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Northwestern University, Feinberg Medical School, Chicago, Illinois, USA
1Correspondence: Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Ward Building, Room 7-334, 303 E Chicago Avenue, Chicago, IL 60611, USA. E-mail: l-lorand{at}northwestern.edu
THIS RETROSPECTIVE IS A SUMMARY of research in the area of remodeling and higher order associations of proteins. Having discovered that thrombin functioned as a protease in unleashing the self-assembly potential of fibrinogen molecules for forming a fibrin clot, our attention shifted to the next phase of clotting which is the stabilization of the clot network by covalent crosslinks. This critically important step in hemostasis requires a transamidating enzyme belonging to the family of transglutaminases, TGs. Deciphering of the physiologic pathway for clotting of fibrinogen laid the foundation for diagnosis and rational treatment of patients with previously unrecognized hereditary and autoimmune hemorrhagic diseases. Protein crosslinking by TGs in cells was shown to be activated by elevation of intra-cellular Ca2+ which often caused permanent membrane and skeletal changes characteristic of cell senescence. The presence of TG-crosslinked polymers in diseased red cells with shorter than normal life spans and in human cataract specimens, further supported the relationship between TG action and a terminal phenotype. Nevertheless, TG is also activated in the fertilized egg and during development. Finally, our finding that TG2, independently of its transamidating enzyme activity, can form tight binary and higher non-covalent complexes with other proteins, such as fibronectin, revealed an unexpected scaffolding function for TG2, which has major implications for the organization of connective tissue and wound healing.
Shortly after completing the last semester of medical school in Budapest and having managed to physically distance myself from the earlier Nazi horrors and the subsequent rise of a communist police state, I joined the Department of Biomolecular Structure of W. T. Astbury, FRS, at Leeds University in 1949. For the two previous years, I had worked long hours in extracurricular research in Albert Szent-Gyorgyi’s Institute of Biochemistry where I was mentored by Koloman Laki. In addition to notable accomplishments pertaining to reactions of the dicarboxylic acid series (cited in Szent-Gyorgyi’s Nobel award, and known now as part of the Krebs cycle), Laki was the first to recognize that thrombin functioned as an enzyme. However, the field of blood clotting was essentially still terra incognita, and two important issues needed to be investigated: the enzymatic nature of thrombin and how it brought about the dramatic conversion of fibrinogen to fibrin. I published a paper in 1948 which provided valuable clues by showing that fibrinogen and its thrombin-clotted product, fibrin, had virtually identical viscosities when dissolved in urea and that, with removal of urea, immediate re-clotting occurred (1)
. The results meant: a) that the fibrin network was held together only by non-covalent secondary chemical forces which could be broken by urea; b) that the polymerization of fibrin was reversible; and c) that, in hydrodynamic terms, fibrinogen and the thrombin-modified fibrin units were quite similar in size and shape. Thus, the change in the primary structure of fibrinogen had to be a relatively minor one and, indeed, it was soon proved that thrombin was a protease which, prior to generating fibrin as the main product, cleaved N-terminal segments from the parent fibrinogen. The released peptide material was named fibrinopeptide (2)
. The notion that limited proteolysis could produce such a profound change in protein behavior as the conversion of fibrinogen to clotted fibrin, was quite novel at the time, but it is now a unifying concept in biochemistry, applicable for the post-translational processing of proteins in general. Reactions as diverse as activating the components of the immune complement and coagulation cascades, activating PAR receptors on platelets and other cells, fall into this category.
