Plasma Transglutaminase Research Articles

Molecular and genetic mechanisms of factor XIII A subunit deficiency.

Factor XIII is a proenzyme for a plasma transglutaminase. Factor XIII in plasma is a tetramer (A2B2) held together by noncovalent bonds, and the A subunit contains the active site. Recently, the three-dimensional structure of the A subunit has been determined by x-ray crystallography. To understand the structure-function relationships of the factor XIII molecule and its clinical implications in factor XIII deficiency, we characterized its genetic defects and closely examined its gene products, including mRNA and protein levels. A variety of missense and nonsense mutations (Arg260-Cys, Tyr283-Cys, Gly562-Arg) and deletions/insertions with or without out-of-frame shift/premature termination and splicing abnormalities (4-bp deletion with 464Stop, T insertion at the exon IV/intron D boundary with exon IV-skipping, 20-bp deletion at the exon I/intron A boundary) has been identified in cases demonstrating A subunit deficiency. In some cases, the A subunit mRNA levels were severely reduced. Their molecular and cellular bases have also been explored by expression experiments in mammalian cells and by molecular modeling. In most cases, impaired folding and/or conformational changes of the mutant A subunits lead to both intra- and extracellular instability, which is responsible for the A subunit deficiency in the patients.

Fibronectin: structure, assembly, and cardiovascular implications.

In the past 2 decades, it has been appreciated that the functions of the extracellular matrix (ECM) are not entirely structural. ECM components interact with specific adhesion receptors on cell surfaces and regulate various cellular functions, including differentiation, proliferation, migration, and apoptosis. Fibronectin (FN) is a paradigm adhesive protein, nonreactive with adhesion receptors in its soluble state but highly adhesive when insoluble. Polymerization of FN into the ECM must be tightly regulated to ensure that the adhesive information in the ECM is appropriate. FN exists in a soluble protomeric form in micromolar concentration in blood plasma and in an insoluble multimeric form in the ECM.1 2 Unlike fibrillar or basement membrane collagens, laminins, actin, and tubulin, circulating FN does not self-polymerize in physiologically relevant solutions. Furthermore, there is little passive accumulation of FN in preexisting ECM. Rather, assembly of FN takes place at specialized areas on the cell surface.3 FN is especially abundant in the ECM of embryonic and regenerating or injured tissues, although it can be found in most ECMs, including basement membranes. FN interacts with cells through integrins, heterodimeric transmembrane receptors linking the ECM to the intracellular cytoskeleton and signaling pathways. The aim of this review is to describe the mechanisms and consequences of FN deposition and give a brief overview of the significance of FN for selected areas of cardiovascular research. In the first section we describe important features of the FN molecule that account for its multiple functions. Next, we focus on the assembly process, ie, the conversion of soluble FN to its active, adhesive, insoluble form. Finally, we discuss several areas of cardiovascular research in which FN may have an important role, exemplifying how the adhesive information of FN can drive pathophysiological processes. Soluble FN is a dimeric glycoprotein. Each subunit is a …

Plasma transglutaminase in hypertrophic chondrocytes: expression and cell-specific intracellular activation produce cell death and externalization.

We previously used subtractive hybridization to isolate cDNAs for genes upregulated in chick hypertrophic chondrocytes (Nurminskaya, M. , and T.F. Linsenmayer. 1996. Dev. Dyn. 206:260-271). Certain of these showed homology with the "A" subunit of human plasma transglutaminase (factor XIIIA), a member of a family of enzymes that cross-link a variety of intracellular and matrix molecules. We now have isolated a full-length cDNA for this molecule, and confirmed that it is avian factor XIIIA. Northern and enzymatic analyses confirm that the molecule is upregulated in hypertrophic chondrocytes (as much as eightfold). The enzymatic analyses also show that appreciable transglutaminase activity in the hypertrophic zone becomes externalized into the extracellular matrix. This externalization most likely is effected by cell death and subsequent lysis-effected by the transglutaminase itself. When hypertrophic chondrocytes are transfected with a cDNA construct encoding the zymogen of factor XIIIA, the cells convert the translated protein to a lower molecular weight form, and they initiate cell death, become permeable to macromolecules and eventually undergo lysis. Non-hypertrophic cells transfected with the same construct do not show these degenerative changes. These results suggest that hypertrophic chondrocytes have a novel, tissue-specific cascade of mechanisms that upregulate the synthesis of plasma transglutaminase and activate its zymogen. This produces autocatalytic cell death, externalization of the enzyme, and presumably cross-linking of components within the hypertrophic matrix. These changes may in turn regulate the removal and/or calcification of this hypertrophic matrix, which are its ultimate fates.

