Correct Processing of the Kiwifruit Protease Actinidin in Transgenic Tobacco Requires the Presence of the C-Terminal Propeptide

Abstract

A 35s cauliflower mosaic virus promoter and a tapetum-specific promoter were used to direct the synthesis in tobacco of preproactinidin and a derivative that lacked a C-terminal extension. Preproactinidin was processed into a form that migrated identically on protein gels with mature actinidin extracted from kiwifruit. This protein was proteolytically active in vitro, and high-leve1 accumulation of this protein appeared to be detrimental to plant growth. Plants expressing an actinidin cDNA construct that lacked the sequence encoding the C-terminal propeptide were phenotypically normal but accumulated N-proactinidin, which was proteolytically active in vitro but did not self-cieave to mature actinidin. In transgenic tobacco, the C-terminal extension of actinidin is therefore required for correct processing.

Structural and Functional Characterization of the Major Allergen Amb a 11 from Short Ragweed Pollen

“…Based on results obtained with the pro-rAmb a 11 ⌬CT variant, we conclude that this C-terminal propeptide does not inhibit the protease activity of rAmb a 11, nor is it required for proper folding and maturation of the molecule. Such observations differ from evidence that the C-terminal propeptide of other plant cysteine proteases, like Act d 1 and ervatamin C, can regulate either the folding or the enzymatic activity of corresponding proforms (43,44). Of note, the presence of a KLLEL motif at the end of this C-terminal propeptide, resembling the (K/H)DEL motif observed in plant cysteine proteases such as vignain (17,45,46), strongly suggests a role for this propeptide in the retention within the endoplasmic reticulum and intracellular transport of the molecule.…”

Applications of Biotechnology in Kiwifruit (Actinidia)

“…Prior to the initiation of generating the Actinidia EST resource in 2000, reports of functional genomics through expression of Actinidia genes in either a heterologous or an Actinidia host were somewhat limited (Guo et al, 1999;Lay et al, 1996;Paul et al, 1995;Schroder et al, 1998; Z.C. Xu et al, 1998).…”

“…Paul et al (1995) expressed A. deliciosa preproactinidin in transgenic Nicotiana tabacum, showing that the protein was correctly processed and detrimental to plant growth when it accumulated to high levels. Yin et al (2010) showed that expression of the A.…”

Isolation and characterization of natural and recombinant allergens from green kiwifruit (Actinidia deliciosa Liang, Ferguson) for allergy diagnosis and therapy

“…Pored jako proteinske detaljne karakterizacije fiziološka funkcija aktinidina nije još uvek poznata [50]. Smatra se da može da služi kao rezervni protein, poput patatina [51], ili da učestvuje u odbrani biljke od insekata i gljiva [52].…”

“…Nakon procesa prečišćavanja nCPI je pokazao inhibitornu aktivnost prema papainu (EC 50 Klinička procena IgE reaktvnosti pojedinačnih proteina iz ekstrakta kivija zahtevala bi imunoblotove sa velikom populacijom pacijenta i takva studija obično se bazira na enzimskim esejima [124,125]. Rezultati do kojih se došlo u ovoj tezi sugerišu da značaj nekih alergena, kao što je nCPI (Act d 4), pri ovakvoj eksperimentalnoj postavci može da bude potcenjen.…”