Transglutaminases: widespread cross-linking enzymes in plants

Abstract

Background: Transglutaminases have been studied in plants since 1987 in investigations aimed at interpreting some of the molecular mechanisms by which polyamines affect growth and differentiation. Transglutaminases are a widely distributed enzyme family catalysing a myriad of biological reactions in animals. In plants, the post-translational modification of proteins by polyamines forming inter- or intra-molecular cross-links has been the main transglutaminase reaction studied.

Characteristics of plant transglutaminases: The few plant transglutaminases sequenced so far have little sequence homology with the best-known animal enzymes, except for the catalytic triad; however, they share a possible structural homology. Proofs of their catalytic activity are: (a) their ability to produce glutamyl-polyamine derivatives; (b) their recognition by animal transglutaminase antibodies; and (c) biochemical features such as calcium-dependency, etc. However, many of their fundamental biochemical and physiological properties still remain elusive.

Transglutaminase activity is ubiquitous: It has been detected in algae and in angiosperms in different organs and sub-cellular compartments, chloroplasts being the best-studied organelles.

Possible roles: Possible roles concern the structural modification of specific protein substrates. In chloroplasts, transglutaminases appear to stabilize the photosynthetic complexes and Rubisco, being regulated by light and other factors, and possibly exerting a positive effect on photosynthesis and photo-protection. In the cytosol, they modify cytoskeletal proteins. Preliminary reports suggest an involvement in the cell wall construction/organization. Other roles appear to be related to fertilization, abiotic and biotic stresses, senescence and programmed cell death, including the hypersensitive reaction.

Conclusions: The widespread occurrence of transglutaminases activity in all organs and cell compartments studied suggests a relevance for their still incompletely defined physiological roles. At present, it is not possible to classify this enzyme family in plants owing to the scarcity of information on genes encoding them.

Fig. 1.

The two-step transamidase reaction of transglutaminase. All mammalian TGases belong to a superfamily of cysteine proteases, have structural homology and possess the catalytic triad of Cys-His-Asp/Asn; the reactivity of this Cys is activated by Ca²⁺, which causes a conformational change in the enzyme, allowing the access of the substrate to the binding site. Step 1: the active Ca²⁺-stabilized conformation of the enzyme forms a covalent intermediate between the active site thiol residue and a glutamyl residue in the protein substrate, releasing ammonia and activating the glutamine acyl moiety. Step 2: the active thioester undergoes an acyl transfer to a primary amine, in this case a polyamine, thus also introducing extra positive charges as PAs are protonated at physiological pH [or (not shown) to the lysyl residue of another protein]. A secondary cross-link might form between the free amine group of the bound polyamine and a glutamyl residue in another protein substrate thus forming bis-(γ-glutamyl)-PA derivatives (see Fig. 2). E, Transglutaminase; C, cysteine residue; PA, polyamine; P, protein substrate of transglutaminase; Q, glutamine residue.

Fig. 2.

Transglutaminase products. The better recognized catalytic activity is the cross-linking activity which leads to N-(γ-glutamyl)-lysine bond formation or to the incorporation of any of various primary amines (e.g. aliphatic PAs) at a glutamyl (former Gln) residue of a substrate protein. (A) The three main aliphatic polyamines present in plants, putrescine (PU), spermidine (SD) and spermine (SM), have different backbone length and numbers of positive charges. (B) PAs (putrescine is the example illustrated) covalently bound to a single protein glutamyl residue by transglutaminase-action, forming mono-(γ-glutamyl)-PA derivatives as shown in Fig 1, or to two glutamyl residues, each located on the same or separate proteins, forming bis-(γ-glutamyl)-PA derivatives. The ‘bridges’ produced in the latter case have a different length according to the molecular length of the PA involved. The formation of mono-derivatives of PAs (the ‘cationization’ of a protein), although not relevant to the thermodynamic properties of the side-chain, causes a very relevant shift in the protein's solubility, stability and conformation, and affects its interaction with other molecules. (C) Glu (former Gln) and Lys residues covalently bound by transglutaminase, forming a cross-link shorter than that formed with PAs.

Fig. 3.

Location of transglutaminases in the plant cell. This class of enzyme has been found in cellular compartments of different plants as here schematically summarized. In chloroplasts, some transglutaminases of different molecular mass have been found both in the stroma, where the substrate is Rubisco, and in thylakoids, where the substrates are mainly the light-harvesting complexes (LHCII). The enzyme is also present in the cytosol, where tubulin and actin have been identified as substrates. The occurrence in the microsomal fraction and in the cell wall of TGases of the same molecular mass suggests the hypothesis that the enzyme was secreted through Golgi vesicles into the cell wall, where polyamines, known to be present, might be conjugated to various unidentified structural or enzymatic wall proteins.