Coagulation Factor XIIIa Undergoes a Conformational Change Evoked by Glutamine Substrate: STUDIES ON KINETICS OF INHIBITION AND BINDING OF XIIIa BY A CROSS-REACTING ANTIFIBRINOGEN ANTIBODY

Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamine:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen Aalpha-chain segment 529-539 was previously observed from analysis of end-stage plasma clots to block fibrin alpha-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit gamma-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope Aalpha-(529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.

Significance of Diminished Factor XIII in Crohn's Disease

Coagulation factor XIII is a plasma transglutaminase involved in crosslinking of fibrin, the last step of the coagulation system and a connective tissue factor contributing to the wound healing process. It circulates as a heterotetrameric molecule consisting of two identical proenzyme subunits (factor XIIIA) and two carrier protein subunits (factor XIIIS). The aim of this study was to determine the disease features associated with the diminution of factor XIII in Crohn's disease. Factor XIIIA and factor XIIIS levels were assessed in patients presenting with Crohn's disease, ulcerative colitis, infectious colitis, or diverticulitis, in patients with rheumatoid arthritis, and in control subjects. Prothrombin fragment 1 + 2 assay, as a marker of the generation of thrombin and measurement of C-terminal telopeptide of type I collagen as an estimate of degradation of collagen type I, were performed. Factor XIIIA was significantly decreased in Crohn's disease, in ulcerative colitis, and in infectious colitis by comparison with subjects presenting with diverticulitis, normal, and rheumatoid subjects p = 0.0001). Factor XIIIS was unmodified in patients with Crohn's disease by comparison with controls but was reduced in those presenting with intestinal bleeding (p = 0.0002). In Crohn's disease, the lowest level of factor XIIIA was observed in patients with intestinal bleeding (p = 0.0003). Factor XIIIA was correlated with the Van Hees index (r = -0.5661; p = 0.0001) and with the C-terminal telopeptide of type I collagen (r = -0.4110; p = 0.0011) but not with prothrombin fragment 1 + 2. The multiple regression analysis showed that only Van Hees index and intestinal bleeding were independent variables for explaining the diminution of Factor XIIIA in Crohn's disease. Factor XIIIA subunit is an indicator of Crohn's disease activity. Our study suggests that a low factor XIIIA level is related to the presence of intestinal lesions and might be linked to intestinal repair mechanisms; loss in intestinal lumen could be also involved, especially in patients with intestinal bleeding.

Significance of Diminished Factor XIII in Crohn’s Disease

Objective: Coagulation factor XIII is a plasma transglutaminase involved in crosslinking of fibrin, the last step of the coagulation system and a connective tissue factor contributing to the wound healing process. It circulates as a heterotetrameric molecule consisting of two identical proenzyme subunits (factor XIIIA) and two carrier protein subunits (factor XIIIS). The aim of this study was to determine the disease features associated with the diminution of factor XIII in Crohn’s disease. Methods: Factor XIIIA and factor XIIIS levels were assessed in patients presenting with Crohn’s disease, ulcerative colitis, infectious colitis, or diverticulitis, in patients with rheumatoid arthritis, and in control subjects. Prothrombin fragment 1 + 2 assay, as a marker of the generation of thrombin and measurement of C-terminal telopeptide of type I collagen as an estimate of degradation of collagen type I, were performed. Results: Factor XIIIA was significantly decreased in Crohn’s disease, in ulcerative colitis, and in infectious colitis by comparison with subjects presenting with diverticulitis, normal, and rheumatoid subjects p = 0.0001). Factor XIIIS was unmodified in patients with Crohn’s disease by comparison with controls but was reduced in those presenting with intestinal bleeding ( p = 0.0002). In Crohn’s disease, the lowest level of factor XIIIA was observed in patients with intestinal bleeding ( p = 0.0003). Factor XIIIA was correlated with the Van Hees index (r = −0.5661; p = 0.0001) and with the C-terminal telopeptide of type I collagen (r = −0.4110; p = 0.0011) but not with prothrombin fragment 1 + 2. The multiple regression analysis showed that only Van Hees index and intestinal bleeding were independent variables for explaining the diminution of Factor XIIIA in Crohn’s disease. Conclusions: Factor XIIIA subunit is an indicator of Crohn’s disease activity. Our study suggests that a low factor XIIIA level is related to the presence of intestinal lesions and might be linked to intestinal repair mechanisms; loss in intestinal lumen could be also involved, especially in patients with intestinal bleeding.

