AtPng1p. The first plant transglutaminase

Computational analysis identified a putative gene in Arabidopsis thaliana coding for a putative peptide N-glycanase, AtPng1p. This protein contains the Cys-His-Asp amino acid residues typical of the catalytic tryad of animal transglutaminase enzymes (E.C. 2.3.2.13), till now not sequenced but functionally active in plants. AtPng1p is a single gene, expressed ubiquitously but at very low level in Arabidopsis (entire plant, different organs, growth stages, and light conditions), as shown by nested RT-PCR. AtPng1p was over-expressed in E. coli, purified and refolded. Western blot analysis with antibodies raised against this protein, detected an 86 kDa band in the Arabidopsis microsomal fraction and some others in cytosol. The recombinant protein, immunodetected by three different antibodies raised against transglutaminases of animal origin, displays a Ca2+-dependent capacity to conjugate, to a decreasing extent, spermine, spermidine and putrescine to dimethyl-casein, as shown by gel autoradiography and determination of glutamyl-derivatives. The synthetic transglutaminase substrate, benzyloxy-carbonyl-L-glutamyl-glycine is also recognised as a substrate of the enzyme. Similarly to guinea pig liver transglutaminase, this enzyme caused cross-linking of bovine serum albumin in the presence of Ca2+ and dithiotreitol, with a maximum at pH 8.5-9, also confirmed by the biotin-cadaverine assay, whereas GTP inhibited and Mg2+, Na+ and K+ were ineffective. This work reports for the first time the characterisation of a recombinant plant transglutaminase, as all the parameters analysed accomplished the typical features of known transglutaminase enzymes of animal origin.