AtPng1p. The first plant transglutaminase

Abstract

Studies have revealed in plant chloroplasts, mitochondria, cell walls, and cytoplasm the existence of transglutaminase (TGase) activities, similar to those known in animals and prokaryotes having mainly structural roles, but no protein has been associated to this type of activity in plants. A recent computational analysis has shown in Arabidopsis the presence of a gene, AtPng1p, which encodes a putative N-glycanase. AtPng1p contains the Cys-His-Asp triad present in the TGase catalytic domain. AtPng1p is a single gene expressed ubiquitously in the plant but at low levels in all light-assayed conditions. The recombinant AtPng1p protein could be immuno-detected using animal TGase antibodies. Furthermore, western-blot analysis using antibodies raised against the recombinant AtPng1p protein have lead to its detection in microsomal fraction. The purified protein links polyamines-spermine (Spm) > spermidine (Spd) > putrescine (Put)-and biotin-cadaverine to dimethylcasein in a calcium-dependent manner. Analyses of the gamma-glutamyl-derivatives revealed that the formation of covalent linkages between proteins and polyamines occurs via the transamidation of gamma-glutamyl residues of the substrate, confirming that the AtPng1p gene product acts as a TGase. The Ca(2+)- and GTP-dependent cross-linking activity of the AtPng1p protein can be visualized by the polymerization of bovine serum albumine, obtained, like the commercial TGase, at basic pH and in the presence of dithiotreitol. To our knowledge, this is the first reported plant protein, characterized at molecular level, showing TGase activity, as all its parameters analyzed so far agree with those typically exhibited by the animal TGases.

Figure 1.

ClustalW alignment of the tripartite TGase domain of AtPng1p. A, Alignment among AtPng1p tripartite domain with those of C. elegans (CePng1p; AAF74721), mouse (mPng1p; NP_067479), and human (hPng1p; AF250924.2) peptide N-glycanases, the human Factor XIIIa (NP_000120), human TGase II (NP_945189), and human TGase III (Q08188). Arrows indicate the amino acids Cys-His-Asp of the catalytic triad. Numbers indicate the AtPng1p amino acidic residue position. In bold, amino acid identity. B, N-terminal peptide signal sequence of AtPng1p: putative Tyr sulfation domain, which is associated to Golgi membranes (based on ScanProsite program prediction).

Figure 2.

Nested RT-PCR assay. Nested RT-PCR from Arabidopsis mRNA extracted from entire plants, leaves, shoots, and roots. 3, 5, 7, 14, 20, and 28 refer to the number of growing days. L refers to plants grown under 16-h-light/8-h-dark conditions. D refers to plants grown under dark conditions. An empty pEt-28a(+) vector was used as a negative control (−), whereas, the AtPng1p cDNA clone was used as a positive one (+). Gen PCR1 and Gen PCR2 indicate the 1,600 and 1,000-bp fragments, respectively, obtained from the first and second couple of primers using Arabidopsis genomic DNA (gDNA) as a template.

Figure 3.

Immunodetection of recombinant AtPng1p by western-blot analysis using different animal TGase antibodies. A, Molecular mass standards. B, Coomassie staining of AtPng1p purified with Ni²⁺-affinity column at 400 mm imidazol. C, Red Poinceau positive bands on nitrocellulose deriving from 10% (w/v) SDS-PAGE (1 μg protein). Immuno-staining with TGase II-Ab 3 and antibodies (D) for C. elegans TGase (E), rat prostatic gland TGase (F). Primary antibodies dilution: 1:1,000; secondary antibodies dilution 1:3,000.

Figure 4.

Immunodetection of AtPng1p by western-blot analysis using AtPng1p antibodies. S refers to 20-d-old Arabidopsis soluble enriched fraction, whereas M refers to microsomal enriched fraction. We ran 100 μg of proteins in 10% (w/v) SDS-PAGE and blotted to a nitrocellulose membrane. The membranes were then treated as described in “Materials and Methods” and incubated with antibodies raised against AtPng1p (used in 1:1,000 dilution). The mass of proteins is indicated in kD.

Figure 5.

TGase conjugation assays. The assays were performed using PAs as substrates. A, [³H]Spd conjugation to DMC catalyzed by AtPng1p incubated at different Ca²⁺ molarity. B, [³H]Spm, -Spd, and -Put conjugation to DMC catalyzed by AtPng1p incubated with 2 mm Ca²⁺ and 10 mm DTT in the presence or absence of 5 mm EGTA. C, Autoradiography of the 15% (w/v) SDS-PAGE of the DMC conjugated to [³H]Spm by catalysis of the AtPng1p as in B, with or without 5 mm EDTA, performed by Fuji imaging [³H]plate. Means significantly differs (1% probability level, Student's t test).

Figure 6.

Glutamyl-derivatives assay. The conjugation of DMC to [³H]Spm produced by the action of AtPng1p were analyzed by HPLC. DMC alone, AtPng1p alone, and denatured AtPng1p incubated with DMC are used as negative control. Arrows indicate standard retention times for bis-γ-glutamyl-Spm and mono-γ-glutamyl-Spm obtained with DMC and guinea pig liver TGase.

Figure 7.

pH-dependent polymerization assay. Relative percentage of cross-linked BSA after incubation with AtPng1p at different pH values. After enzymatic assay, proteins were run in 10% (w/v) SDS-PAGE, and the polymers formation were then measured as O.D. (FLA 3000, Fuji, Milan) of the high molecular mass region (>250 kD) of the SDS-PAGE gel, as shown in the insert. The 100% refers to O.D. of the polymerized zone at pH 8.5. Assay condition: 20 mm Tris-HCl, 30°C, BSA/AtPng1p ratio = 12.5/1.

Figure 8.

BSA polymerization assay. Left, Coomassie staining of 10% (w/v) SDS-PAGE of BSA after incubation with AtPng1p. Ca²⁺, DTT, EGTA, GTP, and Mg²⁺ were assayed on the cross-linking formation as indicated in the lanes. Assay condition: 40 mm MES-NaOH, pH 7.0, 30°C, BSA/AtPng1p ratio = 12.5/1. Right, Polymerization of BSA catalyzed by GPL-TGase. Assay condition: 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 30°C, BSA/GPL-TGase ratio = 12.5/1. Arrow, BSA polymers; asterisk, bands below BSA.