Characterization of the reciprocal binding sites on human alpha-thrombin and factor XIII A-chain.

Solution- and solid-phase techniques were used to probe Factor XIII A-chain-alpha-thrombin interactions. Alpha-thrombin activated Factor XIII more efficiently (Km = 0.83 +/- 0.08 x 10(-7) M; V/K = 14.90 +/- 3.20 x 10(-3) min(-1)) than beta-thrombin (Km = 6.14 +/- 1.26 x 10(-7) M; V/K = 3.30 +/- 1.00 x 10(-3) min(-1)) or gamma-thrombin (Km = 6.25 +/- 1.15 x 10(-7) M; V/K = 3.00 +/- 0.80 x 10(-3) min(-1)). Immobilized FPR-alpha-thrombin bound plasma Factor XIII (Kd = 0.17 +/- 0.04 x 10(-7) M) > Factor XIIIa (Kd = 0.69 +/- 0.18 x 10(-7) M) > liver transglutaminase (Kd = 4.73 +/- 1.01 x 10(-7) M) > Factor XIII A-chain (Kd = 49.00 +/- 9.40 x 10(-7) M). FPR-alpha-thrombin and alpha-thrombin also bound immobilized Factor XIII A-chain with affinities inversely related to protease activity: maximal binding at 1.36 x 10(-7) M and 13.6 x 10(-7) M, respectively. Plasma Factor XIII, transglutaminase, and dithiothreitol competitively inhibited Factor XIII A-chain binding to FPR-alpha-thrombin: IC50 = 1.0 x 10(-7) M, 3.0 x 10(-6) M and 1.52 x 10(-4) M, respectively. Transglutaminase also inhibited Factor XIII binding to alpha-thrombin (IC50 = 2.0 x 10(-6) M). Thrombin-binding site was localized to G38-M731 fragment of Factor XIII A-chain, probably within homologous regions (N72-A493) of transglutaminase. R320-E579 of alpha-thrombin was Factor XIII A-chain binding site. Intra-B-chain disulfides in alpha-thrombin were essential for binding but not catalytic H363 or residues R382-N394 and R443-G475. These studies propose a structural basis for Factor XIII activation, provide a regulatory mechanism for Factor XIIIa generation, and could eventually help in the development of new structure-based inhibitors of thrombin and Factor XIIIa.

Recombinant Polypeptides Derived from the Fibrin Binding Domain of Fibronectin Are Potential Agents for the Imaging of Blood Clots