Specifically, our analysis with Middlebrook (3
4
5
6)
showed that thrombin caused the appearance of new N-termini of Gly in strict proportion to the amount of fibrin produced, with simultaneous loss of some of the end groups originally present in fibrinogen. Removal of the fibrinopeptide moieties is the key for unmasking the self-assembly potential of fibrinogen, because exposure of the new Gly end groups, together with a few downstream amino acid residues, provides the protein-to-protein contacts needed for the non-covalent assembly/polymerization of fibrin molecules.2
However, the two step process of forming a urea-soluble clot (i.e., the thrombin-catalyzed limited proteolytic modification of fibrinogen, followed by a reversible polymerization of fibrin) constitutes only the initial phase of clotting; normal plasma clots cannot be dissolved in 30% urea. While still in England, I had made a good start in purifying the new coagulation factor responsible for this transformation (which I initially named fibrin stabilizing factor or FSF; now known as factor XIII or FXIII); but it was only after moving to the United States that we could embark on the systematic reconstruction of the complex pathway of fibrin stabilization in human blood (Fig. 1
). A great deal of effort was spent on analyzing the intricate choreography of physiologic controls necessary to assure proper timing for generating the enzyme (activated factor XIII or FXIIIa) which stiffens the clot network by insertion of covalent chemical bonds. In brief, we found that the circulating FXIII in plasma was a zymogen which—similarly and concomitantly with the fibrinogen/fibrin transition—also had to be activated by thrombin. Its reaction with thrombin, however, produced only a modified but still inactive zymogen [FXIII’; since the A subunits of the A2B2/FXIII ensemble are susceptible to attack by thrombin (7)
, this is denoted as A2'B2 in Fig. 1
). Thrombin cleavage weakened the affinity between the heterologous subunits which enabled them to dissociate at physiologic Ca2+ concentration; the free A' subunits then undergo a conformational change that unmasks the active center Cys in the FXIIIa enzyme (denoted as A2* in Fig. 1
). FXIII activation is promoted by the clotting substrate, fibrin (broken arrows in Fig. 1
), and the resulting rate advantage for clot stabilization matches the rate difference in thrombin generation between hemophilic and normal plasma.3
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As I was searching for protein modifying reactions to explain the FXIIIa-catalyzed conversion of non-covalent fibrin assembly into a covalently ligated structure, I was attracted to a publication on transglutaminase (TG) from Heinrich Waelsch’s group at Columbia University (8)
. This guinea pig enzyme promoted nucleophilic displacements, such as hydrolysis or amine incorporation involving the 
-carbonylamide functionalities of glutamine (Gln) residues in proteins (e.g., casein, lactoglobulin). Positing that FXIIIa might possess a similar transamidating activity, I tried but was unsuccessful in persuading Waelsch to collaborate in this venture (9)
. Thus, we approached the problem on our own by examining whether small primary amine compounds could inhibit the transformation of the urea-soluble clot to a urea-insoluble one. The results proved immediately that we were on the right track. These substitute nucleophilic donors (Fig. 2
) inhibited the covalent fusion (crosslinking/ligation) of fibrin molecules at the monomeric stage in a highly selective manner without interfering with clotting per se. Moreover, serving as alternate substrates for FXIIIa—tagged, for example, with radioactivity, fluorescence or color—they became incorporated into the relevant Gln (i.e., acceptor) side chains which otherwise would have participated in fibrin-to-fibrin bonding. While the enzyme-driven modification did not appreciably affect the overall morphology of the fibrin network (except that the fibers, themselves, became somewhat tighter), it significantly lowered the stiffness (elastic storage modulus) of the clot. Though not unrelated, perhaps even more striking was to find that, by blocking the introduction of covalent bonds into clots and thrombi, their susceptibilities to digestion by lytic enzymes were remarkably enhanced (3
, 4
, 6)
. I believe these findings provide an adequate rationale for much needed therapeutic trials with inhibitors of FXIIIIa (and of TG2) for preventing the maturation of fibrin deposits, as a general aid to thrombolysis.
