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Apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action.

Physiological deletion of cells ensues programmed death which involves formation of apoptotic bodies with fragmented DNA. Here we report that apoptotic hepatocytes are insoluble in detergents, urea, guanidine hydrochloride, reducing agents and thereby can be isolated from rat liver following collagenase treatment. They are wrinkled, spherical structures similar to cornified envelopes of epidermis by phase‐contrast microscopy and show irregular, globular morphology by scanning‐electron microscopy. Part of their DNA content is cleaved into nueleosomal and oligonucleosomal fragments. Their insolubility, like that of the cornified envelope, is evoked by ε‐(γ‐glutamyl)lysine and N 1,N 8‐bis(γ‐glutamyl)spermidine protein cross‐linking bonds formed by transglutaminase.

Biochemical, structural, and transglutaminase substrate properties of human loricrin, the major epidermal cornified cell envelope protein

Loricrin is the major protein of the cornified cell envelope of terminally differentiated epidermal keratinocytes which functions as a physical barrier. In order to understand its properties and role in cornified cell envelope, we have expressed human loricrin from a full-length cDNA clone in bacteria and purified it to homogeneity. We have also isolated loricrin from newborn mouse epidermis. By circular dichroism and fluorescence spectroscopy, the in vivo mouse and bacterially expressed human loricrins possess no α or β structure but have some organized structure in solution associated with their multiple tyrosines and can be reversibly denatured by either guanidine hydrochloride or temperature. The transglutaminase (TGase) 1, 2, and 3 enzymes expressed during epidermal differentiation utilized loricrin in vitro as a complete substrate, but the types of cross-linking were different. The TGase 3 reaction favored certain lysines and glutamines by forming mostly intrachain cross-links, whereas TGase 1 formed mostly large oligomeric complexes by interchain cross-links involving different lysines and glutamines. Together, the glutamines and lysines used in vitro are almost identical to those seen in vivo. The data support a hypothesis for the essential and complementary roles of both TGase 1 and TGase 3 in cross-linking of loricrin in vivo. Failure to cross-link loricrin by TGase 1 may explain the phenotype of lamellar ichthyosis, a disease caused by mutations in the TGase 1 gene.

Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography–mass spectrometry

A method including protein A purification, limited Lys-C digestion, and mass spectrometry analysis was used in the study to quantify a recombinant monoclonal antibody in cynomolgus monkey serum. The same antibody that was isotopically labeled was used as an internal standard. Interferences from serum proteins were first significantly reduced by protein A purification and then by limited Lys-C digestion of protein A bound IgG, including both monkey and the recombinant IgG. Fab fragment of the recombinant human IgG was analyzed directly by LC-MS, while monkey IgG and the Fc fragment of the recombinant human IgG remained bound to protein A resin. Quantitation was achieved by measuring the peak intensity of the Fab from the recombinant human IgG and comparing it to that of the Fab from the stable isotope-labeled internal standard. The results were in good agreement with the values from ELISA. LC-MS can therefore be used as a complementary approach to ELISA to quantify recombinant monoclonal antibodies in serum for pharmacokinetics studies and it can also be used where specific reagents such as antigens are not readily available for ELISA.

Degradation of cells dying by apoptosis leads to accumulation of ε(γ‐glutamyl)lysine isodipeptide in culture fluid and blood

ε(γ‐Glutamyl)lysine isodipeptide, the end‐product of proteolytic digestion of proteins cross‐linked by transglutaminase, was detected in culture fluid of neonatal rat hepatocytes and plasma of adult rats. The concentration of the isodipeptide was significantly increased in both when high rate of apoptosis with phagocytosis of dying hepatocytes was produced either by epidermal growth factor in the culture or by lead nitrate‐induced hyperplasia with subsequent involution in rats. Specific induction of tissue transglutaminase and the consequent formation of highly cross‐linked protein envelopes in apoptotic cells have been previously demonstrated by us in both systems.

Determination of ϵ(γ-glutamyl)lysine crosslink in proteins using phenylisothiocyanate derivatization and high-pressure liquid chromatographic separation

Abstract A sensitive, reproducible high-performance liquid chromatographic method is reported for the determination of the ϵ(γ-glutamyl)lysine isopeptide bond, which is usually formed by protein-crosslinking transglutaminases between polypeptide chains. The procedure is based on the separation and quantitation of ϵ(γ-glutamyl)lysine isodipeptide following exhaustive proteolytic digestion of the crosslinked peptide. It involves preliminary separation steps on a cation exchanger resin and a silica HPLC column, precolumn derivatization with phenylisothiocyanate, and reversed-phase high-pressure liquid chromatographic separation on a C18 column. The derivatized isodipeptide gave a linear concentration-response relationship, with a detection limit of 10 pmol/mg of protein. The combination of the preliminary separation steps and the sensitive detection system permits the determination of the ϵ(γ-glutamyl)lysine crosslink in complex biological systems including total tissue homogenates.