Thrombus formation in the circulation is accompanied by covalent linkage of fibronectin (FN) through transglutamination of glutamine no. 3 in the fibrin binding amino terminal domain (FBD) of FN. We have exploited this phenomenon for thrombus detection by the employment of radioactively-labelled recombinant polypeptide molecules derived from the 5-finger FBD of human FN. Three recombinant FBD polypeptides, 12 kDa ("2 fingers"), 18.5 kDa ("3 fingers") and 31 kDa FBD ("5 fingers"), were prepared and compared to native FN-derived 31 kDa-FBD with respect to their ability to attach to fibrin clots in vitro and in vivo. The accessibility of Gln-3 in these molecules was demonstrated by the incorporation of stoichiometric amounts of 14C-putrescine in the presence of plasma transglutaminase. Competitive binding experiments to fibrin have indicated that, although the binding affinities of the FBD molecules are lower than that of FN, substantial covalent linkage was obtained in the presence of transglutaminase, and even in the presence of excess FN or heparin. The biological clearance rates of radioactively labelled FBD molecules in rats and rabbits were much higher than those of FN and fibrinogen, thus indicating their potential advantage for use as a diagnostic imaging tool. Of the three molecules, the 12 kDa FBD exhibited the highest rate of clearance. The potential of the 12 kDa and 31 kDa FBDs as imaging agents was examined in a stainless steel coil-induced thrombus model in rats and in a jugular vein thrombus model in rabbits, using either [125I] or [111In]-labelled materials. At 24 h, clot-to-blood ratios ranged between 10 and 22 for [125I]-12 kDa FBD and 40 and 60 for [111In]-12 kDa FBD. In the rat model, heparin did not inhibit the uptake of FBD. Taken together, the results indicate that recombinant 12 kDa FBD is a good candidate for the diagnosis of venous thrombosis.

Location of the Major ∊-(γ-Glutamyl)lysyl Cross-linking Site in Transglutaminase-modified Human Plasminogen

Tissue and plasma transglutaminases cross-link human plasminogen into high molecular weight complexes (Bendixen, E., Borth, W., and Harpel, P. C. (1993) J. Biol. Chem. 268, 21962-21967). A major cross-linking site in plasminogen involved in the tissue transglutaminase-mediated polymerization process has been identified. The epsilon-(gamma-glutamyl)lysyl bridges of the polymer are formed between Lys-298 and Gln-322. Both the acyl donor Gln residue and the acyl acceptor Lys residue are located in the kringle 3 domain of plasminogen, i.e. cross-linking of plasminogen by tissue transglutaminase involves neither the catalytic domain nor the lysine-dependent binding sites of plasminogen. This study documents that kringle 3 contains a novel functional site with the potential to participate in transglutaminase-mediated cross-linking interactions with plasma, cell-surface, and extracellular proteins.

Identification of a substrate site for transglutaminases on the human protein synthesis initiation factor 5A

Protein synthesis initiation factor 5A (eIF-5A) from human erythrocytes was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (GPLTG). When purified eIF-5A was incubated with GPLTG or Factor XIIIa in the presence of succinylated beta-casein, a covalent complex was identified. By isolating and analysing the product of the transglutaminases (TGases) reaction, the site of modification on eIF-5A has been identified as the unique amino acid hypusine. The complex beta-casein.eIF-5A was enzymatically digested with proteinases and the predicted covalent cross-link of gamma-glutamyl-omega-hypusine was isolated from the digests by ion-exchange chromatography and purified by reversed-phase h.p.l.c. Acid hydrolysis of the purified dipeptide yielded equimolar amounts of hypusine and glutamic acid. Furthermore, fast atom bombardment m.s. analysis confirmed the isomer assignment to be gamma-glutamyl-omega-hypusine. These data indicate that hypusine-50 of the eIF-5A chain functions as acyl acceptor substrate for TGases, and reveal that eIF-5A may be cross-linked to intracellular proteins by TGases. Because the precise function of eIF-5A is still unknown, our results appear particularly stimulating in the light of the recent finding of a new biological role for this protein as a cellular factor binding specifically to the human immunodeficiency virus-1 Rev activation domain [Ruhl, Himmelspach, Bahr, Hammerschmid, Jaksche, Wolff, Auschauer, Farrington, Probst, Bevec and Hauber (1993) J. Cell Biol. 123, 1309-1320].

Progressive cross-linking of fibrin gamma chains increases resistance to fibrinolysis.