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The basic research, which led to the formulation of the physiologic framework of clot formation and stabilization (Fig. 1)
, very soon had a clinical impact in that it made accurate diagnosis of previously unrecognized diverse hemorrhagic diseases possible. Fibrin polymerization [n fibrin
(fibrin)n] is used for detecting harmful mutations in the clotting protein (dysfibrinogenemias; for an example, see ref 10
). Clot solubility assays [in 30% urea (1)
or in 1% monochloroacetic acid (11)
4
] are employed routinely for the diagnosis of the variety of molecular disorders of fibrin clot stabilization. Most of these are hereditary deficiencies of FXIII zymogen. By employing an amine incorporation test for measuring the total FXIIIa activity potential of plasma, we could show that the deficiency was an autosomally inherited recessive trait (12
, 13)
. For reasons not yet understood, phenotypes with the deficiency vary from being asymptomatic to having life threatening hemorrhage; however, almost all patients require FXIII maintenance therapy and close monitoring.5
The sudden appearance of an acquired inhibitor of fibrin stabilization in the circulation may present an even more acute challenge, with 
30% mortality (14
, 15)
. We find that most of the inhibitors are autoantibodies directed against one or more of the molecular targets denoted in Fig. 1
(FXIII, FXIII' or FXIIIa). These FXIII-related autoimmune conditions presaged a growing family of diseases (16)
where a TG is the autoantigen which serves as an excellent marker of disease activity.6
Although Heinrich Waelsch was still alive when our early findings were published, I do not know whether he was aware of the fact that FXIIIa would become the first in the family of TGs with a proven biological function; the letter and the reagents I had sent him years before were found in his freezer after he died and were returned to me by his last graduate student. Including FXIIIa, nine products of the human genome (TG1–TG7 and the non-catalytic erythrocyte band 4.2 relative) belong to this family of proteins. I’ll present a few vignettes to show that the seed planted by Waelsch’s biochemical discovery of an amine-incorporating enzyme in guinea pig tissues has grown into a mighty tree. In addition to the work on the human FXIII system, we identified and isolated novel TGs from a variety of species (lobster muscle, sea urchin eggs, and sea sponge) and tissues, including a form of the enzyme (TG4) from the prostate with unusual properties (and partially sequenced its specific substrate, the basic seminal vesicle secretion protein). But, much of our research focused on TG2 which is widely expressed in both the intra- and extracellular milieu where we found it to fulfill important enzymatic as well as non-enzymatic scaffolding functions.
Perhaps because my laboratories were adjacent to those of my friend and colleague, Myron Bender, who did brilliant work on papain, it did not take us long to recognize that there were similarities in the acylation/deacylation pathways of TGs and of the papain Cys proteases. Though TGs can catalyze hydrolytic reactions [including the cleavage of isopeptide bonds (17)
] and papain is effective in promoting amine transfers, we were more curious about the dissimilarities between the two enzyme families. Foremost was the observation that, in sharp contrast to papain, TGs displayed saturation behavior for their amine substrates (18
, 19)
. This meant that the catalytic paths of TGs included an extra step—not present in the reactions with papain—for forming a second Michaelis complex between the acylenzyme intermediate and the amine. As such, TGs show exquisite specificities for the nucleopilic "second or donor" substrate. Among small amines, monosubstituted diaminopentanes (e.g., dansylcadaverine) with an alkyl side chain length similar to that of the Lys side chains in proteins, were found to have the most favorable Km values. While the Cys/His/Asn(Asp) catalytic triad of papain is preserved in all active TGs, suggesting that TGs probably evolved from the papain family of proteases, the enzyme-intermediate stabilizing role of a Gln residue in papain is replaced by a Trp in TGs (20)
. Thus, regarding enzyme function, we ought to think in terms of catalytic tetrads rather than triads (i.e., Cys/His/Asn/Gln in papain vs. Cys/His/Asn/Trp which is conserved in all eukaryotic TGs).
Among the different post-translational reactions of TGs, the crosslinking of proteins (reaction 1 in Fig. 2A
) seems to have attracted greatest interest, and two distinct protein crosslinking motifs could be discerned (16)
. The first is exemplified by the process of fibrin stabilization where the enzyme functions to spotweld a pre-formed protein assembly. Small competitive Lys and Gln analogs (reactions 2 and 3 in Fig. 2A
) or, for that matter, active site directed inhibitors of FXIIIa do not prevent clot formation per se, but only inhibit clot stabilization exclusively (Fig. 2
B1). The second motif is a TG-mediated, de novo polymerization of protein substrates directly from the soluble phase: nProtein crosslinked(Protein)n. Clotting of lobster plasma or the formation of the copulation plug in rodents are prime examples (6)
. In these situations, the inhibitors prevent the generation of a three dimensional polymer network altogether (Fig. 2B2
), demonstrating that a single TG enzyme (released from a compartment separate from its clotting substrate) may shortcut the entire series of multiple reactions cobbled together by evolutionary tinkering for the coagulation of human (vertebrate) blood plasma.7
Geometry of the three-dimensional network will depend on the number and disposition of TG-reactive Gln and Lys groups within the monomeric building blocks. TGs differ significantly in their specificities, and no consensus sequence has emerged for any TG thus far. Though the primary sequence around the TG-reactive residues has some influence, we noticed that exposed location in a flexible segment of the protein is a more important determinant.
