in the plant secretory pathway will require innovative approaches to protect its structural integrity in vivo. Studies are now needed to assess the activity of the different AACT variants, and to identify the molecular determinants for the nuclear localization of AACT expressed in the cytosol.

Tomato cystatin SlCYS8 as a stabilizing fusion partner for human serpin expression in plants

“…Sl CYS8 (10.7 kDa) is about one quarter the size of α1ACT (45 kDa) and was, therefore, more abundant than the human protein on a molar basis. In contrast to the single band detected for the nonglycosylated Sl CYS8, α1ACT was detected as multiple bands (Figure ), presumably due to both proteolytic trimming and variable glycan‐processing in the ER and the Golgi (Benchabane et al ., ).…”

A protease activity–depleted environment for heterologous proteins migrating towards the leaf cell apoplast

“…, 2009a). In a similar way, human α 1 ‐antichymotrypsin targeted to the secretory pathway of BY‐2 tobacco cells could be detected in the culture medium but was cleaved in the C‐terminal region by intracellular and apoplastic resident proteases, within the protease inhibitory site providing biological activity against chymotrypsin (Benchabane et al. , 2009).…”

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

“…benthamiana leaves, and to characterize the antibody-stabilizing effects of co-expressed protease inhibitors at the domain sequence level of a promising therapeutic antibody. Tomato cystatin Sl CYS8 [33] and human serpin α 1 -antichymotrypsin (α 1 -ACT) [34] were used as accessory inhibitor models for the in situ inactivation of Cys and Ser proteases, respectively. H10, a human monoclonal IgG reported to target the tumour-associated antigen tenascin-C [8], was used as a model antibody.…”