In the presence of plasma transglutaminase (factor XIIIa) fibrin first undergoes intermolecular covalent cross-linking between its gamma chains to create gamma dimers followed by slower cross-linking among its alpha chains to form alpha polymers. Progressive cross-linking of gamma chain dimers occurs at the slowest rate, resulting in gamma trimers and gamma tetramers ("gamma multimers"). Most studies indicate that cross-linked fibrin clots become resistant to fibrinolysis, but the basis for this event is not clear. In this study, we explored the role of gamma chain multimerization compared with alpha polymerization as causal factors in time-dependent development of resistance to fibrinolysis. Fibrin clots prepared from native (intact) fibrinogen were incubated for up to 120 h at near physiological ionic strength and a factor XIIIa level approximating that in plasma. These clots were lysed by plasmin at rates that were inversely proportional to the level of gamma multimers, which increased progressively with the time of incubation. In contrast, fibrin cross-linked at high ionic strength (a condition under which only gamma dimers and alpha polymers form) or fibrin formed in the absence of factor XIII showed no time-dependent decrease in lysis rates. Fibrin cross-linked for a fixed time period with increasing amounts of factor XIIIa contained gamma multimer levels that were proportional to the factor XIIIa concentration and lysed at rates that were inversely proportional to the gamma multimer level. Furthermore, cross-linked fibrin formed from fibrinogen fraction I-9, which has limited potential for alpha polymerization, showed the same reduction in the lysis rate as native cross-linked fibrin. These findings indicate that development of resistance to fibrinolysis of cross-linked fibrin is not measurably dependent upon gamma dimer or alpha polymer formation but develops solely as a function of gamma multimerization.

Transglutaminase inhibition by 2-[(2-oxopropyl)thio]imidazolium derivatives: mechanism of factor XIIIa inactivation.

The physiologic role of several transglutaminases could be more precisely defined with the development of specific inhibitors for these enzymes. In addition, specific plasma transglutaminase (fXIIIa) inhibitors may have therapeutic utility in the treatment of thrombosis. For these purposes, the inactivation of fXIIIa and human erythrocyte transglutaminase (HET) by 2-[(2-oxopropyl)thio]imidazolium derivatives, which comprise a novel class of transglutaminase inactivators, was studied. As a specific example, 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (III) inactivated fXIIIa with an apparent second-order rate constant (specificity constant of inactivation) of 6.3 x 10(4) M-1 s-1, corresponding to a rate 4 x 10(7) times greater than its reaction rate with glutathione (GSH). The mechanism of fXIIIa inactivation by this class of compounds was investigated utilizing two [14C]-isotopic regioisomers of 1,3-dimethyl-2-[(2-oxopropyl)thio]imidazolium iodide (II). Structural analyses demonstrated that acetonylation of the active site cysteinyl residue of fXIIIa occurred along with the stoichiometric release of the complementary fragment of the inactivator as the corresponding thione. Kinetic analysis of the inactivation of fXIIIa by nonquarternary analogs of II and III indicated the formation of a reversible complex between the inactivator and fXIIIa prior to irreversible modification of the enzyme. At 1 mM, III displayed no detectable levels of inhibition or inactivation with several serine proteases and thiol reagent-sensitive enzymes. 2-[(2-Oxopropyl)thio]imidazolium derivatives and the related molecule 2-(1-acetonylthio)-5-methylthiazolo-[2,3]-1,3,4-thiadiazo lium perchlorate (I), when present at the time of clot formation at 1-10 microM, enhanced the rates of tissue plasminogen activator catalyzed clot lysis in vitro. These inactivators prevented the fXIIIa-catalyzed covalent incorporation of alpha 2-antiplasmin into the alpha chain of fibrin and the formation of high molecular weight fibrin alpha chain polymers, providing the basis for the observed enhancements in clot lysis rates.

The human tissue transglutaminase gene maps on chromosome 20q12 by in situ fluorescence hybridization.

A cDNA encoding for the human tissue transglutaminase gene has been used to identify the chromosomal localization of the corresponding structural gene. The precise chromosomal and subregional localizations have been established by using in situ fluorescence mapping with a recombinant lambda-Zap phage containing the full cDNA coding sequence. The study showed that the human tissue transglutaminase gene is localized on chromosome 20 and, more precisely, within the band 20q12. To date, this is the third member of the transglutaminase gene family to be mapped. Human factor XIIIa (plasma transglutaminase), human keratinocyte transglutaminase (type I), and human tissue transglutaminase (type II) genes, although codifying for homologous enzymes, are localized on three different chromosomes.

