RESUMEN

Transglutaminase (TGase) is a family of enzymes that catalyzes cross-linking reaction between glutamine- and lysine residue of substrate proteins in several mammalian biological events. Substrate proteins for TGase and their physiological relevance have been still in research, continuously expanding. In this study, we have established a novel screening system that enables identification of cDNA sequence encoding favorable primary structure as a substrate for tissue-type transglutaminase (TGase 2), a multifunctional and ubiquitously expressing isozyme. By the screening, we identified several T7 phage clones that displayed substrate peptides for TGase 2 as a translated product from human brain cDNA library. Among the selected clones, the C-terminal region of IKAP, IkappaB kinase complex associated protein, appeared as a highly reactive substrate sequence for TGase 2. This system will open possibility of rapid identification of substrate sequences for transglutaminases at a genetic level.

RESUMEN

Transglutaminases (TGases) are a family of enzymes that catalyze cross-linking reactions between proteins. During epidermal differentiation, these enzymatic reactions are essential for formation of the cornified envelope, which consists of cross-linked structural proteins. Two main transglutaminases isoforms, epidermal-type (TGase 3) and keratinocyte-type (TGase 1), are cooperatively involved in this process of differentiating keratinocytes. Information regarding their substrate preference is of great importance to determine the functional role of these isozymes and clarify their possible co-operative action. Thus far, we have identified highly reactive peptide sequences specifically recognized by TGases isozymes such as TGase 1, TGase 2 (tissue-type isozyme) and the blood coagulation isozyme, Factor XIII. In this study, several substrate peptide sequences for human TGase 3 were screened from a phage-displayed peptide library. The preferred substrate sequences for TGase 3 were selected and evaluated as fusion proteins with mutated glutathione S-transferase. From these studies, a highly reactive and isozyme-specific sequence (E51) was identified. Furthermore, this sequence was found to be a prominent substrate in the peptide form and was suitable for detection of in situ TGase 3 activity in the mouse epidermis. TGase 3 enzymatic activity was detected in the layers of differentiating keratinocytes and hair follicles with patterns distinct from those of TGase 1. Our findings provide new information on the specific distribution of TGase 3 and constitute a useful tool to clarify its functional role in the epidermis.

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A large number of substrate proteins for tissue transglutaminase (TGase 2) have been identified in vivo and in vitro. Preference in primary sequence or secondary structure around the reactive glutamine residues in the substrate governs the reactivity for TGase 2. We established a screening system to identify preferable sequence as a glutamine-donor substrate using a phage-displayed peptide library. The results showed that several peptide sequences have higher reactivity and specificity to TGase 2 than those of preferable sequences previously reported. By analysis of the most reactive 12-amino acid sequence, T26 (HQSYVDPWMLDH), residues crucial to the enzymatic reaction were investigated. The following review summarizes the screening system and also the preference in substrate sequences that were obtained by this method and those previously reported.

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Transglutaminase 1 (TGase 1) is an essential enzyme for cornified envelope formation in stratified squamous epithelia. This enzyme catalyzes the cross-linking of glutamine and lysine residues in structural proteins in differentiating keratinocytes. To gain insight into the preferred substrate structure of TGase 1, we used a phage-displayed random peptide library to screen primary amino acid sequences that are preferentially selected by human TGase 1. The peptides selected as glutamine donor substrate exhibited a marked tendency in primary structure, conforming to the sequence: QxK/RpsixxxWP (where x and psi represent non-conserved and hydrophobic amino acids, respectively). Using glutathione S-transferase (GST) fusion proteins of the selected peptides, we identified several sequences as preferred substrates and confirmed that they were isozyme-specific. We generated GST-fused alanine mutants of the most reactive sequence (K5) to determine the residues that were critical for reactivity. Even in peptide form, K5 appeared to have high and specific reactivity as substrate. In situ analysis of mouse skin sections using fluorescence-conjugated K5 peptide resulted in detection of TGase 1 activity with high sensitivity, but no signal was detected in a TGase 1-null mouse. In conclusion, we were successful in generating a novel substrate peptide for sensitive detection of endogenous TGase 1 activity in the skin.

