Producing transglutaminases by molecular farming in plants: minireview article.

Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities, both in terms of biology and biotechnology, is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases in research, medicine and industry has increased the demand for an inexpensive, efficient and safe source of recombinant enzymes. We describe initial results concerning the production of a mammalian transglutaminase in transgenic rice plants as a first step towards the large-scale molecular farming of this enzyme.

Suborganellar localisation and effect of light on Helianthus tuberosus chloroplast transglutaminases and their substrates.

The light stimulation of transglutaminase (TGase EC 2.3.2.13) activity was verified by incubating isolated chloroplasts of Helianthus tuberosus L. continuously or for alternate periods of light or dark (light/dark and dark/light). The first 10 min of incubation always represented the critical period. Light-harvesting complexes of photosystem II (LHCII) were more intensely labelled by (14)C-polyamines under light and light/dark than under dark and dark/light conditions. Chloroplasts were fractionated into thylakoid- and stroma-enriched fractions in which multiple TGase forms and substrates were found. Antibodies against TGase recognised 58- and 24-kDa bands in thylakoids and a 150-kDa band in the stroma. The latter, and its 150-kDa fraction, catalysed the conjugation of 14C-polyamines to Rubisco. In both fractions (thylakoid-pre and stroma-pre) the analysis of polyamine glutamyl derivatives showed a significant light-affected conjugation of polyamines to endogenous proteins. Alternatively, entire chloroplasts were incubated and afterwards their sub-fractions were isolated (thylakoid-post and stroma-post). The PSII and LHCII complexes were more intensely immunodetected in thylakoid-post than in thylakoid-pre, especially under dark conditions. Conversely, the conjugation of polyamines to thylakoid proteins was clearly light-stimulated in thylakoid-post, and much less in thylakoid-pre. Stroma-pre proteins were poorly polyamine-conjugated and not light-affected; on the contrary, stroma-post proteins were much more polyamine-modified and strongly light-stimulated. Thus, the light-activated conjugation depends mainly on the presence of the thylakoid fraction during the assay. The protective effect on chloroplasts under photo-damage, stress or senescence conditions attributed in the literature to free polyamines is discussed with regard to the occurrence of polyamine conjugates catalysed by TGases.

Immunogold localization of a transglutaminase related to grana development in different maize cell types.

A comparative study of the subcellular localization of a plant transglutaminase (TGase; EC 2.3.2.13) in various in vivo and in vitro maize cell types was carried out with a polyclonal antibody raised against a 58 kDa TGase purified from Helianthus tuberosus leaves. Immunocytochemical staining, followed by electron microscopy, showed that this enzyme was markedly present in the grana-appressed thylakoids of mature chloroplasts of the light-exposed cells. Moreover, during embryogenic callus chloroplast differentiation, the abundance of TGase in the grana-appressed thylakoids depended on the degree of grana development and was greater than in mature leaf chloroplasts. In addition to the 58 kDa form, two other forms of the protein (of 77 and 34 kDa) were obtained by Western blot. The 77 kDa form might correspond to the inactive form and was immunodetected in dense vesicles observed in dark-grown embryogenic callus cells. In adult leaves, the enzyme was also markedly present in the grana-appressed thylakoids of the mesophyll cell chloroplasts, though very scarce and dispersed in the bundle-sheath cell chloroplasts (which do not contain grana). The concordance of these localizations with those described for the light-harvesting antenna proteins of the photosystem II suggests that it is possible that this TGase has a functional role in photosynthesis, perhaps modulating the photosynthetic efficiency and the absorption of excess light by means of polyamine conjugation to the antenna proteins.