Intramolecular crosslinking of monomeric fibrinogen by tissue transglutaminase.

Abstract

In addition to generating polymeric products from human fibrinogen, human erythrocyte transglutaminase (protein-glutamine:amine y-glutamyltransferase, EC 2.3.2.13) was shown to catalyze the intramolecular reaction of crosslinking two of the constituent chains within monomeric fibrinogen itself. This internally fused protein derivative contains appreciable amounts of the N6-(y-glutamyl)lysine bridge peptide and displays the Aa-y hybrid chain pattern of crosslinking, characteristic for the actions of tissue transglutaminases on fibrinogen. Diagnostic analysis in pathological situations, where such enzymes might have escaped from cells into the plasma environment, should include a search for the internally crosslinked soluble fibrinogen monomer.It is known that human fibrinogen, upon reaction with a tissue transglutaminase (protein-glutamine:amine y-glutamyltransferase, EC 2.3.2.13) from human erythrocytes or from guinea pig liver, forms high molecular weight multimeric products by a program of polymerization different from that seen with coagulation factor XIIIa (1, 2). Factor XIIIa promotes first y-to-y and then Aa-to-Aa chain homologous crosslinking (3), whereas Aacy type of hybrid crosslinking is the unique feature of the action of transglutaminase on fibrinogen. The question arises (1) whether these unusual crosslinks, apart from participating in the intermolecular process of polymerization, might also be introduced intramolecularly between the constituent Aa and r chains of monomeric fibrinogen itself. This issue became even more pertinent when we found that, even after extended exposure of fibrinogen to transglutaminase, a sizeable portion of the protein remained in the unpolymerized Mr 340,000 form. The present report shows that the monomeric species of fibrinogen, reisolated from the mixture after reaction with erythrocyte transglutaminase, contains significant amounts of the N'-(y-glutamyl)lysine bridge peptide and shows the characteristic Aay hybrid chain pattern of crosslinking (1). MATERIALS AND METHODSThe contents of one vial of human fibrinogen (40 mg, IMCO, American Diagnostica, Greenwich, CT) were dissolved in 3 ml of buffer (50 mM Tris HCl, pH 7.5/150 mM NaCl/1 mM EDTA) and were dialyzed against 4 liters of the same buffer for 16 hr at 4'C. The concentration of fibrinogen (11.6 mg/ml) was estimated using A 1% = 15.1 (4). Human erythrocyte transglutaminase was purified by David Schilling to apparent SDS/PAGE homogeneity based on a published procedure (5). Concentration of the erythrocyte enzyme [1.7 mg/ml of 20 mM imidazole HCI, pH 6/1 mM EDTA/1 mM dithiothreitol/0.35 M KCI/10 units of Trasylol per ml/5% (vol/vol) glycerol] was estimated by using the BCA assay kit (Pierce) according to the manufacturer's instructions. Erythrocyte transglutaminase was stored at -80'C.Crosslinking of fibrinogen was typically carried out at 370C for 1 hr in reaction mixtures of about 24 ml containing approximately 2.3 ,M fibrinogen, 0.1 M NaCl, 50 mM Tris HCI (pH 7.5), and ca. 0.3 AM erythrocyte transglutam...

Transglutaminase-catalyzed crosslinking of the Aα and γ constituent chains in fibrinogen

“…1) was separated from the polymeric forms of the protein by gel filtration as described (10). The N (␥-glutamyl)lysine isopeptide content of the material was 1.6 moles per 340,000 g (i.e., 1 mole) of fibrinogen.…”

“…The digest was concentrated by lyophylization (to 5 ml), which was followed by several runs of HPLC (Beckman, Ultrasphere octyl) at room temperature (280 min) with a 0-45% gradient of acetonitrile in 0.1% trifluoroacetic acid (TFA). Combined fractions of the eluates were analyzed for N (␥-glutamyl)lysine contents (10), and two pools that were particularly rich in isopeptide content (I Ϸ 29%; II Ϸ 22% of total) were further purified. Processing of pool I required various acetonitrile͞0.1% TFA gradients: 8.8-22.8% (180 min); 13.1-17.7% (120 min), and a 14% isocratic separation on C 8 columns followed by 25% of the same on a C 18 column.…”

“…After ammonium sulfate precipitation, the crosslinked fibrinogen was fractionated by gel filtration on Sepharose 4B (2.6 ϫ 96 cm), and the last peak on the elution profile, found to comprise predominantly monomeric fibrinogen, was used for detailed analysis. N (␥-glutamyl)lysine crosslink contents were measured after sequential, total degradation by a series of proteases, as previously (10).…”

“…[A␣B␤␥] 2 , themselves (10). We have now investigated in greater detail this latter mode of crosslinking of fibrinogen and identified regions of the molecule that are involved in the reactions of its A␣ and ␥ chains with the red cell TG.…”

Prolactin-immunoglobulin G complexes from human serum act as costimulatory ligands causing proliferation of malignant B lymphocytes.

“…A 1.1-ml sample of the PRLIgG complex purified without the use of protease or microbial growth inhibitors by affinity chromatography (eluted with 100 mM glycine at pH 2 and neutralized) was treated with 10% trichloroacetic acid (4°C; 30 min); the centrifuged sediment was washed with 0.5 ml each of 5% trichloroacetic acid (three times in succession), a 1:1 mixture of ethanol/acetone (twice), and acetone (twice) and dried (Speed-Vac concentrator; Savant). Serial digestion by Pronase (three additions of 6 ,ug), carboxypeptidase (two additions of 10 ,g), leucine aminopeptidase (two additions of 3 jig), and prolidase (two additions of 3 ,ug), as well as analysis ofNe--(y-glutamyl)lysine by HPLC was then carried out essentially as published (25). were pulse labeled with [3H]thymidine (0.5 ACi per well; 1 Ci = 37 GBq) for 4 hr, and thymidine incorporation into DNA was measured by scintillation counting as described (27).…”