Characterization of a Monoclonal Antibody Directed Against the Carboxyl-Terminus of Human Factor XIII

By deriving an anti-peptide monoclonal antibody, mAb 7A4, we characterized the relatively unstudied carboxyl-terminal end of the a-chain of human factor XIII, the plasma transglutaminase. MAb 7A4 was directed against the last eight amino acids (Gln-Ile-Gln-Arg-Arg-Pro-Ser-Met) and bound with a dissociation constant of 3.4 x 10(-8)M. In a solid assay format, mAb 7A4 bound equally well to factor XIII obtained from human plasma, platelets or placenta. However, in a solution-phase assay format, the epitope was largely unavailable but could be readily exposed by heat denaturation. Immunoblotting showed that this epitope is conserved among all species of plasma factor XIII tested except rabbit suggesting that the carboxyl-terminus might be an important structural element. Other competitive binding experiments with synthetic peptides as inhibitors pointed toward the final carboxyl-terminal amino acid, Met-731, as an immunochemically important determinant. This was used advantageously to confirm the finding that the carboxyl-terminal Met-731 is largely absent from placental factor XIII (1) as compared to platelet or plasma factor XIII.

Wheat .gamma. gliadin as substrate for bovine plasma factor XIII

For the first time, a transglutaminase (EC 2.3.2.13) activity was investigated in water/solvent mixtures. This study highlighted that bovine plasma transglutaminase (factor XIII) was active in the presence of dioxane, acetonitrile, and ethanol up to 15%, 20%, and 20%, respectively. It allowed enzymatic modification of wheat prolamins. The transfer and hydrolytic activities of factor XIII were investigated on a purified gamma 46 gliadin considered as a prolamin model substrate. In optimum conditions of pH and dioxane concentration, 15% of the glutaminyl residues present in gamma gliadin were deamidated after 24 h of enzymatic treatment. The nonrepetitive region of the gamma gliadin was preferentially modified by factor XIII. The transglutaminase was able to produce soluble polymeric complexes of gliadins, through intermolecular epsilon-(gamma-glutamyl)lysyl cross-links. The two lysyl residues of gamma gliadin were supposed to act as acyl acceptor sites.

Human Erythrocyte Protein 4.2: Isoform Expression, Differential Splicing, and Chromosomal Assignment

Abstract Human protein 4.2 (P4.2) is a major membrane skeletal protein in erythrocytes. Individuals with P4.2 deficiency exhibit spherocytosis and experience various degrees of hemolytic anemia, suggesting a role for this protein in maintaining stability and integrity of the membrane. Molecular cloning of P4.2 cDNAs showed that P4.2 is a transglutaminaselike molecule in erythrocytes but lacks the essential cysteine for cross-linking activity. Two cDNA isoforms have been identified from a human reticulocyte cDNA library, with the long isoform containing a 90-base pair (bp) in-frame insertion encoding an extra 30 amino acids near the N-terminus. Characterization of the P4.2 gene suggests differential splicing as the mechanism for generating these two cDNA isoforms. The donor site for the short isoform (P4.2S) agrees better with the consensus than the donor site for the long isoform (P4.2L) does. Expression of P4.2L was detected by a long- isoform-specific antibody raised against a peptide within the 30-amino acid insert. Western blot analyses showed P4.2L to be a minor membrane skeletal protein in human erythrocytes with an apparent molecular weight (mol wt) of approximately 3 Kd larger than the major protein 4.2, P4.2S. By in situ hybridization of a full-length 2.4-kilobase (kb) cDNA to human metaphase chromosomes, the gene for P4.2 was mapped to bands q15-q21 of chromosome 15, and it is not linked to the gene for coagulation factor XIIIa (plasma transglutaminase, TGase).