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Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between glutamine residues in various substrate proteins and lysine residues or primary amines. To better understand the sequence preference around the reactive glutamine residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.

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Transglutaminase is a Ca2+-dependent enzyme catalyzing protein cross-linking reactions. We investigated the contribution of this enzyme to the resealing of the injured plasma membrane in animal cells, using a lung cancer-derived cell line, A549. After mechanical injury by razor-scratching, the level of membrane resealing was estimated by differential incorporation of dextrans labeled with two distinct fluorescent dyes. The recovery level was decreased in the presence of excess primary amine, as a competitive inhibitor of transglutaminase. We established a cell line that stably expresses shRNA (short hairpin RNA) to specifically inhibit the expression of TGase 2 (tissue-type isozyme of transglutaminase) and confirmed the suppressed resealing level in the cell. Furthermore, additional expression of TGase 2 rescued the ability for membrane resealing. These results show that, after mechanical damage, this enzyme appeared to contribute to membrane resealing.

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Transglutaminase (TGase) catalyzes the formation of a covalent cross-link between a peptide-bound glutamine residue and a lysine residue or primary amine. We have recently identified specific preferred sequences as glutamine-donor substrates in TGase 2 and Factor XIII reactions. By taking advantage of preference of the 12-amino acid sequence for the enzymatic reaction, an efficient immobilization method was established using two different model proteins, glutathione S-transferase (GST) and single-chain fragment antibody (scFv). Both proteins were genetically attached with the preferred substrate sequence to produce a fusion protein. Attachment of the sequence enables the recombinant proteins to act as prominent TGase-substrates and enables them to be immobilized onto chemically amine-terminated gels. Investigation of the biological activities of the two proteins demonstrated their effective immobilization in comparison with that by using a chemically immobilizing method. This established system, which we designated as Transglutaminase-mediated site-specific immobilization method (TRANSIM), would provide site-specific and biologically active conjugation between proteins and several non-protein materials.

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Transglutaminases are Ca(2+)-dependent enzymes that post-translationally modify proteins by crosslinking or polyamination at specific polypeptide-bound glutamine residues. Physarum polycephalum, an acellular slime mold, is the evolutionarily lowest organism expressing a transglutimase whose primary structure is similar to that of mammalian transglutimases. We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage. With use of a biotin-labeled primary amine, three major proteins constituting possible transglutimase substrates were affinity-purified from the damaged slime mold. The purified proteins were Physarum actin, a 40 kDa Ca(2+)-binding protein with four EF-hand motifs (CBP40), and a novel 33 kDa protein highly homologous to the eukaryotic adenine nucleotide translocator, which is expressed in mitochondria. Immunochemical analysis of extracts from the damaged macroplasmodia indicated that CBP40 is partly dimerized, whereas the other proteins migrated as monomers on SDS/PAGE. Of the three proteins, CBP40 accumulated most significantly around injured areas, as observed by immunofluoresence. These results suggested that transglutimase reactions function in the response to mechanical injury.

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Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPphiD(P), QxPphi, and QxxphiDP; Factor XIIIa, QxxphixWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.

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The solvent effects of cyclopentyl methyl ether (CPME) on the reaction rates and enzyme enantioselectivity in the enantioselective transesterifications of racemic 6-methyl-5-hepten-2-ol (racemic sulcatol: SUL) and racemic 2,2-dimethyl-1,3-dioxolane-4-methanol (racemic solketal: SOL) with a series of enol esters catalyzed by Pseudomonas cepacia lipase co-lyophilized with cyclodextrins (alpha-, beta-, gamma-, partially methylated beta-, and 2,3,6-tri-O-methyl-beta-cyclodextrin: alphaCyD; betaCyD; gammaCyD; Me1.78betaCyD; Me3betaCyD) were investigated and compared with those in diisopropyl ether (IPE). In the case of SUL, enzyme activities of the co-lyophilizate with Me1.78betaCyD in CPME were lower than those in IPE with every acyl source, however, the absolute enantiopreference was shown in the transesterification with vinyl butyrate (VBR) in IPME. When the substrates were SOL and VBR, the enzyme activities in CPME were greatly enhanced as high as 1.6-9.8-fold, while the enantioselectivities in CPME were comparable to those in IPE.