To examine the protein remodeling roles of TGs in cells, one could rely again on the approach of probing protein crosslinking with competitive inhibitors (Fig. 2)
. We showed that, depending on the time of exposure, latent TGs in mammalian cells could be activated by 0.01 mM Ca2+ (21)
, and initiated a search for TG-crosslinked protein polymers in Ca2+-stressed human red cells as the first paradigm (22
, 23)
. The ionophore-mediated influx of Ca2+ induced a permanent fixation of cell shape and an irreversible loss of membrane deformability (24)
. The abnormalities arose as a result of the TG2-catalyzed crosslinking of the membrane skeleton (spectrin, ankyrin, band 4.1 and the anion-transporter band 3 protein, among others) by N
(-glutamyl)lysine bonds. As with fibrin crosslinking, all the morphological, physical and chemical changes could be prevented by including small amines (e.g., cystamine,8
cysteamine) in the Ca2+-containing incubation medium; thus, it was evident in this case, too, that the amines blocked the chain of polymerization by virtue of becoming incorporated into the Gln residues of the participating protein subunits. Inasmuch as isopeptide-containing membrane skeletal polymers were found in Hb-Koln cells and in sickle cells (perhaps because of the presence of the irreversibly sickled cells) with shortened life spans (16
, 21)
, but not in normal erythrocytes, it was suggested that the TG2-catalyzed reaction—possibly by exposing surface epitopes that are recognized by macrophages—hastened the removal of the affected cells from the circulation. Because the Ca2+-ions must also overcome the inhibition of some of the intracellular TGs, including TG2, by GTP/GDP, it might be further surmised that TG-catalyzed reactions might be triggered at even less than 0.01mM concentration of Ca2+ in cells (and not just in the erythrocytes) of patients with hereditary disorders of purine metabolism (26)
; [molecular aspects of the allosteric switch which, upon nucleotide binding, converts TG2 from an open active configuration into a compact inactive conformation were recently clarified (27)
]. As a further corroboration of the idea that the TG-dependent formation of crosslinked polymers was a terminal phenomenon in cells, such structures were found in abundance in specimens of human cataracts (28)
and in thrombin-activated platelets (29)
. Yet, somewhat paradoxically, the protein remodeling activity of TG could be demonstrated not only in cells in extremis but also in the fertilized egg (sea urchin) and during development, where blocking of TG action produced abnormal embryos (30
31
32)
.
The approach of triggering TG actions in cells by influx of Ca2+, while probing protein crosslinking reactions with inhibitors, has been widely adopted for a variety of studies in other laboratories. Prime examples include the formation of cornified envelope in terminally differentiating keratinocytes (33)
and the angiotensinII-induced, cellular FXIIIa-dependent dimeric crosslinking of the type I angiotensinII receptor in monocytes of patients with essential hypertension and in those of apoE–/– dyslipidemic mice. The modified monocytes adhere to endothelial cells and are thought to initiate the atherosclerotic process (34)
.
A new perspective on the biological roles of TGs was revealed with the finding that TG2, independently of its enzymatic and nucleotide binding ability, formed tight non-covalent binary and higher complexes with fibronectin (35
36
37
38)
, such as [TG2:fibronectin] and [TG2:fibronectin:collagen]. This has important implications for the disposition and function of TG2 in tissues, in general, and also for wound healing where TG2 is discharged from lysed erythrocytes. We also found that TG2-specific antibodies (Ab) in patients with celiac disease did not interfere with the binding of TG2 to fibronectin; as such, large [Ab:TG2:fibronectin:...] deposits can form in various organs. More recently, it has been shown that TG2 binds to ß1-integrin which seems to be essential for cell adhesion, spreading and mobility (39)
. Thus TG2 serves the function of an essential bridge molecule in the scaffold [ß1integrin:TG2:fibronectin:collagen] between the cell surface and connective tissue matrix. This, along with the observation that TG2 binds to a protein (GPCR56) on the melanoma cell surfaces and that its down-regulation correlates with an aggressive metastatic phenotype (40)
, is likely to be relevant to cancer metastasis. In the intracellular milieu, TG2 co-localizes with intermediate filaments in the cytoskeleton [vimentin (41)
, keratin (42)
] and associates with phospholipases, exerting regulatory influence on the activities of the cytosolic PLC
(43
, 44)
and the membrane-bound PLC (Murthy, S. N. P., Chung, P. H., Belkin, A. M. Lorand, L., and Lomasney, J. W., unpublished data). But, the exact role of TG2 (also denoted as a high molecular weight G protein, Gh) in signal transmission still awaits clarification (45)
.
