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Expression of different forms of transglutaminases by immature cells of Helianthus tuberosus sprout apices

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Abstract

Immature cells of etiolated apices of sprouts growing from Helianthus tuberosus (H. t.) tubers showed Ca2+-dependent transglutaminase (TG, EC 2.3.2.13) activity on fibronectin (more efficiently) and dimethylcasein as substrates. Three main TG bands of about 85, 75 and 58 kDa were isolated from the 100,000×g apices supernatant through a DEAE-cellulose column at increasing NaCl concentrations and immuno-identified by anti-TG K and anti-rat prostate gland TG antibodies. These three fractions had catalytic activity as catalyzed polyamine conjugation to N-benzyloxycarbonyl-L-γ-glutaminyl-L-leucine (Z-L-Gln-L-Leu) and the corresponding glutamyl-derivatives were identified. The amino acid composition of these TG proteins was compared with those of several sequenced TGs of different origin. The composition of the two larger bands presented great similarities with annotated TGs; in particular, the 75 kDa form was very similar to mammalian inactive EPB42. The 58 kDa form shared a low similarity with other TGs, including a maize sequence of similar molecular mass, which, however, did not present the catalytic triad in the position of all annotated TGs. A 3D model of the H. t. TGs was built adopting TG2 as template. These novel plant TGs are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells. These data suggest that in plants, multiple TG forms are active in the same organ and that plant and animal enzymes probably are very close not only for their catalytic activity but also structurally.

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Abbreviations

aa:

Amino acid

aac:

Amino acid composition

AtPng1p:

Arabidopsis thaliana peptide N-glycanase

BCIP/NBT:

5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium

DEAE:

Diethylaminoethyl

DMC:

N,N’-dimethylcasein

DTT:

Dithiotreitol

FXIIIA:

Factor XIIIA subunit

FN:

Fibronectin

H. t :

Helianthus tuberosus

PAs:

Polyamines

PNG1_ARATH:

Peptide: N-glycanase_Arabidopsis thaliana

Put:

Putrescine

PVPP:

Polyvinylpolypyrrolidone

Q6KF61 and Q6KF70:

Cloned maize sequences

SDS-PAGE:

Sodium dodecyl-sulphate-gel electrophoresis

SN:

Supernatant

Spd:

Spermidine

TBST:

Saline Tris-buffer containing 0.05 % Tween 80

TCA:

Trichloroacetic acid

TGAS_STRMB:

TG from Streptomyces mobaraensis

TGs:

Transglutaminases

TGM2:

Mammalian TG2

Z-L-Gln-L-Leu:

N-benzyloxycarbonyl-L-γ-glutaminyl-L-leucine

EPB42:

Erythrocyte membrane protein band 4.2

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Acknowledgments

We acknowledge the financial support provided by RFO 2009, 2010. Rat prostate gland TG was a kind gift of prof. C. Esposito, Salerno University.

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Correspondence to Stefano Del Duca.

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Beninati, S., Iorio, R.A., Tasco, G. et al. Expression of different forms of transglutaminases by immature cells of Helianthus tuberosus sprout apices. Amino Acids 44, 271–283 (2013). https://doi.org/10.1007/s00726-012-1411-y

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