Isolation of transglutaminase-reactive sequences from complex biological systems: a prominent lysine donor sequence in bovine lens.

Abstract

The transglutaminase (protein-glutamine: amine 'y-glutamyltransferase, EC 2.3.2.13)-catalyzed crosslinking of proteins in biological systems can often be inhibited by inclusion of small primary amines or glutamine-containing peptides, which act as site-specific blockers of the relevant acceptor (i.e., glutamine) and donor (i.e., lysine) fumctlonalIties of the natural substrates. Compounds such as dansylcadaverine and dansyl-e-aminocaproyl-Gln-Gln-Ile-Val are particularly useful in sorting out acceptor-donor relationships among lens crystallins. Apart from its fluorescent properties, the dansyl hapten offered special advantages as a "handle" for the rapid isolation of transglutaminase targets even in the complex system oflens cortical homogenate. The dansylated peptide was incorporated into bovine lens proteins under the influence of the Ca2+-activated intrinsic transglutaminase and, after digestion by endoproteinase Glu-C, the tracer-containing fragments were isolated by affinity chromatography on an anti-dansyl antibody column. The major fluorescent peak was isolated by HPLC and sequenced by Edman degradation, which yielded phenylthiohydantoin amino acid derivatives for the first 10 cycles, EKPAVTAAPK, and none for the next 2. The sequence, corresponding to residues 165-174 of aB-crystallin, unambiguously identifies the known carboxyl-terminal domain, EK-PAVTAAPKK, as the prominent lysine-donating fragment in bovine lens.The introduction of short peptides containing a transglutaminase (protein-glutamine:amine y-glutamyltransferase, EC 2.3.2.13)-reactive glutamine residue and an easily recognizable reporter group (e.g., dansyl) made it possible to identify the lysine-carrying (i.e., donor) protein substrates of the enzyme in biological systems (1-4). The peptides fulfilled the dual role of inhibiting protein-protein cross-linking phenomena as they, themselves, became incorporated into the natural donors. Use ofthe compounds in lens homogenates (1-3) revealed a remarkably high degree of selectivity for labeling of proteins under the influence of the Ca2+-activated intrinsic transglutaminase. Moreover, the enzyme-directed, sitespecific labeling reaction could be accomplished with minimum perturbation of the native system under the simple experimental conditions of incubating the tissue homogenate with the tracer peptide in the presence of Ca2+. Following electrophoretic separation, identification ofproteins could be accomplished by detection of fluorescence of the dansyl marker or by blotting with an anti-dansyl antibody. Similar procedures were developed previously for identifying the complementary glutamine-carrying (i.e., acceptor) protein substrates of the enzyme with a fluorescent lysine analogue, dansylcadaverine, as tracer (5).We have now explored in greater depth the potentialities of this general approach of specific tracer recognition and applied it to the isolation of a prominent lysine donor sequence in bovine lens. MATERIALS AND METHODSDansyl-e-aminocaproyl-Gln-Gln-Ile-Val (dns-E-aca-QQIV) was...

Transglutaminases and Disease: Lessons From Genetically Engineered Mouse Models and Inherited Disorders

“…It has long been accepted that TG2-mediated cross-linking of some of the ␤-crystallins (169,195,297), ␣B-crystallin (104,174), and the intermediate filament protein vimentin (62) is likely involved. Very high (unphysiological) levels of oxidative stress were shown to stimulate the in situ transamidase activity of TG2 in human lens epithelial cells (249) via transforming growth factor (TGF)-␤2 activation of the Smad3 signaling pathway, resulting in TG2-mediated cross-linking and aggregation of lens proteins (250) (Fig.…”

The Intermediate Filament Protein, Vimentin, in the Lens Is a Target for Cross-linking by Transglutaminase

“…Dansyl-labeled peptides were isolated on a monoclonal anti-dansyl Affi-Gel 10 column (6,19) and concentrated by lyophilization. Separation was carried out by HPLC on a Ultrasphere C8 column (Beckman Instruments) with a gradient of acetonitrile (5-40% for separating the dansylcadaverine-labeled peptides or 10 -60% for the dansyl-⑀-aca-QQIV-labeled peptides) in H 2 O, 0.1% trifluoroacetic acid over 90 min at a flow rate of 1 ml/min.…”

“…Enzyme-specific probes, such as dansylcadaverine, dansyl-⑀-aca-QQIV, or similar biotinylated analogues were synthesized for the identification and analysis of the substrates of the enzyme. These compounds compete against the TGase-catalyzed cross-linking of proteins, and by becoming incorporated in the enzyme-driven reaction into the biological substrates themselves, they could be employed for locating the Gln (or acceptor) and the Lys (or donor) functionalities exhibiting the unique specificities (1)(2)(3)(4)(5)(6)(7)(8).…”

Cross‐Linking Sites of the Human Tau Protein, Probed by Reactions with Human Transglutaminase

“…After termination of the reactions by the addition of 1~il of 200 mg/ml phenylmethylsulfonyl fluoride (prepared in dimethyl sulfoxide), the resultant dansyl-decorated peptides were isolated by immunoaffinity chromatography as described previously (Lorand et al, 1992b;Murthy et al, 1994;Clement et al, 1998).…”