Human erythrocyte protein 4.2: isoform expression, differential splicing, and chromosomal assignment

Human protein 4.2 (P4.2) is a major membrane skeletal protein in erythrocytes. Individuals with P4.2 deficiency exhibit spherocytosis and experience various degrees of hemolytic anemia, suggesting a role for this protein in maintaining stability and integrity of the membrane. Molecular cloning of P4.2 cDNAs showed that P4.2 is a transglutaminaselike molecule in erythrocytes but lacks the essential cysteine for cross-linking activity. Two cDNA isoforms have been identified from a human reticulocyte cDNA library, with the long isoform containing a 90-base pair (bp) in-frame insertion encoding an extra 30 amino acids near the N-terminus. Characterization of the P4.2 gene suggests differential splicing as the mechanism for generating these two cDNA isoforms. The donor site for the short isoform (P4.2S) agrees better with the consensus than the donor site for the long isoform (P4.2L) does. Expression of P4.2L was detected by a long-isoform-specific antibody raised against a peptide within the 30-amino acid insert. Western blot analyses showed P4.2L to be a minor membrane skeletal protein in human erythrocytes with an apparent molecular weight (mol wt) of approximately 3 Kd larger than the major protein 4.2, P4.2S. By in situ hybridization of a full-length 2.4-kilobase (kb) cDNA to human metaphase chromosomes, the gene for P4.2 was mapped to bands q15-q21 of chromosome 15, and it is not linked to the gene for coagulation factor XIIIa (plasma transglutaminase, TGase).

Factor XIII-dependent generation of 5th complement component(C5)-derived monocyte chemotactic factor coinciding with plasma clotting

Blood coagulation or plasma clotting caused generation of a monocyte chemotactic factor(s) in vitro. The chemotactic factor, of which the apparent molecular mass was 75 kDa, shared antigenicity with complement C5 and possessed the affinity to monocytes, but not to polymorphonuclear leukocytes. The generation of the chemotactic factor was hindered in the presence of a thiol enzyme inhibitor, p-chloromercuriphenyl sulfonic acid, at the concentration of 1 mmol/l, although the gelation of plasma was apparently completed. Furthermore, the generation of chemotactic factor was not observed when a plasma deficient in blood coagulation factor XIII, which is a precursor of a thiol enzyme, plasma transglutaminase, was used; and the activity normally appeared when the deficient plasma was reconstituted with purified factor XIII or with a tissue transglutaminase prior to clotting. When the human sera were injected into guinea pig skin, the serum derived from normal plasma or from the reconstituted factor XIII deficient one caused mononuclear cell filtration, however, the serum from the deficient plasma without reconstitution infiltrated to a significantly smaller extent. These results indicated that the complement system was initiated somehow during the clotting process resulting in the generation of the C5-derived monocyte chemotactic factor in cooperation with factor XIIIa (activated factor XIII).

Transglutaminases: multifunctional cross‐linking enzymes that stabilize tissues

Transglutaminases catalyze the posttranslational modification of proteins by transamidation of available glutamine residues. This action results primarily in the formation of epsilon-(gamma-glutamyl)lysine cross-links but includes the incorporation of polyamines into suitable protein substrates as well. The covalent isopeptide crosslink is stable and resistant to proteolysis, thereby increasing the resistance of tissue to chemical, enzymatic, and mechanical disruption. The plasma transglutaminase, factor XIIIa, is formed at sites of blood coagulation and impedes blood loss by stabilizing the fibrin clot. The squamous epithelium constituting the protective callus layer of skin is formed by the action of keratinocyte transglutaminase (TGK) and epidermal transglutaminase (TGE). The tissue transglutaminase (TGC) is a cytoplasmic enzyme present in many cells including those in the blood vessel wall. TGC function is unknown, although it could function to stabilize intra- and extra-cellular molecules in a wide variety of physiologic or pathologic processes. The amino acid sequences of factor XIII, TGC, and TGK establish them as a homologous gene family and also reveal a striking homology to the erythrocyte membrane protein, band 4.2. This review summarizes the current information on structures, functions, and evolution of the most prominent members of this gene family.

Osteopontin, a substrate for transglutaminase and Factor XIII activity

Osteopontin (OPN), an extracellular matrix cell adhesion protein, was found to serve as a substrate for the incorporation of radiolabelled putrescine mediated by a commercial preparation of guinea pig liver transglutaminase. Preliminary evidence also suggests that OPN serves as a substrate for the plasma transglutaminase, Factor XIIIa. While the protein substrates to which OPN is linked in vivo have not been identified, it is reasonable to speculate that this capacity of OPN may dictate its extracellular location and thereby affect its role in bone homeostasis, tumorigenesis, metastasis, resistance to bacterial infections or, perhaps, wound repair.