Identification of Protein Substrates for Transglutaminase in Caenorhabditis elegans Andra´s Ma´ di,* Zolta´ n Kele,² Tama´s Jana´ky,² Ma´ ria Punyiczki,‡ and La´ szlo´ Fe´su¨ s* , ‡ ,1 *Signal Transduction and Apoptosis Research Group of the Hungarian Academy of Sciences, University of Debrecen, Nagyerdei krt. 98., H-4012 Debrecen, Hungary; ²Department of Medical Chemistry, Szeged University of Sciences, Do´m te´r 8., H-6720 Szeged, Hungary; and ‡Department of Biochemistry and Molecular Biology, University of Debrecen, Nagyerdei krt. 98., H-4012 Debrecen, Hungary Received April 12, 2001 Transglutaminase-dependent cross-linking of pro- teins leads to protein polymerisation that confers stabil- ity as well as resistance to mechanical disruption and chemical attack. Various transglutaminases have been implicated in a wide range of biological phenomena oc- curring in both extracellular and intracellular compart- ments, but further clarification of the physiological role of these enzymes requires identification of possible sub- strate molecules. Here we report the detection, purifica- tion, and identification of two proteins, enolase and ATP synthase a subunit as glutamine donor protein sub- strates for the transglutaminase of the nematode Caeno- rhabditis elegans. © 2001 Academic Press Key Words: transglutaminase; substrate; Caenorhab- ditis elegans; enolase; mitochondrial ATP synthase. The calcium-dependent protein cross-linker trans- glutaminases (TGases, EC 2.3.2.13) act in an acyl transfer reaction where the g-carboxamide group of a peptide bound glutamine serves as acyl donor and the h-amino group of peptide bound lysine residue acts as acyl donor. The result of this reaction is a covalent h(g-glutamyl)lysine isopeptide bond between proteins (1, 2). The lysil residue can be replaced by primer amino groups as it is applied in activity measurement methods (3). In order to clarify the physiological role of TGases, numerous techniques were used for detection of protein substrates with the help of labeled primer amines. In case of tissue TGase and mammalian or- ganisms the presence of some intracellular protein sub- strates were identified in vitro, among them there are b-crystallines (4 – 6), lipocortin I (7), fructose-1,6- bisphosphate aldolase (8), actin (9) in addition to sev- eral extracellular proteins (10) which can be glutamine donor substrates. Actin was demonstrated to be a TGase substrate during programmed cell death using a new substrate detection procedure in living cells (11). It was also proved that glutathione S-transferase acts as an efficient acyl donor as well as acceptor substrate both in cells and in vitro (12). Complex mammalian cell phenomena, such as cell death, can be studied in the well-known model organ- ism of developmental genetics, the nematode Caeno- rhabditis elegans (13–15). TGase activity could be mea- sured in the worm extracts and it must be active in cells, since significance level of TGase generated cova- lent cross-link content was detected in adult animals (16). Biochemical characterization of the enzyme re- vealed that it was different from the known mamma- lian TGases but very similar to enzymes investigated in other nematode species (17–19). Our previous data obtained by studying cell-death mutants of C. elegans suggested that the action of a TGase activity was pre- sumably part of the molecular events occurring during the programmed deaths of cells (16). Here we demon- strate the existence of two enzymes, the cytosolic eno- lase and the mitochondrial ATP synthase a subunit as the most pronounced glutamine donor protein sub- strates of the C. elegans TGase by an amine incorpo- ration approach. MATERIALS AND METHODS Materials. The wild-type (N2, Bristol) of C. elegans and Esche- richia coli XI666 strain were obtained from the Caenorhabditis Ge- netics Center (University of Minnesota, MN). Triptone, yeast ex- tract, PhMeSO 2 F, 1,4-dithiotreitol, polyoxyethanyl-cholesteryl sebacate, Chaps, streptavidin alkaline phosphatase, nitro-blue tet- razolium, 5-bromo-4-chloro-3-indolyl phosphate, monomeric avidin sepharose, trypsin, Nonidet P-40, urea, iodoacetamide were all pur- chased from Sigma. Amine donor substrate N-(5-aminopentyl)- biotinamide was from Molecular Probes, protein assay reagent was from Bio-Rad Laboratories, Centricon YM-30 and Immobilon-P Abbreviations used: PhMeSO 2 F, phenylmethylsulfonyl fluoride; ced, cell death abnormal, BSA, bovine serum albumin; Chaps, 3-[(3- cholamidopropyl)-dimethylammonio]-1-propansulfonate. 1 To whom correspondence should be addressed. Fax: 136 52 416432. E-mail: fesus@indi.biochem.dote.hu. Biochemical and Biophysical Research Communications 283, 964 –968 (2001) doi:10.1006/bbrc.2001.4872, available online at http://www.idealibrary.com on 964 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.