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Five gemini-type amphiphiles bearing cyclitol head groups, which have abundance of axial hydroxy groups, are newly synthesized. The syntheses are based on a common mixed anhydride method utilizing N,N'-[iminobis(trimethylene)]bisquinamide, prepared from iminobispropylamine and quino-1,5-lactone, and dialkyl N-(3-carboxypropanoyl)-L-glutamates as polar and hydrophobic components, respectively. Candida rugosa lipase (CRL) and Pseudomonas cepacia lipase (PCL) are co-lyophilized with these synthesized gemini-type amphiphiles, and their transesterification activities in organic solvents are evaluated. The modified PCL and CRL prepared by using each amphiphile showed highly enhanced and moderately enhanced enzyme activity, respectively. These results are discussed in terms of the increased preferential exclusion of the hydrophilic heads of the amphiphile and of the topological view of the amphiphile.

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Modified Candida rugosa and Pseudomonas cepacia lipase (CRL and PCL) were co-lyophilized with two pairs of synthetic diastereoisomeric amphiphiles, D- and L-2-(3-[bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl]-propionylamino)-pentanedioic acid didodecyl ester (D- and L-BIG2C12CA); D- and L-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-pentanedioic acid didodecyl ester (D- and L-2C12GE). Enzyme activities of the modified lipase in the transesterification in organic solvent were evaluated. Both pairs of the diastereoisomeric amphiphiles showed enhanced enzyme activity in the transacetylation between racemic sulcatol and isopropenyl acetate in diisopropyl ether, catalyzed by the PCL-co-lyophilizate, by 19-48 fold when compared to the native lipase lyophilized from buffer alone independent of the stereochemistry of the amphiphiles, while in the case of the CRL-co-lyophilizate only the L-BIG2C12CA showed enhanced enzyme activity in the transbutyrylation between racemic solketal and vinyl butyrate in cyclohexane as high as 68-78 fold.

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Lipases from Candida rugosa (CRL) and Pseudomonas cepacia (PCL) were co-lyophilized with cyclic oligoethers including four crown ethers and nine cyclodextrins (CyDs), and their transesterification activity and enantioselectivity in organic solvents were evaluated. The PCL co-lyophilized with each additive showed simultaneously enhanced enzyme activity and enantioselectivity when compared to the native lipase lyophilized from buffer alone; in contrast, such enhancement was not observed for the co-lyophilized CRL. Among the cyclic oligoethers examined, permethylated betaCyD (Me1.78betaCyD), as the most suitable additive, was used for the optimization of both the co-lyophilized PCL preparation and reaction conditions by determining the effects of varying the additive/lipase ratio, aqueous pH, the nature of organic solvents, and temperature. The initial rate determined for the transesterification between racemic 2,2-dimethyl-1,3-dioxolane-4-methanol and vinyl butyrate in diisopropyl ether at 30 degrees C increased by up to 17-fold and the enantioselectivity represented by E could be doubled. While there was an inverse correlation between temperature and enantioselectivity, with the Me1.78betaCyD-PCL co-lyophilizate, the reaction rate even at 0 degrees C was much higher than that at higher temperatures in the native PCL-catalyzed reaction. Hence, this method seems to be of practical use for the large-scale production of optically active compounds.

RESUMEN

Lipases co-lyophilized with water-soluble gemini-type amphiphiles were found to have high enzyme activity in nonaqueous media without washing out of the amphiphile with anhydrous organic solvent. In this study, we obtained freeze-dried complexes of Candida rugosa lipase (CRL) with six water-soluble twin glusitol-headed amphiphiles bearing different types of hydrophobic tails, including newly synthesized ones, and their transesterification activity in organic solvent was evaluated. The results indicate that the increased enzyme activity upon CRL modification at 200 molar ratio of amphiphile/CRL, which are restricted to the ester-containing amphiphiles, is probably due to the surface activation by the interaction between ester-carbonyl of the amphiphile and phenyl group of the tyrosine residue situated on the surface of the lid in the CRL.