In tune with the present excitement with regard to three-dimensional nanostructures for biological applications, it might be of interest to remind ourselves how natural superstructures are generated either by a protease (thrombin) or a transglutaminase, or, as in the case of human blood clotting, by the two acting in succession. Mechanical and physiologic properties of the polymeric networks depend on the relative contributions of these enzymes. Hemorrhagic diseases of fibrin stabilization highlight the importance of the system for normal hemostasis. Our research also showed that the protein crosslinking activity of TGs was triggered by a rise of intracellular Ca2+ concentration to levels seen only with excessive agonist stimulation, failure or blockade of the Ca2+ pump. Frequently, this correlates with a terminal/senescent phenotype but occurs also during embryogenesis. Independently of its crosslinking activity, TG2 was found to form tight complexes with other proteins, of which the [TG2:fibronectin] and [TG2:fibronectin:collagen] ensembles were the fist examples, opening up new avenues for studies on the scaffolding and regulatory roles of TG2.
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FOOTNOTES
2 Historical details and primary references pertaining to statements in this article may be found in references 3
4
5
6
, 9
, and 16
. 
3 Because many clinical tests are designed to override the important activating effects of fibrin, I believe that a number of cases of hemorrhagic disorders of fibrin stabilization may be overlooked.
4 Solubility in monochloroacetic acid, a reagent I borrowed from a textile laboratory, appears to be the fastest means for diagnosing FXIII-related disorders.
5 Other TG deficiency diseases include lamellar ichtyosis (TG1) and spherocytosis (band 4.2 protein).
6 TG2 is the autoantigen in celiac disease, TG2 and TG3 in dermatitis herpetiformis.
7 So much for the myth of irreducible complexity of the Intelligent Design doctrine. TGs seem to be remarkably interchangeable in some systems; e.g. the guinea pig liver TG2 and the 0.5 billion year old sponge Microciona proliferans TG are potent enzymes for clotting lobster blood (6)
. TG2s obtained from a variety of sources can stabilize human fibrin, though reacting at sites in the protein other than those utilized by FXIIIa (4
, 16)
.
8 Given the widely held assumption that TGs contribute either to the formation or the maturation of protein deposits in a variety of neurodegenerative diseases, including the polyglu extension diseases, injections of cystamine (for a caveat regarding cystamine, see ref 25
) and cysteamine have been shown to ameliorate the neurological symptoms in mice transgenic for Huntington’s disease. However, as it often happens, drugs designed for interfering with the action of a specific enzyme may produce pharmacological benefits in an entirely unpredictable manner, and this seems to be the case with these compounds in relation to Huntington’s disease.
The opinions expressed in editorials, essays, letters to the editor, and other articles comprising the Up Front section are those of the authors and do not necessarily reflect the opinions of FASEB or its constituent societies. The FASEB Journal welcomes all points of view and many voices. We look forward to hearing these in the form of op-ed pieces and/or letters from its readers addressed to journals@faseb.org.
REFERENCES
IVS4+1G>T mutation and an AGln328 truncation (fibrinogen Keokuk). Blood 103,2571-2576: isopeptides by cytosolic transglutaminases and by coagulation of factor XIIIa. J. Biol. Chem. 272,10311-103171 is a guanine nucleotide exchanging factor for transglutaminase II (Gh) and promotes 1B-adrenoreceptor-mediated GTP binding and intracellular calcium release. J. Biol. Chem. 276,5591-55971 and with GTP. Proc. Natl. Acad. Sci. USA 96,11815-11819Related Articles
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