Transglutaminase crosslinking and structural studies of the human small proline rich 3 protein

The cell envelope (CE) is a vital structure for barrier function in terminally differentiated dead stratified squamous epithelia. It is assembled by transglutaminase (TGase) cross-linking of several proteins, including SPR3 in certain specialized epithelia normally subjected to mechanical trauma. We have expressed recombinant human SPR3 in order to study its cross-linking properties. It serves as a complete substrate for, and is cross-linked at similar efficiencies by, the three enzymes (TGases 1, 2 and 3) that are widely expressed in many epithelia. Multiple adjacent glutamines (4, 5, 16, 17, 18, 19 and 167) and lysines (6, 21, 164, 166 and 168) of only head and tail domain sequences are used for cross-linking. However, each enzyme preferentially uses certain residues on the head domain. Moreover, our in vitro data suggest a defined temporal order of cross-linking of SPR3 in vivo: It is first cross-linked by TGase 3 into short intra- and inter-chain oligomers which are later further cross-linked to the CE by TGase 1. To investigate the absence of cross-linking in the central domain (e.g. lysine in position 2 of each of the 16 repeats) we performed structural studies on recombinant SPR3 and on a synthetic peptide containing three repeats of the central domain. 2D H-1 NMR spectroscopy, TOCSY and ROESY, shows strong and medium intensity NOEs connectivities along the amino acid sequence with one weak long range NOE contact between Thr and Cys of subsequent repeats. Distance geometry computation on the basis of intensities of NOEs found generated 50 compatible structures grouped in three main families differing by the number of H-bonds. These measurements were repeated at different concentrations of trifluoroethanol (TFE)-water mixture, an α-helical promoting solvent, in order to check the stability of the conformations determined; no changes were observed up to 50% TFE in solution. Also temperature changes did not produce any variation in the ROESY spectrum in the same condition as above. The NMR and circular dichroism data strongly indicate the presence of an ordered (not α-helix nor β-sheet) highly flexible structure in the eight amino acids repetitive units of SPR3, confirming the prediction of one possible β-turn per each repeating unit. Thus, biochemical and biophysical data, strongly support SPR3 to function as a flexible cross-bridging protein to provide tensile strength or rigidity to the CE of the stratified squamous epithelia in which it is expressed.

The presence of a covalently cross-linked matrix in human platelets

Exhaustive extraction of human platelets with 6 M guanidine-HCl, and 5% beta-mercaptoethanol, followed by 5% SDS resulted in a sedimentable material which showed fibrous structure by transmission electron microscopy. When platelets treated with 8 M urea, 50 mM DTT and 2% SDS were applied on a 3% solubilizable acrylamide gel a high molecular weight material could be also isolated which was highly crosslinked by epsilon(gamma-glutamyl)lysine bonds. Its amino acid composition was Asp 110, Glu 119, Ser 55, Gly 70, Arg 33, Thr 41, Ala 112, Pro 93, Tyr 35, Val 18, Met 55, Cys 46, IIe 47, Leu 71, Phe 27, Lys 76 expressed as residue per 1000. The quantity of platelet polymer material as well as the amount of epsilon(gamma-glutamyl)lysine bond was slightly higher in thrombin activated platelets. The insoluble matrix of resting platelets reacts with antibodies against spectrin, alpha-actinin, actin, myosin, tropomyosin. The matrix from activated platelets has shown reaction with additional antibodies including ones against blood coagulation factor XIIIa, fibrinogen, von Willebrand factor, thrombospondin, tubulin and filamin. The presence of an epsilon(gamma-glutamyl)lysine cross-linked cell matrix in platelets is consistent with the observation of a similar structure in other cells.

Enzymatic fluorimetric assay for ϵ(γ-glutamyl)lysine isodipeptides A new assay for proteolytic fragments of proteins cross-linked by factor XIII and other transglutaminases

Abstract A fluorescent method for the quantitation of ϵ(γ-glutamyl)lysine isodipeptides is described which is applicable to the quantitation of the free isodipeptides in human plasma and in proteolytic digest of human platelet lysates. The possible diagnostic importance of free ϵ(γ-glutamyl)lysine isodipeptides in plasma revealed the necessity of developing a relatively simple method suitable for the fast screening of large numbers of samples. Samples are deproteinised by Centrifree micro partition system, then purified by cation-exchange chromatography and lyophilised. Aliquots of the reconstituted samples are incubated with γ-glutamylamine cyclotransferase, the enzyme which specifically catalyses the conversion of ϵ(γ glutamyl)lysine to free lysine and 5-oxo-L-praline. The L-lysine is decarboxylated and the cadaverin formed is extracted by pentane-l-ol. The primary amine produces a stable product with fluorescamine which is measured fluorimetrically. The ϵ(γ-glutamyl)lysine isodipeptide standard curves were linear in a concentration range of 0–100 μmol/l. Imprecision of measurement was 34% at 1.4 gmmol/l and 18% at 24.6μmol/l concentrations. Results correlated with the HPLC technique described previously (r=0.798). Median (and ranges) of the isodipeptide levels in plasma were 2.4 (0–9.2) gmmol/l (n=14) in apparently healthy individuals, 11.8 (0–80.7) μmol/l(n=23) in patients with deep vein thrombosis and 0.9 (0.1–7.9) μmol/l (n=6) in patients with acute myocardial infarction.

Role of Tissue Transglutaminase in the Formation of Apoptotic Bodies

Recent developments in programmed cell death research have shown that the induction and activation of a transglutaminase (EC 2.3.2.13; glutaminyl-peptide γ-glutamyltransferase) may play an important role in the apoptotic program1.