TRANSGLUTAMINASE-MEDIATED REMODELING OF THE HUMAN ERYTHROCYTE MEMBRANE SKELETON: RELEVANCE FOR ERYTHROCYTE DISEASES WITH SHORTENED CELL LIFESPAN

The human red blood cell transglutaminase (hRBC TG2) was the first in this family of enzymes for which an important role in cell–matrix interaction was found by demonstrating that the protein—when released from cells—could form an extremely tight complex with human fibronectin (FN). The binding, with a stoichiometry of 2TG2:FN (i.e., 1TG2 per constituent chain of FN), is independent of the catalytic activity of TG2 and occurs in the absence as well as in the presence of Ca2+ ions [1–3]. Residues 81–106 of TG2, located at the extended hairpin between antiparallel β strands 5 and 6 of the first domain of the protein, seem to be essential for binding to FN; mutations of Asp94 and Asp97 to Ala reduce the binding affinity of TG2 to FN significantly. A synthetic peptide, corresponding to the sequence 88WTATVVDQQDCTLSLQLTT106 in TG2, inhibits the TG2–FN interaction, and also TG2-dependent cell adhesion and spreading [4]. The complementary binding sites of FN are located in a 42-kDa collagen-binding domain of the protein, comprising motifs I6-II1-II2-I7-I8-I9. This fragment shows as high an affinity for TG2 as the individual parent FN chains themselves [5]; furthermore, the 42-kDa fragment of FN can neutralize the functions of TG2 on cell surfaces [6]. Binding to TG2 is so specific that an affinity column made by coupling the 42-kDa fragment of FN to a gel matrix can be used for isolating hRBC TG2 to the highest purity with a single passage of hemoglobin-depleted erythrocyte lysate [5] (Figure 1A). This procedure was employed for purifying the TG2 protein on which nucleotide-binding studies were carried out [7], and on which the large conformational change—attendant to binding GTP—could be demonstrated by transition from a slow-moving, extended structure to a faster moving, compact configuration in nondenaturing electrophoresis [8] (Figure 1B). FIGURE 1 (A) Affinity purification of TG2 by single passage of the hemoglobin-depleted lysate of hRBCs through a column of the 42-kDa gelatin-binding fragment of human fibronectin. The Hb-depleted cell lysate was applied to the affinity column. After extensive ... TG2s of different species vary in sensitivities to inhibition by GTP, but hRBC TG2 binds tightly to the nucleotide (measured by a fluorescently labeled analog), with an association constant of 4 × 107 M−1 [7]. Even in the highly purified form, this TG2 seems to exist preponderantly in the closed compact, inactive configuration of the enzyme, corresponding to the electrophoretically fast-moving GDP-bound form (Figure 1B). It is perhaps more relevant to the present discussion that human red cells provided the paradigm for showing that TG2—though inactive in the intracellular milieu—becomes rapidly converted by entry of Ca2+ to an active transamidase, producing profound alterations in the structural organization and physical properties of the cell [10–14]. It is remarkable that the changes brought about by treating normal hRBCs with Ca2+ plus ionophore closely parallel those seen in some erythrocyte diseases in which the lifespans of the cells are appreciably shortened. Therefore, the sequence of events in the hRBC diseases, and also in the experimental model with Ca2+ overload, may be illustrated by Scheme 1. SCHEME 1 The Ca2+-triggered, transglutaminase-mediated protein crosslinking cascade in cells. In the resting cell, TG2 is kept in the inactive, latent form by virtue of its tight binding to GTP. However, the entry of Ca2+ ions removes the inhibition by GTP and allows expression of transamidating activity. The enzyme catalyzes the cross-linking of protein substrates (P1, P2, P3, …, Pn) by covalent γ:e isopeptide bridges and the concomitant formation of large-molecular-weight polymeric structures in the cell membrane. This activity causes a stiffening of the membrane and irreversible fixation of cell shape [15], which are thought to promote the premature removal of the affected hRBCs from the circulation. Moreover, competitive and noncompetitive inhibitors of TG2 prevent the protein cross-linking by the enzyme (i.e., the formation of abnormal protein polymers) and also block the physical consequences of membrane stiffening and fixation of cell shape. It is important to bear in mind that the polymeric structures created by TG2 action are not conventional molecular aggregates; they cannot be separated into their original building blocks by protein-solubilizing agents (such as urea, weak acids and alkalis, ionic or nonionic detergents, including sodium dodecylsulfate, SDS) or their combinations. The portions of the polymers soluble in a mixture of SDS and a reducing agent, such as dithiothreitol (DTT), contain a fraction of the Ne (γ-glutaminyl)lysine-bonded constituent chain framework—essentially the backbone of a branched structure with ill-defined geometry—onto which other polypeptides may be attached in the cell, for example, by disulfide interchain linkages.