Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting

Abstract

Hordothionins (HTHs) are small anti-bacterial proteins present in barley endosperm which are processed from larger precursor proteins, consisting of an amino-terminal signal peptide (SP), the mature highly basic HTH and a carboxy-terminal acidic peptide (AP). Different HTH precursor proteins were expressed in tobacco to study the effects of the pre-sequences (SP) and pro-sequences (AP) on expression, processing, sorting and biological activity and hence the feasibility of engineering bacterial disease resistance into crops which lack these proteins. Maximum HTH expression levels of approximately 0.7% (11 mumol/kg) of total soluble protein in young tobacco leaves were obtained using a semi-synthetic gene construct encoding a complete chimaeric HTH precursor protein. Tenfold lower HTH expression levels (maximum 1.3 mumol/kg) were obtained using synthetic gene constructs without the AP-coding sequence and no expression was found in plants containing synthetic HTH gene constructs without SP- and AP-coding sequences. In both cases where expression was found, the precursors were apparently correctly processed, although the HTH produced in plants containing a construct without AP sequence appeared to be slightly modified. No effect on plant phenotype was observed. Localization studies indicated that the HTH was in identical fractions of plants expressing the two different precursors, albeit at a different ratio, and was not secreted into the intercellular spaces of leaves or culture medium by protoplasts. Our results indicated that the AP is not involved in sorting and suggested that it might facilitate transport through membranes. The in vitro toxicity of HTH isolated from transgenic tobacco plants expressing the two different precursor proteins for the bacterial plant pathogen Clavibacter michiganensis subsp. michiganensis appeared similar to that of the HTH purified from barley endosperm.

A plant signal peptide–hepatitis B surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells

“…If HBsAg signal I does not fully function in plant cells, some of the nascent chains may fold incorrectly in the reducing environment of the cytoplasm. Therefore, inefficient ER targeting could result in poor HBsAg accumulation in the plant system, as was observed with hordothionins expressed in transgenic tobacco (21). There is evidence in other systems that the host signal peptide functions more efficiently than a transgene's native signal peptide (22), findings consistent with our data on HBsAg expression.…”

Latarcins, Antimicrobial and Cytolytic Peptides from the Venom of the Spider Lachesana tarabaevi (Zodariidae) That Exemplify Biomolecular Diversity

“…We suppose that the functional role of the latarcin precursor prosequence is to neutralize the high positive charge of the mature peptide and thus prevent its interactions with proteins/lipids and inhibit its cytolytic activity during biosynthesis. These functions were also suggested previously for acidic prosequences in precursors of insect and mammalian defensins and plant thionins (42,52,53). Subsequent latarcin precursor processing in the venom gland results in mature chain separation and functional activation.…”

Production of recombinant proteins in transgenic plants: Practical considerations

“…Tobacco and Brassica napus have successfully performed a several-step proteolytic processing of recombinant thionins (Carmona et al, 1993;Florack et al, 1994), Brazil nut 2S albumin (Altenbach et al, 1989(Altenbach et al, , 1992 and human protein C (Cramer et al, 1996). The cleavage of both the aminoterminal signal peptide and the carboxyterminal peptide of prepro-thionins appeared to be important for mediating the transition of the recombinant protein into the endoplasmic reticulum and the correct folding of mature thionins, respectively (Florack et al, 1994).…”

“…The cleavage of both the aminoterminal signal peptide and the carboxyterminal peptide of prepro-thionins appeared to be important for mediating the transition of the recombinant protein into the endoplasmic reticulum and the correct folding of mature thionins, respectively (Florack et al, 1994).…”